Heterogeneity of Ghrelin/Growth Hormone Secretagogue Receptors

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Neuroendocrinology DOI: 10.1159/000105141

Heterogeneity of Ghrelin/Growth Hormone Secretagogue Receptors Toward the Understanding of the Molecular Identity of Novel Ghrelin/GHS Receptors

Giampiero Muccioli a Alessandra Baragli a Riccarda Granata b Mauro Papotti c

Ezio Ghigo b a

Division of Pharmacology, Department of Anatomy, Pharmacology and Forensic Medicine, b Division of

Endocrinology and Metabolism, Department of Internal Medicine, and c Division of Pathology, Department of

Clinical and Biological Sciences, University of Turin, Turin , Italy

ceptor is that ghrelin and GHS do not always share the same biological activities and activate a variety of intracellular sig-nalling systems besides G q . The biological actions on the heart, adipose tissue, pancreas, cancer cells and brain shared by ghrelin and the non-acylated form of ghrelin (des-oc-tanoyl ghrelin), which does not bind GHS-R1a, represent the best evidence for the existence of a still unknown, function-ally active binding site for this family of molecules. Finally, located in the heart and blood vessels is the scavenger re-ceptor CD36, involved in the endocytosis of the pro-athero-genic oxidized low-density lipoproteins, which is a pharma-cologically and structurally distinct receptor for peptidyl GHS and not for ghrelin. This review highlights the most re-cently discovered features of GHS-R1a and the emerging ev-idence for a novel group of receptors that are not of the GHS1a type; these appear involved in the transduction of the multiple levels of information provided by GHS and ghrelin. Copyright © 2007 S. Karger AG, Basel

Introduction

Ghrelin is the result of a story of reverse pharmacol-ogy, which started more than a quarter of a century ago with the discovery of synthetic, non-natural growth hor-mone-releasing peptides (GHRP). The latter are now

Key Words

Ghrelin � Growth hormone secretagogues � Receptor types

Abstract

Ghrelin is a gastric polypeptide displaying strong GH-releas-ing activity by activation of the type 1a GH secretagogue receptor (GHS-R1a) located in the hypothalamus-pituitary axis. GHS-R1a is a G-protein-coupled receptor that, upon the binding of ghrelin or synthetic peptidyl and non-peptidyl ghrelin-mimetic agents known as GHS, preferentially cou-ples to G q , ultimately leading to increased intracellular cal-cium content. Beside the potent GH-releasing action, ghrelin and GHS influence food intake, gut motility, sleep, memory and behavior, glucose and lipid metabolism, cardiovascular performances, cell proliferation, immunological responses and reproduction. A growing body of evidence suggests that the cloned GHS-R1a alone cannot be the responsible for all these effects. The cloned GHS-R1b splice variant is appar-ently non-ghrelin/GHS-responsive, despite demonstration of expression in neoplastic tissues responsive to ghrelin not expressing GHS-R1a; GHS-R1a homologues sensitive to ghrelin are capable of interaction with GHS-R1b, forming heterodimeric species. Furthermore, GHS-R1a-deficient mice do not show evident abnormalities in growth and diet-induced obesity, suggesting the involvement of another re-ceptor. Additional evidence of the existence of another re-

Received: January 5, 2007 Accepted after revision: May 21, 2007 Published online: July 2, 2007

Ezio Ghigo Division of Endocrinology and Metabolism Department of Internal Medicine, University of Turin Corso Dogliotti 14, IT–10126 Turin (Italy) Tel. +39 011 633 4317, Fax +39 011 664 7421, E-Mail [email protected]

© 2007 S. Karger AG, Basel 0028–3835/07/0000–0000$23.50/0

Accessible online at: www.karger.com/nen

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classified as ghrelin-mimetics. In their pioneering works, Bowers et al. [1, 2] demonstrated that small synthetic D -Trp 2 pentapeptides derived from the natural opiate Met enkephalin, acted at hypothalamic and pituitary sites to stimulate the release of GH. Accordingly, it was hypoth-esized that the GH-releasing effect of the D -Trp 2 GHRP reflected the activity of an unknown endogenous factor or hypophysiotropic hormone distinct from growth hor-mone-releasing hormone (GHRH). The ability of the hexapeptide GHRP-6 to synergize with GHRH with re-markable potency in men [3] , prompted the development of other GHRP-6 analogs (GHRP-1, GHRP-2 and hexare-lin) with high potency [4–6] and some orally active pep-tido-mimetic GH secretagogues (GHS) such as the spiro-peridine derivative MK-0677 [7–12] . Particularly re-markable is the broad range of chemistries of the GHS developed in the last few years by several pharmaceutical groups. They consist of low-molecular-weight peptides, partial peptides and non-peptide molecules [6, 13–15] . Several GHRP/GHS candidates were studied clinically, but none have reached the market [6, 11, 12] . GHS bind to specific sites in the rat forebrain [16] and other brain regions such as the cerebral cortex, hippocampus, me-dulla oblongata and choroid plexus, although the greatest density of binding sites is in the hypothalamus and pitu-itary gland [17–20] . The GHS receptor was in fact cloned in 1996 from tissues of the hypothalamo-pituitary axis [18] : the codified protein became known as the ‘type 1a GHS receptor’ (GHS-R1a). Three years later, in 1999, Ko-jima et al. [21] isolated and characterized a natural bioac-tive ligand for this receptor, which was found to be a small polypeptide primarily secreted by the stomach. This pep-tide was named ‘ghrelin’ and was described as a ‘GH-re-leasing substance’. Ghrelin was shortly identified as a mo-tilin homologue or the ‘motilin-related polypeptide’ [22, 23] , which had been found capable of stimulating gut mo-tility [24, 25] . Since this time, numerous other neuroen-docrine effects have been attributed to ghrelin, such as stimulation of CRH, ACTH, PRL secretion and inhibi-tion of GnRH and gonadotropin release [26–28] . In addi-tion, non-endocrine and metabolic activities of ghrelin have also been described, notably stimulation of food in-take and gut motility [27, 29–33] , influences on body weight and energy balance [34] , insulin release, glucose and lipid metabolism [35–43] , sleep [44, 45] , behavioral responses to stress [46–48] , learning and memory [49] , improvements of cardiovascular performances [50–54] , effects on cell proliferation, differentiation, survival and fetal development [55–61] , besides influencing immuno-logical responses [ 62 , see also for reviews 63–66 ]. The

ghrelin gene resides on chromosome 3p25-26, has five exons and four introns and produces two distinct mRNAs, which start at position –80 or –555. The main mRNA codifies for the 117-amino-acid precursor polypeptide ‘preproghrelin’ which, through enzymatic processing, leads to ‘proghrelin’ and subsequently to mature ghrelin and a C-terminal polypeptide [67, 68] .

Mature ghrelin consists of 28 amino acids and it is es-terified by an octanoyl group at a serine 3 residue; this is a special feature of this hormone which is conserved across species. Acylation is necessary for ghrelin binding to GHS-R1a [69] , GH-releasing activity in vivo [70] and other central and peripheral endocrine and non-endo-crine actions [71–73] of the peptide. The modification also determines the extent and the direction of ghrelin transport across the blood-brain barrier [74] .

Although the active site for ghrelin action appears to be 4–5 amino acids of the amino terminal end of the mol-ecule including the acyl group [69, 75, 76] , short ghrelin peptides consisting of residues 1-8 neither displace ghre-lin binding to pituitary and hypothalamic membranes nor stimulate GH release in vivo [70] . Quite on the con-trary, in GHS-R1a-transfected cells and in adipocytes, the acylated fragments longer than 4 amino acids all maintain affinity and potency similar to that of ghrelin [69, 77, 78] .

In humans, multiple ghrelin-derived molecules dif-fering in their acyl groups on serine 3 (decanoyl or dec-enoyl-esterified molecules) have also been isolated from the stomach and found in the bloodstream [79] . These acyl-modified ghrelins are capable of stimulating GH re-lease in rats to a degree similar to that of octanoylated ghrelin [79] . In addition to the acylated form, unacylated ghrelin (UAG) is found in circulation at far greater con-centrations than ghrelin (octanoylated/des-octanoylat-ed ratio 1: 4) [80] suggesting physiological relevance. UAG, however, does not bind to GHS-R1a [80] , does not displace ghrelin binding to rat hypothalamus or pitu-itary membranes and it is unable to stimulate GH release in vivo [70, 80] , so it was initially considered inactive [21] . A conspicuous number of recent reports, however, describe non-endocrine activities of UAG [63–65] break-ing a paradigm lasting from 1999. Besides ghrelin, a wide variety of polypeptides is encoded by the ghrelin gene. Alternate splicing phenomena, post-translational modi-fications and differential processing create a number of products, some of which, e.g. des-Gln 14 -ghrelin, are found in circulation with the same biological activities as ghrelin [81, 50] . Other products originating in central and peripher al tissues are devoid of any endocrine effect

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[82–87] . Amongst these is the 23-amino-acid polypep-tide obe statin that may be an appetite suppressant [84] , although this is widely debated [86, 87] and various exon-deleted proghrelin mRNAs, which are expressed in neoplastic tissues [82, 83] . The multiplicity of ghrelin/GHS effects prompted the question as to whether GHS-R1a is the single receptor that mediates action or wheth-er other receptors are involved [54, 65] . In particular, UAG appears to possess biological activities arguing for the role of other receptors to mediate effects of the ghre-lin family.

Type 1a GHS Receptor

The GHS receptor gene is located on chromosome 3q26.2 and encodes for two transcripts, 1a which encodes a full-length receptor (GHS-R1a) and 1b which codifies for a shortened version (GHS-R1b) [88] . In human fetus-es, GHS-R1a mRNA is detected at 18 and 31 weeks of ges-tation, indicating that ghrelin might be active early in development [59, 89] . Significant expression of GHS-R1a mRNA is evident in the pituitary gland and several en-docrine and non-endocrine tissues [90] as well in the cen-tral nervous system (CNS), where GHS-R1a is found both in the cortex and in the midbrain [17, 91–93] . This is con-sistent with observations that ghrelin affects synaptogen-esis and development of neuronal circuits, as well as mod-ulation of memory processes, sleep patterns and behavior [44–49, 94–96] . Within the hypothalamus-pituitary axis, GHS-R1a mediates ghrelin/GHS modulation of GH, PRL, CRH/ACTH and GnRH/gonadotropin secretion [26–28, 97–102] . Orexigenic effects of ghrelin/GHS are likely me-diated by GHS-R1a expressed by hypothalamic neurons containing neuropeptide Y (NPY)/Agouti-related pro-tein [34, 103–106] . Additionally, centrally located GHS-R1a was found to mediate ghrelin activity on gastric acid secretion [107] . Peripheral GHS-R1a is responsible for ghrelin inhibitory effect on pancreatic insulin secretion [42, 43] , for the prevention of oxidative stress [108, 109] and for GHS control of pro-inflammatory and immune responses [62, 110, 111] . Finally, GHS-R1a has also been found in neoplastic tissues, where it mediates the stimu-latory effect of ghrelin on neoplastic cell growth [ 58, 83 ].

Structure The human GHS-R1a is a polypeptide of 366 amino

acids with a molecular mass of approximately 41 kDa [18, 112, 113] and belongs to family A of G-protein-coupled

receptors (GPCRs) [114] . GPCRs span the membrane with seven � -helix hydrophobic domains forming the re-ceptor core, joint each other by three alternate intra- and extracellular domains, beginning with an extracellular N-terminal domain and ending with an intracellular C-terminal domain [114] . GHS-R1a possesses the three conserved residues Glu-Arg-Tyr at the intracellular end of transmembrane 3 (TM3) domain, in position 140–142 (ERY/DRY motif), which are important for the isomeri-zation between the active and inactive conformation (see below), and the two cysteine (Cys) residues on the extra-cellular loop 1 and 2 forming a disulfide bond [113, 114] . Based on its deduced peptide sequence, GHS-R1a is not obviously related to known subfamilies of GPCRs, al-though it is often included in a small family of receptors for small polypeptides comprising the receptor for moti-lin (52% homology), neurotensin receptor-1 (NTS-R1) and NTS-R2 subtype (33–35% homology), neuromedin U receptor-1 (NMU-R1) and NMU-R2 subtype ( ; 30% homology), and the orphan receptor GPR39 (27–32% ho-mology) [115, 116] , named GHS-R1a homologues. Also, the thyrotropin-releasing hormone receptor possesses high homology (56%) to GHS-R1a [113] . Comparison of the predicted human rat, pig and sheep GHS-R1a amino acid sequences reveals 91.8–95.6% sequence homology [117] .

Ligands and Ligand Binding Domains Ghrelin and its natural acylated variants, as well as

synthetic GHS bind with high affinity to the GHS-R1a. Their efficacy in displacing radiolabeled non-peptidyl GHS ([ 35 S]MK-0677) or [ 125 I]Tyr 4 -ghrelin binding from pituitary membranes or the cloned GHS-R1a correlates well with concentrations required to stimulate GH re-lease [17, 69, 118] . By contrast, UAG does not bind GHS-R1a [69, 70, 118] . Adenosine was initially proposed to be a partial agonist for GHS-R1a [119–121] , but this has been questioned [122, 123] . A series of other molecules appar-ently unrelated to ghrelin have also been shown to bind GHS-R1a, such as the natural SRIH-like neuropeptide cortistatin, some synthetic SRIH-mimetic octapeptides (octreotide, lanreotide and vapreotide) [124–126] and the atypical L-type Ca 2+ channel blocker diltiazem [127] . Our own findings support the hypothesis that cortistatin (CST) acts as an endogenous antagonist for GHS-R1a [124–126] . In fact, CST not only binds all five SRIH re-ceptor subtypes (SRIH-Rs), but also displaces radiola-beled ghrelin from its pituitary-hypothalamic binding sites and diminishes ghrelin secretion in humans [124, 128] . Recently, a synthetic CST-derived octapeptide,

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CST-8, which does not bind to SRIH-Rs, was shown to interact with GHS-R and to remove the inhibitory action of ghrelin on gastric acid volume and acid output [107] . An inverse agonist for GHS-R1a is the synthetic D -Arg 1 - D -Phe 5 - D -Trp 7,9 -Leu 11 -substance P, a substance P and bombesin antagonist [129] ; this is able to reduce constitu-tive signalling of GHS-R1a overexpressed in COS-7 cells [130, 131] , but an endogenous counterpart remains to be identified. Most recently, shorter peptides derived from D -Arg 1 - D -Phe 5 - D -Trp 7,9 -Leu 11 -substance P were shown to display inverse agonist activity on GHS-R1a as well [131] . Finally, GHS-R1a antagonists are also available, such as D -Lys 3 -GHRP-6, L765-867 [6, 7, 120] , isoxazole, diaminopyrimidine and triazole derivatives [132–134] . Antibodies and RNA-Spiegelmers have been also devel-oped for the blockade of GHS-R1a activity [135, 136] .

Ligand binding to receptors is believed to stabilize the active conformation. While the main binding pocket for small amines is deep in the cavity created by the TM do-mains, small peptides also bind to extracellular epitopes. According to the general model based on the � 2 -adrener-gic receptor and the rhodopsin receptor, either methods of binding result in an alteration of receptor molecular structure leading to a reciprocal re-arrangement of the � -helices with vertical seesaw movements of TM6 and TM7 around a pivot represented by the proline residues in the middle of these helices [137] . Hence the intracel-lular end of TM6 and TM7 move away from the center of the receptor toward TM3, exposing the sites subsequent-ly recognized by G-protein and � -arrestin [137] . The ‘toggle switch model’, as it is known, is applicable to GHS-R1a, with a binding domain for the natural ligand ghrelin involving six amino acids located in TM3, TM6 or TM7 [131] . According to Pedretti et al. [138] , ligand binding and activation of GHS-R1a by ghrelin requires the ligand to interact with one pocket formed by polar amino acids and one formed by non-polar amino acids found in TM2/TM3 and TM5/TM6, respectively. On the contrary, the inverse agonist D -Arg 1 - D -Phe 5 - D -Trp 7,9 -Leu 11 -substance P requires a wider binding pocket, dis-persed across the main binding crevice [131] . Concerning the synthetic peptidyl and non-peptidyl GHS, they share a common binding pocket in the TM3 region of the GHS-R1a, although there are other distinct binding sites in the receptor that appear to be selective for particular classes of agonists [139] . The basic amine common to peptidyl (GHRP-6) and non-peptidyl (MK-0677) GHS likely es-tablishes an electrostatic interaction with Glu124 in the TM3 domain [139] , as substitution of glutamine for glu-tamic acid [Glu124Gln mutant] in human GHS-R1a in-

activates receptor function. Also, mutating Arg283 in TM6, which interacts with Glu124 eliminates both ago-nist-induced activation and constitutive signalling [130, 139] . Finally, the activity of all agonists can be complete-ly abolished by disrupting the disulfide bond between Cys116 and Cys198 in the extracellular portion of the re-ceptor, by mutating Cys116 into alanine [Cys116Ala mu-tant] [117, 139] .

Prototypical and Alternative Signalling Once bound to GHS, activated GHS-R1a normally

binds the G � q/11 subunit of a G-protein, which leads to activation of phospholipase C (PLC) and consequent hy-drolysis of membrane-bound phospholipids to generate inositol (1,4,5)-triphosphate (IP 3 ) and diacylglycerol (DAG) [18, 19] . The intracellular free calcium (Ca 2+ ) con-centration increases because of the rapid, though tran-sient, release of Ca 2+ from IP 3 -responsive cytoplasmatic storage pools and because of a more sustained accumula-tion of Ca 2+ due to the activation of L-type Ca 2+ channels. Together with the blockade of potassium (K + ) channels, the intracellular rise in free Ca 2+ provokes depolarization of the somatotropes and release of GH [18, 19, 140] . In addition to the ‘prototypical signalling’ of GHS-R1a, spe-cies-, tissue- and ligand-specific second messenger path-ways are activated by various GHS in an ‘alternative-sig-nalling mode’. In sheep, but not in rat pituitary cells, GHRP-2 activates the adenylyl cyclase/cyclic adenosine monophosphate/protein kinase A (AC/cAMP/PKA) pathway, while GHRP-6 and non-peptidyl GHS act through the PLC pathway to enhance GH release [141] . In porcine somatotropes, ghrelin-stimulated GH secre-tion depends on activation of PLC/PKC, AC/PKA and extracellular Ca 2+ influx through L-type voltage-sensi-tive channels, as well as on nitric oxide/cyclic guanosine monophosphate (cGMP) signalling, suggesting that mul-tiple signalling pathways originate from GHS-R1a [142–144] . In GH3 cells, ghrelin modulates K + currents through cGMP production [145] , while in primary cultures of rat pituitary cells GHRP-6 stimulates Na + conductance [146] . Because of the opening of N-type Ca 2+ channels, which are modulated by cAMP-dependent PKA activation, GHS-R1a was suggested to couple to Gs in NPY neurons [147] . Although there is evidence linking GHS-R1a to Gs, protein/protein interaction between GHS-R1a and Gs was never shown in those cells or in heterologously ex-pressing ones. A GHS-R1a homologue also displays sim-ilar pleiotropy, NTS-R1: it preferentially couples to Gq/11, although, depending on the cell type, it is capable of in-creasing cGMP, cAMP and IP 3 , as well as modulating ex-

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tracellular-signal-regulated kinase-1 and -2 (ERK1/2) signalling [148–151] .

Besides pleiotropy, a cross-talk between Gq and Gs might be responsible for the multiple effects of GHS-R1a, as demonstrated for diacylglycerol-activated PKC stimu-lation of adenylyl cyclase [152, 153] . Different ligands may also induce different conformations of GHS-R1a, favor-ing coupling to different G-proteins and activation of dis-similar signal transduction pathways, similarly to other GPCRs [154] ; nevertheless, a more appealing hypothesis is that more than one receptor for GHS is endogenously expressed [141] and that GHS-R1a can heterodimerize with other GPCRs or membrane proteins creating new binding sites distinct in terms of pharmacological and functional properties (see below).

Although ghrelin can induce ERK phosphorylation in cells overexpressing GHS-R1a [130] involving novel iso-forms of PKC [155] and increase the activity of both the transcription factor cAMP-responsive element (CRE)-binding protein and of serum-responsive element (SRE) [130, 156] , several reports indicate a role for ghrelin in cell survival through such pathways, but independently from GHS-R1a activation [56, 60, 61, 157, 158] .

Constitutive Activity The number of GPCRs displaying constitutive activity

with clear physiological implications is not vast, but it is steadily growing [159] . Several reports have now demon-strated the physiological relevance of GHS-R1a constitu-tive activity. In a study by Wang et al. [160] , Phe279Leu (corresponding to PheVI:16) and a Ala204Glu polymor-phisms were found in one obese and one short stature child. A recent study has re-stated that an Ala204Glu missense allele segregates with both short stature and obesity. Surprisingly the derived GHS-R1a receptor dis-played reduced basal activity and lower expression at the plasma membrane when transfected to HEK-293 cells [161] . It should be noted that, although constitutive activ-ity of the receptor had been lost, responsiveness to ghrelin was intact [161, 162] . Both studies indicate that constitu-tive activity of GHS-R1a in vivo might be mandatory for proper growth and development of the human body [162] .

When overexpressed in COS-7 cells, GHS-R1a pos-sesses a constitutive activity in terms of the turnover of IP 3 , which is approximately 50% of the maximal agonist-induced activity [130, 131] . In HEK-293 cells, GHS-R1a transfection led to constitutively stimulated CRE and SRE activity [130] . Intriguingly, GHS-R1a did not show any constitutive activity in the pituitary cell line RC-4B/

C40: this might indicate that GHS-R1a activity may be turned off or on depending on the cellular context [140] ; so far the only known ligand blocking GHS-R1a constitu-tive activity is D -Arg 1 - D -Phe 5 - D -Trp 7,9 -Leu 11 -substance P [130, 131] .

The molecular basis of such constitutive activity ap-pears to relate to three aromatic residues located in TM6 and TM7, namely PheVI:16, PheVII:06 and PheVII:09. This region promotes the formation of a hydrophobic core between helices 6 and 7, to ensure proper docking of the extracellular end of TM7 into TM6, mimicking ago-nist activation and stabilizing the receptor in the active conformation [156] .

Modulators of Receptor Signalling In the past 2 years a blossoming of papers has ad-

dressed the issue of ‘fine tuning’ of GHS-R1a signalling through multiple mechanisms. In this section, three dis-tinct aspects of this regulation are discussed: ago-alloste-ric modulation, heterodimerization and receptor inter-nalization. Allosteric modulators increase (positive allo-sterism) or decrease (negative allosterism) the potency (EC 50 ) of the agonist, shifting the dose-response curve to the left or to the right, respectively. Most recently, the term ‘ago-allosterism’ has been used to indicate com-pounds, which not only modulate potency, but also ago-nist efficacy (E max ), being classified as full or partial ago-nists: two non-peptidyl (L-692,429, MK-0667) and one peptidyl agonists (GHRP-6) of GHS-R1a fall into this cat-egory, L-692,429 improves ghrelin potency and efficacy (positive ago-allosterism), MK-0667 has no effect on ghrelin potency but increases its efficacy (neutral ago-al-losterism), while GHRP6 reduces ghrelin potency, though increasing its efficacy (negative ago-allosterism) [163] . Since ago-allosterism has been recently described for di-meric GPCRs [164, 165] , likewise GHS-R1a may behave as dimeric receptor, with one ‘orthosteric’ protomer bind-ing ghrelin and one ‘allosteric’ protomer binding the ‘ago-allosteric’ compound resulting in a modulation of both ghrelin potency or efficacy [163, 166] .

With regard to heterodimerization, the synergistic ef-fect of ghrelin and GHRH on GH secretion [167] , the lack of effect of peripheral ghrelin once GHRH antagonists are administered in men [168–170] and the modulation of GHS-R1a expression by GHRH both in hypothalamic neurons and pituitary cells [171–175] , suggest a strong interplay between these two hormonal systems which may involve heterodimer formation [176] . Although GHS- and GHRH-activated intracellular signalling path-ways differ in pituitary cells, in that GHS increases free

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intracellular Ca 2+ , while GHRH increases cAMP levels, when applied together, intracellular accumulation of cAMP raises above the GHRH-stimulated level [177] . Al-though PKC appears involved [153] , heterodimer forma-tion between GHRH-R and GHS-R1a mainly triggers this potentiation [177] . Similarly, Jiang et al. [178] have recently reported about the heterodimeric interaction be-tween GHS-R1a and dopamine D 1 receptor subtype, which resulted in enhanced signalling through the AC/cAMP pathway. Thus, GHS-R1a heterodimers represent novel receptors, which bind ghrelin but may display dif-ferent pharmacological and functional properties. This likely accounts for at least some of the ‘alternative signal-ling’ previously described.

In response to agonists, GPCRs undergo desensitiza-tion through phosphorylation, uncoupling from G-pro-teins and internalization. This may lead, depending on the receptor, to their recycling back to the plasma mem-brane or passage into lysosomes for degradation [179] . In cells overexpressing GHS-R1a, Camina et al. [180] demonstrated that prolonged administration of ghrelin or hexarelin determined a rapid attenuation of receptor-mediated Ca 2+ accumulation and a conspicuous recep-tor internalization through clathrin-coated pits within 20 min. On the other hand, Holst et al. [156] reported constitutive internalization of the ghrelin receptor, which could be prevented by the presence of its inverse agonist D -Arg 1 - D -Phe 5 - D -Trp 7,9 -Leu 11 -substance P. Al-though modulation of constitutive receptor internaliza-tion by inverse agonists is common to other receptors [181–184] , the above-mentioned experimental discrep-ancies suggest that additional studies are needed to clar-ify the situation. Conversely, in in vivo studies, GHS-R1a upregulated during fasting [185, 186] , and during the hyperdynamic phases of sepsis in male adult rats [187] .

Non-Type 1a GHS Receptors

If heterodimeric receptor complexes may partially ac-count for the GHS-R1a ‘alternative signalling’ in response to ghrelin, it is likely that other, as yet unidentified recep-tors exist. In fact, remarkable differences in the binding profile among ghrelin, synthetic peptidyl (hexarelin) and non-peptidyl (MK-0677) GHS have been reported [188–191] , mostly in tissues that do not express GHS-R1a or express the receptor at a low level. For instance, the heart possesses GHS-binding sites specific for peptidyl GHS only [190–192] . The existence of various GHS-R1a homo-

logues and of a splice variant GHS-R1b, the lack of a clear phenotype in GHS-R1a knockout mice and female rats genetically deficient of GHS-R1a, as well as the presence of multiple endogenous ghrelin-like ligands, strongly suggest the existence of multiple receptors for ghrelin and GHS.

GPCR Homologues of GHS-R1a All GHS-R1a homologues bind gastrointestinal or

neuronal peptides except for GPR39, for which a ligand is still unknown [87, 116] . The highest homology is shared by GHS-R1a and the GPR38 receptor for motilin [113, 115] . The ghrelin and motilin genes are also structurally similar, despite the encoded polypeptides being different. GRP38 is less widely expressed in the neuroendocrine tis-sues than GHS-R1a, being mostly confined to the thyroid gland, bone marrow, stomach and gastrointestinal smooth muscles. Nevertheless, both receptors mediate pulsatile biological effects upon continuous stimulation and increase gastrointestinal motility. In response to mo-tilin, GRP38 increases cytosolic free Ca 2+ , consistently with IP 3 -dependent Ca 2+ release, mediated by its cou-pling to G � q /G � 13 [193] .

The other GHS-R1a homologue, GRP39, remains an orphan receptor since the recent controversy whether or not it binds obestatin, a sub-product of the ghrelin gene, has been solved [84, 85, 194–197] . Its structure is obvi-ously similar to the GHS-R1a with the fundamental ami-no acids being conserved in the key sites for receptor function [115] . GRP39 transcripts are detected in many tissues, but mostly in brain regions [115] . In contrast to GHS-R1a and GRP38, GRP39 has a very long C-terminal and two potential palmitoylation sites (C360-1), which creates a 4th intracellular loop [115, 198] . Activation of GRP39 by zinc (Zn 2+ ) leads to PLC signalling, activation of CRE- and SRE-dependent transcriptional activity, and also cAMP production [87] . Besides receptor sequence homology with GHS-R1a, GPR39’s relationship with the ghrelin system remains elusive and unexplored yet.

Neuromedin U is a 23- to 25-amino-acid polypeptide which is the ligand for other members of this receptor family, the NMU-R1 and NMU-R2 receptors. NMU-R1 and NMU-R2 have a 40–50% homology and both are about 30% homologous with GHS-R1a and the neuroten-sin receptors [199] . While the NMU-R1 subtype is most-ly expressed in the periphery, NMU-R2 is mostly ex-pressed in the brain [199] . Both receptors are implicated in smooth muscle contraction, in the regulation of gastric acid secretion, insulin secretion, ion transport in the gut, feeding behavior and stress [199] . Both NMU-R1 and

Ghrelin/GHS Receptors Neuroendocrinology 019-T1 7

NMU-R2 signal through activation of Gq/11, PLC and Ca 2+ , with the R2 subtype stimulating arachidonic acid production through activation of PLA 2 [199] . NMU is highly conserved among species, is widely distributed in the body and expressed at high levels in the brain where it mediates effects on food intake opposite to those of ghrelin [200] , probably by cross-talking with the anorec-tic polypeptide leptin system [200, 201] . NMU also ap-pears to play some function in the growth of neoplastic cells with expression being downregulated in cancer [202] ; in fact it was demonstrated to inhibit esophageal squamous cell carcinoma cell growth [203] .

A most intriguing discovery was that in absence of its receptors, neuromedin was capable of stimulating the heterodimeric receptor GHS-R1b/NTS-R1 in order to in-duce lung cancer cell proliferation [204] . Although it is difficult to make conclusions on the basis of one single work, the hypothesis of heterodimeric assortment be-tween ghrelin receptor homologues as a mean to create pharmacological and functional diversity is an extremely appealing one. Neuromedin might thus function as a me-diator of the cross-talk between ghrelin receptor homo-logues, which, at least in cancerous cells, leads to aberrant signalling.

The other group of receptors included in the ‘ghrelin superfamily’ are those for NTS, namely NTS-R1 and NTS-R2. NTS-R1 mainly functions through Gq/11-PLC, but it is also capable of activating cGMP, stimulating cAMP, inositol phosphate signalling and ERK1/2 phos-phorylation [148–151, 205, 206] , while NTS-R2 activation signals via increased Ca 2+ accumulation and ERK phos-phorylation [207–209] . Similar to GHS-R1a, a splice vari-ant of the NTS-R2 has been recently found, bearing only the first five TM domains, albeit pharmacologically and functionally active [210] .

NTS is a 13-amino-acid polypeptide, with biological function being ascribed to the C-terminal portion. The peptide has high sequence homology with neuromedin N. In the CNS, NTS-Rs have been found in the hypothal-amus, amygdala and nucleus accumbens, being involved in modulation of the dopaminergic system, but neuroten-sin also acts in the small intestine endocrine cells where it increases acid secretion and regulates smooth muscle contraction [205] .

Besides their sequence homology and signalling simi-larities, to date heterodimerization of GHS-R1b and NTS-R1 is the only available evidence for a functional cross-talk amongst GHS-R homologues with physiologi-cal implications [204] .

Type 1b GHS Receptor Type 1b GHS receptor (GHS-R1b) is a splice variant

of the GHS-R1a. GHS-R1b is a truncated receptor, con-taining 298 amino acids corresponding to the first five TM domains (encoded by exon 1), plus a unique 24-ami-no-acid ‘tail’ encoded by an alternatively spliced intron-ic sequence. In GHS-R1b-transfected cells, the receptor did not bind ghrelin or GHS and the cells did not respond to these ligands [19] . It was concluded, therefore, that this receptor was not of biological significance. Never-theless, since it is widely expressed in many normal GHS-R1a-positive or -negative tissues [90] , it is likely that this receptor possesses biological functions. A recent report has revealed that GHS-R1b acts as a repressor of the con-stitutive activity of GHS-R1a when overexpressed in HEK-293 cells [211] ; thus GHS-R1b may represent an en-dogenous candidate for GHS-R1a modulation. The pres-ence of the 1b form in neoplastic tissues and its overex-pression in growing and differentiating cells [57, 212–214] supports a significant role in tumor progression. In lung cancer-derived cell lines, NMU induced cell prolif-eration through binding to the heterodimeric receptor GHS-R1b/NTS-R1 [204] ; it is then apparent that GHS-R1b is at least capable of forming heterodimers with full-length GPCRs receptors, causing altered biological prop-erties compared to the original receptor, a fascinating behavior which is indeed shared by neurotensin trun-cated receptor 2 [210] .

Ghrelin/GHS-Binding Sites Shared by Non-Acylated Ghrelin An early report on the physiological role of non-acyl-

ated ghrelin (UAG) demonstrated that, together with ghrelin and other GHS, this natural ghrelin variant pre-vented cell death of cultured cardiomyocytes and endo-thelial cells by activation of ERK1/2 and kinase B/AKT [56] . This evidence implies that UAG is a biologically ac-tive peptide, which exerts its function through a receptor that is not GHS-R1a [56] . UAG is also active on isolated guinea pig papillary muscle where exerts negative inotro-pic effects similar to those of ghrelin and peptidyl GHS [50] . The potency of these compounds correlates with their ability to compete with [ 125 I]Tyr 4 -ghrelin, indicat-ing a common binding site [50] . In the past 3 years an increasing number of ghrelin-sensitive tissues or cell lines have been shown to be UAG-responsive, occasion-ally with different pharmacological and functional pro-files. Both ghrelin and UAG were shown to stimulate neurogenesis of rat fetal spinal cord [215] and to augment osteoblast proliferation through the ERK/PI 3 K pathways

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Neuroendocrinology 019-T1 8

during the differentiation period, despite the undetect-able expression of GHS-R1a [61] . Similarly, both ghrelin and UAG modulate the survival and proliferation of pan-creatic � cells and human islet cells [60] . Interestingly, in bone marrow of GH-deficient rats, both ghrelin and UAG, but not the potent GHS-R1a agonists such as L163-255, stimulated adipogenesis, thus excluding a ghrelin/GHS-R1a-mediated effect [39] . Similarly, rat adipocytes isolated from epididymal adipose tissue that did not ex-press mRNA for GHS-R1a, specifically bound radiola-beled ghrelin, which was equally displaced by both unla-belled ghrelin and UAG and slightly less potently by hexarelin and MK-0667 [78] . Moreover, UAG inhibited isoproterenol-stimulated lipolysis in rat epididymal adi-pocytes in a manner similar to ghrelin [78] . If ghrelin, UAG and GHS mostly mediate cell proliferation and/or differentiation of normal tissues by activating this ‘shared receptor’, they exert opposite effects on the growth of some neoplastic cell lines derived from human prostate carcinomas [58] , raising the issue as to whether the re-ceptor is the same but the environment has evidently changed, or if a different receptor is involved [57] . With a comparable pattern, ghrelin, UAG and some GHS (MK-0667 and hexarelin) displace 125 I-Tyr 4 -ghrelin binding to membranes of cells from human prostate carcinomas and the PC-3 prostate cancer cell line, which do not express GHS-R1a or GHS-R1b. This pattern of competition, however, is also seen in DU-145 prostate cancer cells, which do express GHS-R1a and GHS-R1b [58] . These binding studies indicate the presence of a specific bind-ing moiety, common for ghrelin, UAG and GHS, which is likely to be involved in mediating the antiproliferative effects of ghrelin/GHS molecules, which is not the 1a or 1b receptor. The involvement of non-GHS-R1a receptors in the control of neoplastic cell growth is consistent with their presence only in the tumoral stages of tissues de-rived from organs that normally do not express them, e.g. breast [55] . In breast tumors, the highest binding activity is present in well-differentiated invasive breast carcino-mas and is progressively reduced in moderately to poorly differentiated tumors [55, 57] . GHS-R are also present in both estrogen-dependent (MCF7 and T47D) and estro-gen-independent (MDA-MB231) breast cancer cell lines, in which ghrelin, synthetic GHS and EP-80317 (a hexare-lin analog devoid of GH-releasing effect) inhibit cell pro-liferation at concentrations close to their binding affinity [55] . The above evidence clearly demonstrates that at least one receptor exists, different from 1a or 1b which is shared by the acylated and non-acylated form of ghrelin, eventually recognized by GHS.

Receptors for Non-Acylated Ghrelin That Are Not Shared by Ghrelin The existence of receptors which distinguish between

ghrelin, UAG and selective GHS was suggested by the finding that in isolated pig primary hepatocytes, lacking GHS-R1a mRNA expression, ghrelin increased glucose release, hexarelin was ineffective, while UAG antago-nized not only ghrelin-induced but also glucagon-in-duced glucose release [40] . In humans, the combination of ghrelin and UAG prevented ghrelin-induced reduction in the peripheral sensitivity to insulin [216] and elimi-nated the negative ghrelin effect on insulin release and elevation of glucose levels [217] . These findings suggest potentially antagonistic roles of the two molecules. Two receptors may be responsible for such opposite effects, nevertheless it is possible that both ghrelin and UAG bind to the same receptor in a competitive manner, activating two different signalling pathways. The existence of a UAG receptor not shared by ghrelin was strongly sug-gested by Gauna et al. [218] . These data indicated that both ghrelin and UAG stimulated insulin release in insu-linoma INS-1E cells, but while ghrelin acted through GHS-R1a because its effect was completely blocked by the GHS-R1a antagonist D -Lys 3 -GHRP-6, UAG appeared to interact with a different receptor, since its effect on insu-lin secretion was not blocked by D -Lys 3 -GHRP-6 [218] . Recently, the capability of GHS-R1a only (and conse-quently of acylated ghrelin only) to influence food intake has been revised since UAG was shown capable of modu-lating appetite itself in several experimental models. If centrally administered, UAG stimulates appetite in GHS-R1a knockout mice insensitive to the ghrelin orexigenic activity, probably through stimulation of an unidentified receptor localized on orexin neurons [219] . On the con-trary, UAG has been reported to prevent ghrelin-induced food intake in goldfish [220] .

GHS-R not only include receptors for ghrelin and/or UAG, but there is evidence of receptors which do not bind ghrelin/UAG and are specific for synthetic peptidyl GHS only. The only one of these receptors that is well charac-terized is CD36 (see below). Nevertheless, it is worth mentioning the existence of binding sites for hexarelin shared by other peptides structurally related to hexarelin such as GHRP-6 and EP-80317, but not by ghrelin, MK-0667 and EP9399 (a cyclic derivative of hexarelin), which were found in CALU-1 lung carcinoma cell line, and may be capable of reducing cell growth stimulated by insulin-like growth factors (IGFs) [221] .

Ghrelin/GHS Receptors Neuroendocrinology 019-T1 9

CD36 Receptor for Synthetic Peptidyl GHS Specific binding sites for Tyr-Ala-hexarelin and other

peptidyl GHS, which appear to exist in greater amounts than GHS-R1a, have been found in some endocrine glands (pituitary, thyroid and adrenal) and in a wide range of non-endocrine peripheral animal and human tissues such as heart, lung, arteries, skeletal muscle, kid-ney, liver, uterus and adipose tissue [190, 191] . These binding sites are presumably different to GHS-R1a be-cause they show a very low binding affinity for non-pep-tidyl GHS such as MK-0677 and no binding affinity for ghrelin. Studies utilizing a hexarelin derivative, Tyr-Bpa-Ala-hexarelin, that is photoactive and has the same bio-logical activities as the native molecule, demonstrated the existence of a GHS-R subtype in human and bovine pi-tuitary with a molecular weight of 57 kDa [188] and a second receptor subtype for peptidyl GHS in rat heart with a molecular weight of 84 kDa [192] . The tripeptide EP-51389 is as effective as hexarelin in stimulating GH secretion in the rat, but is far less effective in protecting the heart from ischemia [222] . Binding experiments re-vealed that EP-51389 effectively displaced hexarelin from hypothalamic binding sites, but poorly from cardiac membranes [223] , confirming that different GHS-R are present at the two levels. Recently, CD36, a multifunc-tional class B scavenger receptor expressed in many tis-sues, including microvascular endothelium, skeletal and smooth muscle cells and monocytes/macrophages [224] , was identified as a peptidyl GHS cardiac binding site [192, 225] . CD36 is implicated in multiple physiological functions (i.e. antigen presentation, cellular adhesion, fatty acid/lipid transportation and modulation of vascu-lar tone), as well as pathophysiological processes related to the formation of macrophage foam cell and atheroscle-rotic lesions [224] . In a perfused heart preparation, hexarelin elicited vasoconstriction perhaps via CD36, since no similar effect occurred in CD36 null mice and rats [225] . Since ghrelin and MK-0677 do not share all the cardiotropic actions of peptidyl GHS [222, 226–228] , it has been suggested that the reason for this discrepancy could reflect a different interaction of ghrelin, hexarelin and MK-0677 with a heterogeneous population of cardio-vascular GHS-R: some (GHS-R1a) recognized by ghrelin, hexarelin and MK-0677 [19, 21] , some specific for ghre-lin, UAG and hexarelin [50, 56, 226, 227] and others spe-cific (CD36) for hexarelin alone [188, 190–192] . It has also been demonstrated recently that hexarelin inhibits accu-mulation of cholesterol as oxidized low-density lipopro-tein (oxLDL) in macrophages through CD36 by interfer-ing with the binding of oxLDL on the same interaction

site on CD36 [229] . In addition, hexarelin acting through both CD36 and GHS-R1a enhances expression of the ABCA1 and ABCG1 transporters which improve choles-terol efflux from macrophages [229] . In a manner similar to hexarelin, EP-80317, a hexarelin analog devoid of GH-releasing effect, also reduced internalization of oxLDL and increased cholesterol efflux in macrophages, result-ing in a decreased number of atherosclerotic lesions in apolipoprotein E-deficient mice fed with atherogenic diet [230] .

Conclusions

The physiological role of the ghrelin system has been matter of debate for at least three decades. The GHS and ghrelin appear to play a major role in the control of the GH/IGF-I axis, as well as in the regulation of appetite and energy expenditure. The first studies in mouse knockout models for either ghrelin or its receptor (GHS-R1a) showed that these animals were not anorectic dwarves as expected. This evidence contributed to re-dimension the expectations that ghrelin and its analogs, acting as ago-nists or antagonists, might be potential therapeutical agents for treatment of GH deficiency, eating disorders, cachexia or obesity. Quite apart from these potential clin-ical applications, ghrelin physiology has continued to at-tract the interest of many researchers, progressively in-crementing the knowledge about the molecular aspects underlying its involvement in different physiological and pathophysiological conditions. From the basic point of view, the past few years have witnessed the recognition of a number of endogenous ligands (des-Glu 14 -ghrelin, de-canoyl ghrelin) originating from the ghrelin gene, which act on a pleiotropic receptor, the GHS-R1a. In addition, the possible existence of an endogenous inverse agonist has been suggested by the fact that GHS-R1a possesses a high constitutive activity, modulable by synthetic inverse agonists [131] (see also table 1 ). Most of all, the re-evalu-ation of the role played by the non-acylated form of ghre-lin, which shares some of ghrelin effects or elicits oppo-site ones, has strongly indicated the presence of still un-identified multiple receptors subtypes ( table 1 ). Although the cloning of the gene(s) for such receptors has proven difficult, uncovering their molecular identity would rep-resent a major step forwards in this field. On the other hand, GHS-R1a has been demonstrated capable of het-erodimerization and susceptible to allosteric modulation by synthetic GHS. These characteristics may explain, at least in part, the GHS-R1a pleiotropy in different tissues

Muccioli /Baragli /Granata /Papotti /Ghigo

Neuroendocrinology 019-T1 10

and in response to different ligands. Thus, far from being simply a ‘saginary hormone’ [231] , ghrelin and its natural and synthetic analogs possess a wide variety of activities, sometimes ligand- and receptor-specific ( table 1 ). Re-cently, transgenic and knockout animal models, as well as in vitro and in vivo studies, provided evidence for a major role of the ‘ghrelin/GHS orchestra’ in peripheral metabolism. For instance, it is noteworthy to remind that

knocking out ghrelin improves glucose tolerance in ob/ob mice [43] , while mice lacking ghrelin or GHS-R1a are resistant to diet-induced obesity [232, 233] . There is great interest in the hypothesis that both the acylated and non-acylated ghrelin may influence endocrine pancreatic ac-tivity, by improving � -cell survival and regulating insu-lin secretion [86, 218] , thus revealing useful in the treat-ment of diabetes mellitus and metabolic syndrome.

Table 1. Survey of ghrelin/GHS receptor types

Receptor type andmolecular features

Ligands Signalling Biological significance

GHS-R1aGPCR family AHeptahelical (TM1–7) with366 amino acids [18, 112, 113]Constitutive activity[130, 131, 156]Homo*/heterodimeric withfull-length GPCRs [163, 177, 178, 211]

Agonists: ghrelin and short ghrelinfragments [69] synthetic peptidyl (GHRP-6) [3, 102, 223, 234],partial peptidyl and non-peptidyl GHS [6, 7, 15, 120, 127]

Partial agonists: adenosine* [119–121]

Antagonists: D-Lys3-GHRP-6 [234], L765–867 [120], isoxazole, diaminopyrimidine and triazole derivatives [132–134], cortistatin and its octapeptide analogue CST-8 [107]

Inverse agonists: D-Arg1-D-Phe5-D-Trp7,9-Leu11-substance P [130, 131]

G�q: d Ca2+, PLC, IP3, DAG, PKC [18, 19]

d ERK1/2 , CREB and SRE [130]

blockade of K+

channels [145]

d cAMP [141, 147]Heterodimeric formation with GHRH and dopamine D1 receptors [177, 178]

d GH, PRL, CRH, ACTH and glucocorticoid secretion [21, 26, 27]

f GnRH and gonadotropin released Appetite [30, 105] and imagination of food [27]d Synaptogenesis and memory performance

[49, 96], dopaminergic neurotransmission, locomotor activity and motivation to feed[95, 96]

d Neoplastic cell growth [57]d Gastrointestinal motility and gastric acid

secretion [25]d Hepatic glucose output [40]f Glucose-stimulated insulin secretion [36*, 42*]f Peripheral insulin sensitivity [72*, 41*]f Pro-inflammatory and immune responses [111]

GHS-R1bTruncated (TM1–5) with298 amino acids [88]. Heterodimeric withfull-length GPCRs [211, 204]

Agonists: neuromedin in the neurotensin receptor 1 (NTS-R1)/GHS-R1b heterodimer [204]

Heterodimer formation with GHS-R1a and NTS-R1 [211, 204]

fd

GHS-R1a constitutive activity [211]Neoplastic cell growth [204]

GHS-RxaUncloned Agonists: ghrelin, UAG and synthetic

GHS [50, 56, 58, 60, 61, 78, 215]d cAMP/PKA,PI3K/AKT, ERK1/2[56, 60, 61]

dd

dff

Neurogenesis [215]Cell proliferation [60, 61] and survival under pro-apoptotic conditions [56, 60]Adipogenesis [39]Lipolysis under stimulated conditions [78]Neoplastic cell growth [55, 57, 58]

GHS-RxbUncloned Agonists: UAG [40, 218, 219] Unknown d Food intake [219]

Hepatic glucose output [40, 217]Glucose-stimulated insulin secretion[217, 218]Peripheral insulin sensitivity [216]

fd

d

CD36Scavenger receptor familyclass B glycoprotein, largeextracellular domain withtwo TMs and 471 amino acids

Agonists: oxidized low-densitylipoproteins [224]Antagonists: peptidyl GHS (hexarelinand its analog EP-80317) [229, 230]

Uptake of oxidizedlow-density lipoproteins [224]

d

d

Foam cell formation and pro-atherogenic processes [224]Angiogenesis and immune cells recognition [224]

* Supposed; d and f indicate increased or decreased activity.TM = Transmembrane domain; UAG = unacylated ghrelin; CREB = cAMP-responsive element-binding protein; SRE = serum-responsive element.

References are shown in brackets.

Ghrelin/GHS Receptors Neuroendocrinology 019-T1 11

Interestingly, a number of studies are also focusing on the role of the ghrelin system in the control of neoplastic cell growth through heterodimerization of the truncated splice variant GHS-R1b with full-length GPCRs [204] , as well as in modulating immune responses, cardiovascular performances and brain functions ( table 1 ). Finally, we should not forget that ghrelin was discovered as a ‘moti-lin-related gastric peptide’, and ghrelin as well as ghrelin-mimetic agents have potential clinical applications in the treatment of gastrointestinal motility disorders. Almost 30 years after the ‘invention’ of synthetic peptidyl GHS by Cyril Bowers [234] , the ghrelin system is yet not com-

pletely understood. We may expect exciting aspects of ghrelin biology to emerge, including the identification of multiple receptors for multiple ligands.

Acknowledgments

This review was supported by grants to G.M. (Regione Piemon-te A58/2004), to E.G. (European Community Sixth Framework Programme – LSHM-CT-2003-503041) and to E.G. and G.M. (MIUR, Rome, Italy – project No. 2005060517, year 2005) and to E.G. (European Community Sixth Framework Programme – LSHM-CT-2003-503041).

References

1 Bowers CY, Momany F, Reynolds GA, Chang D, Hong A, Chang K: Structure-activity re-lationships of a synthetic pentapeptide that specifically releases growth hormone in vi-tro. Endocrinology 1980; 106: 663–667.

2 Bowers CY, Momany FA, Reynolds GA, Hong A: On the in vitro and in vivo activity of a new synthetic hexapeptide that acts on the pituitary to specifically release growth hormone. Endocrinology 1984; 114: 1537–1545.

3 Bowers CY, Reynolds GA, Durham D, Bar-rera CM, Pezzoli SS, Thorner MO: Growth hormone (GH)-releasing peptide stimulates GH release in normal men and acts synergis-tically with GH-releasing hormone. J Clin Endocrinol Metabol 1990; 70: 975–982.

4 Deghenghi R, Cananzi MM, Torsello A, Bat-tisti C, Muller EE, Locatelli V: GH-releasing activity of hexarelin, a new growth hor-mone-releasing peptide, in infant and adult rats. Life Sci 1994; 54: 1321–1328.

5 Camanni F, Ghigo E, Arvat E: Growth hor-mone-releasing peptides and their analogs. Front Neuroendocrinol 1998; 9: 47–72.

6 Isidro ML, Cordido F: Growth hormone se-cretagogues. Comb Chem High Throughput Screen 2006; 9: 175–180.

7 Smith RG: Development of growth hormone secretagogues. Endocr Rev 2005; 26: 346–360.

8 Smith RG, Cheng K, Schoen WR, Pong SS, Hickey G, Jacks T, Butler B, Chan WW, Chaung LY, Judith F, et al: A nonpeptidyl growth hormone secretagogue. Science 1993; 260: 1640–1643.

9 Patchett AA, Nargund RP, Tata JR, Chen MH, Barakat KJ, Johnston DB, Cheng K, Chan WW, Butler B, Hickey G, et al.: Design and biological activities of L-163,191 (MK-0677): a potent, orally active growth hor-mone secretagogue. Proc Natl Acad Sci USA 1995; 92: 7001–7005.

10 Chapman IM, Bach MA, Van Cauter E, Farmer M, Krupa D, Taylor AM, Schilling LM, Cole KY, Skiles EH, Pezzoli SS, Hart-man ML, Veldhuis JD, Gormley GJ, Thorner MO: Stimulation of the growth hormone (GH)-insulin-like growth factor I axis by daily oral administration of a GH secreta-gogue (MK-677) in healthy elderly subjects. J Clin Endocrinol Metab 1996; 81: 4249–4257.

11 Smith RG, Sun Y, Betancourt L, Asnicar M: Growth hormone secretagogues: prospects and potential pitfalls. Best Pract Res Clin En-docrinol Metab 2004; 18: 333–347.

12 Smith RG, Jiang H, Sun Y: Developments in ghrelin biology and potential clinical rele-vance. Trends Endocrinol Metabol 2005; 16: 436–442.

13 Broglio F, Boutignon F, Benso A, Gottero C, Prodam F, Arvat E, Ghe C, Catapano F, Tor-sello A, Locatelli V, Muccioli G, Boeglin D, Guerlavais V, Fehrentz JA, Martinez J, Ghigo E, Deghenghi R: EP1572: a novel peptido-mimetic GH secretagogue with potent and selective GH-releasing activity in man. J En-docrinol Invest 2002; 25:RC26–RC28.

14 Guerlavais V, Boeglin D, Mousseaux D, Oiry C, Heitz A, Deghenghi R, Locatelli V, Tor-sello A, Ghè C, Catapano F, Muccioli G, Gal-leyrand JC, Fehrentz JA, Martinez J: New ac-tive series of growth hormone secretagogues. J Med Chem 2003; 46: 1191–1203.

15 Nagamine J, Kawamura T, Tokunaga T, Hume WE, Nagata R, Nakagawa T, Taiji M: Synthesis and pharmacological profile of an orally-active growth hormone secretagogue SM-130686. Comb Chem High Throughput Screen 2006; 9: 187–196.

16 Codd EE, Yellin T, Walker RF: Binding of growth hormone-releasing hormone and en-kephalin-derived growth hormone-releas-ing peptides to � and � opioid receptors in forebrain of rat. Neuropharmacology 1988; 27: 1019–1025.

17 Sethumadavan K, Veeraragavan K, Bowers CY: Demonstration and characterization of the specific binding of growth hormone-re-leasing peptide to rat anterior pituitary and hypothalamus. Biochem Biophys Res Com-mun 1991; 178: 31–37.

18 Howard AD, Feighner SD, Cully DF, Arena JP, Liberator PA, Rosenblum CI, Hamelin M, Hreniuk DL, Palyha OC, Anderson J, Paress PS, Diaz C, Chou M, Liu KK, McKee KK, Pong SS, Chaung LY, Elbrecht A, Dashkevicz M, Heavens R, Rigby M, Sirinathsinghji DJ, Dean DC, Melillo DG, Patchett AA, Nar-gund R, Griffin PR, DeMartino JA, Gupta SK, Schaeffer JM, Smith RG, Van der Ploeg LH: A receptor in pituitary and hypothala-mus that functions in growth hormone re-lease. Science 1996; 273: 974–977.

19 Smith RG, Van der Ploeg LH, Howard AD, Feighner SD, Cheng K, Hickey GJ, Wyvratt MJ Jr, Fisher MH, Nargund RP, Patchett AA: Peptidomimetic regulation of growth hor-mone secretion. Endocr Rev 1997; 18: 621–645.

20 Muccioli G, Ghè C, Ghigo MC, Papotti M, Arvat E, Boghen MF, Nilsson MH, Deghen-ghi R, Ong H, Ghigo E: Specific receptors for synthetic GH secretagogues in the human brain and pituitary gland. J Endocrinol 1998; 157: 99–106.

21 Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth hormone-releasing acylated peptide from stomach. Nature 1999; 402: 656–660.

22 Tomasetto C, Karam SM, Ribieras S, Masson R, Lefebvre O, Staub A, Alexander G, Che-nard MP, Rio MC: Identification and charac-terization of a novel gastric peptide hor-mone: the motilin-related peptide. Gastro-enterology 2000; 119: 395–405.

23 Coulie BJ, Miller LJ: Identification of moti-lin-related peptide. Gastroenterology 2001; 120: 588–589.

Muccioli /Baragli /Granata /Papotti /Ghigo

Neuroendocrinology 019-T1 12

24 Masuda Y, Tanaka T, Inomata N, Ohnuma N, Tanaka S, Itoh Z, Hosoda H, Kojima M, Kan-gawa K: Ghrelin stimulates gastric acid se-cretion and motility in rats. Biochem Bio-phys Res Commun 2000; 276: 905–908.

25 Asakawa A, Inui A, Kaga T, Yuzuriha H, Na-gata T, Ueno N, Makino S, Fujimiya M, Nii-jima A, Fujino MA, Kasuga M: Ghrelin is an appetite-stimulatory signal from stomach with structural resemblance to motilin. Gas-troenterology 2001; 120: 337–345.

26 Mozid AM, Tringali G, Forsling ML, Hen-dricks MS, Ajodha S, Edwards R, Navarra P, Grossman AB, Korbonits M: Ghrelin is released from rat hypothalamic explants and stimulates corticotrophin-releasing hormone and arginine-vasopressin. Horm Metab Res 2003; 35: 455–459.

27 Schmid DA, Held K, Ising M, Weikel JC, Stei-ger A: Ghrelin stimulates appetite, imagina-tion of food, GH, ACTH, and cortisol, but does not affect leptin in normal controls. Neuropsychopharmacology 2005; 30: 1187–1192.

28 Fernandez-Fernandez R, Tena-Sempere M, Navarro VM, Barreiro ML, Castellano JM, Aguilar E, Pinilla L: Effects of ghrelin upon gonadotropin-releasing hormone and go-nadotropin secretion in adult female rats: in vivo and in vitro studies. Neuroendocrinol-ogy 2006; 82: 245–255.

29 Nakazato M, Murakami N, Date Y Kojima M, Matsuo H, Kangawa K, Matsukura S: A role for ghrelin in the central regulation of feeding. Nature 2001; 409: 194–198.

30 Wren AM, Seal LJ, Cohen MA, Brynes AE, Frost GS, Murphy KG, Dhillo WS, Ghatei MA, Bloom SR: Ghrelin enhances appetite and food intake in humans. J Clin Endocri-nol Metab 2001; 86: 5992–5995.

31 Date Y, Nakazato M, Murakami N, Kojima M, Kangawa K, Matsukura S: Ghrelin acts in the central nervous system to stimulate gas-tric acid secretion. Biochem Biophys Res Commun 2001; 280: 904–907.

32 Inui A, Asakawa A, Bowers CY Mantovani G, Laviano A, Meguid MM, Fujimiya M: Ghrelin, appetite, and gastric motility: the emerging role of stomach as an endocrine or-gan. FASEB J 2004; 18: 439–456.

33 Date Y, Shimbara T, Koda S, Toshinai K, Ida T, Murakami N, Miyazato M, Kokame K, Ishizuka Y, Ishida Y, Kageyama H, Shioda S, Kangawa K, Nakazato M: Peripheral ghrelin transmits orexigenic signals through the noradrenergic pathway from the hindbrain to the hypothalamus. Cell Metab 2006; 4: 323–331.

34 Cumming DE: Ghrelin and the short- and long-term regulation of appetite and body weight. Physiol Behav 2006; 89: 71–84.

35 Tschöp M, Smiley DL, Heiman ML: Ghrelin induces adiposity in rodents. Nature 2000; 407: 908–913.

36 Broglio F, Arvat E, Benso A, Gottero C, Muc-cioli G, Papotti M, van der Lely AJ, Deghen-ghi R, Ghigo E: Ghrelin, a natural GH secre-tagogue produced by the stomach, induces hyperglycemia and reduces insulin secretion in humans. J Clin Endocrinol Metab 2001; 86: 5083–5086.

37 Murata M, Okimura Y, Iida K, Matsumoto M, Sowa H, Kaji H, Kojima M, Kangawa K, Chihara K: Ghrelin modulates the down-stream molecules of insulin signaling in hepatoma cells. J Biol Chem 2002; 277: 5667–5674.

38 Poykko SM, Kellokoski E, Horkko S, Kauma H, Kesaniemi YA, Ukkola O: Low plasma ghrelin is associated with insulin resistance, hypertension, and the prevalence of type 2 diabetes. Diabetes 2003; 52: 2546–2553.

39 Thompson NM, Gill DA, Davies R, Love-ridge N, Houston PA, Robinson IC, Wells T: Ghrelin and des-octanoyl ghrelin promote adipogenesis directly in vivo by a mecha-nism independent of the type 1a growth hor-mone secretagogue receptor. Endocrinology 2004; 145: 234–242.

40 Gauna C, Delhanty PJ, Hofland L, Janssen JA, Broglio F, Ross RJ, Ghigo E, van der Lely AJ: Ghrelin stimulates, whereas des-octano-yl ghrelin inhibits, glucose output by prima-ry hepatocytes. J Clin Endocrinol Metab 2005; 90: 1055–1060.

41 Heijboer AC, van den Hoek AM, Parlevliet ET, Havekes LM, Romijn JA, Pijl H, Corssmit EP: Ghrelin differentially affects hepatic and peripheral insulin sensitivity in mice. Diabe-tologia 2006; 49: 732–738.

42 Dezaki K, Sone H, Koizumi M, Nakata M, Kakei M, Nagai H, Hosoda H, Kangawa K, Yada T: Blockade of pancreatic islet-derived ghrelin enhances insulin secretion to pre-vent high-fat diet-induced glucose intoler-ance. Diabetes 2006; 55: 3486–3493.

43 Sun Y, Asnicar M, Saha PK, Chan L, Smith RG: Ablation of ghrelin improves the diabet-ic but not obese phenotype of ob/ob mice. Cell Metab 2006; 3: 379–386.

44 Gluck EF, Stephens N, Swoap SJ: Peripheral ghrelin deepens torpor bouts in mice through the arcuate nucleus neuropeptide Y signal-ing pathway. Am J Physiol 2006; 291:R1303–R1309.

45 Steiger A: Ghrelin and sleep-wake regula-tion. Am J Physiol 2007; 292:R573–R574.

46 Asakawa A, Inui A, Kaga T, Yuzuriha H, Na-gata T, Fujimiya M, Katsuura G, Makino S, Fujino MA, Kasuga M: A role of ghrelin in neuroendocrine and behavioral responses to stress in mice. Neuroendocrinology 2001; 74: 143–147.

47 Carlini VP, Varas MM, Cragnolini AB, Schioth HB, Scimonelli TN, de Barioglio SR: Differential role of the hippocampus, amyg-dala, and dorsal raphe nucleus in regulating feeding, memory, and anxiety-like behavior-al responses to ghrelin. Biochem Biophys Res Commun 2004; 313: 635–641.

48 Kanehisa M, Akiyoshi J, Kitaichi T, Matsu-shita H, Tanaka E, Kodama K, Hanada H, Isogawa K: Administration of antisense DNA for ghrelin causes an antidepressant and anxiolytic response in rats. Prog Neuro-psychopharmacol Biol Psychiatry 2006; 30: 1403–1407.

49 Diano S, Farr SA, Benoit SC, McNay EC, da Silva I, Horvath B, Gaskin FS, Nonaka N, Jaeger LB, Banks WA, Morley JE, Pinto S, Sherwin RS, Xu L, Yamada KA, Sleeman MW, Tschöp MH, Horvath TL: Ghrelin con-trols hippocampal spine synapse density and memory performance. Nat Neurosci 2006; 9: 381–388.

50 Bedendi I, Alloatti G, Marcantoni A Malan D, Catapano F, Ghè C, Deghenghi R, Ghigo E, Muccioli G: Cardiac effect of ghrelin and its endogenous derivatives des-octanoyl ghrelin and des-Gln 14 -ghrelin. Eur J Phar-macol 2003; 476: 87–95.

51 Nagaya N, Uematsu M, Kojima M Ikeda Y, Yoshihara F, Shimizu W, Hosoda H, Hirota Y, Ishida H, Mori H, Kangawa K: Chronic administration of ghrelin improves left ven-tricular dysfunction and attenuates develop-ment of cardiac cachexia in rats with heart failure. Circulation 2001; 104: 1430–1435.

52 Tritos NA, Kissinger KV, Manning WJ, Danias PJ: Association between ghrelin and cardiovascular indexes in healthy obese and lean men. Clin Endocrinol (Oxf) 2004; 60: 60–66.

53 Muccioli G, Broglio F, Tarabra E, Ghigo E: Known and unknown growth hormone se-cretagogue receptors and their ligands; in Ghigo E (ed): Ghrelin. Boston, Kluwer Aca-demic, 2004, pp 27–46.

54 Broglio F, Prodam F, Me E, Riganti F, Lu-catello B, Granata R, Benso A, Muccioli G, Ghigo E Ghrelin: Endocrine, metabolic and cardiovascular actions. J Endocrinol Invest 2005; 28: 23–25.

55 Cassoni P, Papotti M, Ghè C, Catapano F, Sapino A, Graziani A, Deghenghi R, Reiss-mann T, Ghigo E, Muccioli G: Identification, characterization and biological activity of specific receptors for natural (ghrelin) and synthetic growth hormone secretagogues in human breast carcinomas and cell lines. J Clin Endocrinol Metab 2001; 86: 1738–1745.

56 Baldanzi G, Filigheddu N, Cutrupi S, Cata-pano F, Bonissoni S, Fubini A, Malan D, Baj G, Granata R, Broglio F, Papotti M, Surico N, Bussolino F, Isgaard J, Deghenghi R, Siniga-glia F, Prat M, Muccioli G, Ghigo E, Graziani A: Ghrelin and des-acyl ghrelin inhibit cell death in cardiomyocytes and endothelial cells through ERK1/2 and PI 3 -kinase/AKT. J Cell Biol 2002; 159: 1029–1037.

57 Papotti M, Ghè C, Volante M, Muccioli G: Ghrelin and tumors; in Ghigo E (ed): Ghre-lin. Boston, Kluwer Academic, 2004, pp 143–164.

Ghrelin/GHS Receptors Neuroendocrinology 019-T1 13

58 Cassoni P, Ghè C, Marrocco T, Tarabra E, Allia E, Catapano F, Deghenghi R, Ghigo E, Papotti M, Muccioli G: Expression of ghrelin and biological activity of specific receptors for ghrelin and des-acyl ghrelin in human prostate neoplasms and related cell lines. Eur J Endocrinol 2004; 150: 173–184.

59 Nakahara K, Nakagawa M, Baba Y, Sato M, Toshinai K, Date Y, Nakazato M, Kojima M, Miyazato M, Kaiya H, Hosoda H, Kangawa K, Murakami N: Maternal ghrelin plays an important role in rat fetal development dur-ing pregnancy. Endocrinology 2006; 147: 1333–1342.

60 Granata R, Settanni F, Biancone L, Trovato L, Nano R, Bertuzzi F, Destefanis S, Annun-ziata M, Martinetti M, Catapano F, Ghè C, Isgaard J, Papotti M, Ghigo E, Muccioli G: Acylated and unacylated ghrelin promote proliferation and inhibit apoptosis of pan-creatic � cells and human islets. Involvement of cAMP/PKA, ERK1/2 and PI 3 K/AKT sig-nalling. Endocrinology 2007; 148: 512–529.

61 Delhanty PJ, van der Eerden BC, van der Vel-de M, Gauna C, Pols HA, Jahr H, Chiba H, van der Lely AJ, van Leeuwen JP: Ghrelin and unacylated ghrelin stimulate human os-teoblast growth via mitogen-activated pro-tein kinase/phosphoinositide 3-kinase path-ways in the absence of GHS-R1a. J Endocrinol 2006; 188: 37–47.

62 Dixit VD, Taub DD: Ghrelin and immunity: a young player in an old field. Exp Gerontol 2005; 40: 900–910.

63 Van der Lely AJ, Tschöp M, Heiman ML, Ghigo E: Biological, physiological, patho-physiological, and pharmacological aspects of ghrelin. Endocr Rev 2004; 25: 426–457.

64 Kojima M, Kangawa K: Ghrelin: structure and function. Physiol Rev 2005; 85: 495–522.

65 Ghigo E, Broglio F, Arvat E, Maccario M, Pa-potti M, Muccioli G: Ghrelin: more than a natural GH secretagogue and/or an orexi-genic factor. Clin Endocrinol (Oxf) 2005; 62: 1–17.

66 Nogueiras R, Perez-Tilve D, Wortley KE, Tschöp M: Growth hormone secretagogue (ghrelin) receptors – a complex drug target for the regulation of body weight. CNS Neu-rol Disord Drug Targets 2006; 5: 335–343.

67 Gualillo O, Lago F, Casanueva FF, Dieguez C: One ancestor, several peptides post-trans-lational modifications of preproghrelin gen-erate several peptides with antithetical ef-fects. Mol Cell Endocrinol 2006; 256: 1–8.

68 Zhu X, Cao Y, Voodg K, Steiner DF: On the processing of proghrelin to ghrelin. J Biol Chem 2006; 281: 38867–38870.

69 Bednarek MA, Feighner SD, Pong SS, McKee KK, Hreniuk DL, Silva MV, Warren VA, Howard AD, Van Der Ploeg LH, Heck JV: Structure-functions studies on the new growth hormone-releasing peptide, ghrelin: minimal sequence of ghrelin necessary for activation of growth hormone secretagogue receptor 1a. J Med Chem 2000; 43: 4370–4376.

70 Torsello A, Ghè C, Bresciani E, Catapano F, Ghigo E, Deghenghi R, Locatelli V, Muccioli G: Short ghrelin peptides neither displace ghrelin binding in vitro nor stimulate GH re-lease in vivo. Endocrinology 2002; 143: 1968–1971.

71 Muccioli G, Tschöp M, Papotti M, Deghen-ghi R, Heiman M, Ghigo E: Neuroendocrine and peripheral activities of ghrelin: implica-tions in metabolism and obesity. Eur J Phar-macol 2002; 440: 235–254.

72 Broglio F, Prodam F, Riganti E, Muccioli G, Ghigo E: Ghrelin: From somatotrope secre-tion to new perspectives in the regulation of peripheral metabolic functions. Front Horm Res 2006; 35: 102–114.

73 Hosoda H, Kojima M, Kangawa K: Biologi-cal, physiological, and pharmacological as-pects of ghrelin. J Pharmacol Sci 2006; 100: 398–410.

74 Banks WA, Tschöp M, Robinson SM, Hei-man ML: Extent and direction of ghrelin transport across the blood-brain barrier is determined by its unique primary structure. J Pharmacol Exp Ther 2002; 302: 822–827.

75 Matsumoto M, Hosoda H, Kitajima Y, Moro-zumi N, Minamitake Y, Tanaka S, Matsuo H, Kojima M, Hayashi Y, Kangawa K: Struc-ture-activity relationship of ghrelin: phar-macological study of ghrelin peptides. Bio-chem Biophys Res Commun 2001; 287: 142–146.

76 Matsumoto M, Kitajima Y, Iwanami T, Hayashi Y, Tanaka S, Minamitake Y, Hosoda H, Kojima M, Matsuo H, Kangawa K: Struc-tural similarity of ghrelin derivatives to pep-tidyl growth hormone secretagogues. Bio-chem Biophys Res Commun 2001; 284: 655–659.

77 Tolle V, Zizzari P, Tomasetto C, Rio MC, Epelbaum J, Bluet-Pajot MT: In vivo and in vitro effects of ghrelin/motilin-related pep-tide on growth hormone secretion in the rat. Neuroendocrinology 2001; 73: 54–61.

78 Muccioli G, Pons N, Ghè C, Catapano F, Granata R, Ghigo E: Ghrelin and des-acyl ghrelin both inhibit isoproterenol-induced lipolysis in rat adipocytes via a non-type 1a growth hormone secretagogue receptor. Eur J Pharmacol 2004; 498: 27–35.

79 Hosoda H, Kojima M, Mizushima Y, Shi-mizu S, Kangawa K: Structural divergence of human ghrelin. Identification of multiple ghrelin-derived molecules produced by post-translational processing. J Biol Chem 2003; 278: 64–70.

80 Hosoda H, Kojima M, Matsuo H, Kangawa K. Ghrelin and des-octanoyl ghrelin: two major forms of rat ghrelin peptide in gastro-intestinal tissue. Biochem Biophys Res Com-mun 2000; 279: 909–913.

81 Hosoda H, Kojima M, Matsuo H, Kangawa K: Purification and characterization of rat des-Gln 14 -ghrelin, a second endogenous li-gand for the growth hormone secretagogue receptor. J Biol Chem 2000; 275: 21995–22000.

82 Jeffery PL, Murray RE, Yeh AH, McNamara JF, Duncan RP, Francis GD, Herington AC, Chopin LK: Expression and function of the ghrelin axis, including a novel preproghrelin isoform, in human breast cancer tissues and cell lines. Endocr Relat Cancer 2005; 12: 839–850.

83 Yeh AH, Jeffery PL, Duncan RP, Herington AC, Chopin LK: Ghrelin and a novel pre-proghrelin isoform are highly expressed in prostate cancer and ghrelin activates mito-gen-activated protein kinase in prostate can-cer. Clin Cancer Res 2005; 11: 8295–8303.

84 Zhang JV, Ren PG, Avsian-Kretchmer O, Luo CW, Rauch R, Klein C, Hsueh AJ: Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin’s effects on food in-take. Science 2005; 310: 996–999.

85 Dun SL, Brailoiu GC, Brailoiu E, Yang J, Chang JK, Dun NJ: Distribution and biolog-ical activity of obestatin in the rat. J Endocri-nol 2006; 191: 481–489.

86 Samson WK, White MM, Price C, Ferguson AV: Obestatin acts in brain to inhibit thirst. Am J Physiol 2007; 292:R637–R643.

87 Holst B, Egerod KL, Schild E, Vickers SP, Cheetham S, Gerlach LO, Storjohann L, Stidsen CE, Jones R, Beck-Sickinger AG, Schwartz TW: GPR39 signaling is stimulat-ed by zinc ions but not by obestatin. Endocri-nology 2007; 148: 13–20.

88 McKee KK, Palyha OC, Feighner SD, Hre-niuk DL, Tan CP, Phillips MS, Smith RG, Van der Ploeg LH, Howard AD: Molecular analysis of rat pituitary and hypothalamic growth hormone secretagogue receptors. Mol Endocrinol 1997; 11: 415–423.

89 Hayashida T, Nakahara K, Mondal MS, Date Y, Nakazato M, Kojima M, Kangawa K, Mu-rakami N: Ghrelin in neonatal rats: distribu-tion in stomach and its possible role. J Endo-crinol 2002; 173: 239–245.

90 Gnanapavan S, Kola B, Bustin SA, Morris DG, McGee P, Fairclough P, Bhattacharya S, Carpenter R, Grossman AB, Korbonits M: The tissue distribution of the mRNA of ghre-lin and subtypes of its receptor, GHS-R, in humans. J Clin Endocrinol Metab 2002; 87: 2988–2991.

91 Guan XM, Yu H, Palyha OC, McKee KK, Feighner SD, Sirinathsinghji DJ, Smith RG, van der Ploeg LH, Howard AD: Distribution of mRNA encoding the growth hormone se-cretagogue receptor in brain and peripheral tissues. Brain Res Mol Brain Res 1997; 48: 23–29.

92 Sun Y, Garcia JM, Smith RG: Ghrelin and growth hormone secretagogue receptor ex-pression in mice during aging. Endocrinol-ogy 2007; 148: 1323–1329.

Muccioli /Baragli /Granata /Papotti /Ghigo

Neuroendocrinology 019-T1 14

93 Zigman JM, Jones JE, Lee CE, Saper CB, Elmquist JK: Expression of ghrelin receptor mRNA in the rat and the mouse brain. J Comp Neurol 2006; 494: 528–548.

94 Jaszberenyi M, Bujdoso E, Bagosi Z, Teleg-dy G: Mediation of the behavioral, endo-crine and thermoregulatory actions of ghrelin. Horm Behav 2006; 50: 266–273.

95 Jerlhag E, Egecioglu E, Dickson SL, Ander-sson M, Svensson L, Engel JA: Ghrelin stim-ulates locomotor activity and accumbal do-pamine-overflow via central cholinergic systems in mice: implications for its in-volvement in brain reward. Addict Biol 2006; 11: 45–54.

96 Abizaid A, Liu ZW, Andrews ZB, Shana-brough M, Borok E, Elsworth JD, Roth RH, Sleeman MW, Picciotto MR, Tschöp MH, Gao XB, Horvath TL: Ghrelin modulates the activity and synaptic input organiza-tion of midbrain dopamine neurons while promoting appetite. J Clin Invest 2006; 116: 3229–3239 .

97 Arvat E, Ramunni J, Bellone J, Di Vito L, Baffoni C, Broglio F, Deghenghi R, Barto-lotta E, Ghigo E: The GH, prolactin, ACTH and cortisol responses to hexarelin, a syn-thetic hexapeptide, undergo different age-related variations. Eur J Endocrinol 1997; 137: 635–642.

98 Ghigo E, Arvat E, Camanni F: Growth hor-mone secretagogues as corticotrophin-re-leasing factors. Growth Horm IGF Res 1998; 8(suppl B):145–148.

99 Ghigo E, Arvat E, Giordano R, Broglio F, Gianotti L, Maccario M, Bisi G, Graziani A, Papotti M, Muccioli G, Deghenghi R, Ca-manni F: Biologic activities of growth hor-mone secretagogues in humans. Endocrine 2001; 14: 87–93.

100 Popovic V, Miljic D, Micic D, Damjanovic S, Arvat E, Ghigo E, Dieguez C, Casanueva FF: Ghrelin main action on the regulation of growth hormone release is exerted at hy-pothalamic level. J Clin Endocrinol Metab 2003; 88: 3450–3453.

101 Enomoto M, Nagaya N, Uematsu M, Oku-mura H, Nakagawa E, Ono F, Hosoda H, Oya H, Kojima M, Kanmatsuse K, Kangawa K: Cardiovascular and hormonal effects of subcutaneous administration of ghrelin, a novel growth hormone-releasing peptide, in healthy humans. Clin Sci (Lond) 2003; 105: 431–435.

102 Rubinfeld H, Hadani M, Taylor JE, Dong JZ, Comstock J, Shen Y, DeOliveira D, Dat-ta R, Culler MD, Shimon I: Novel ghrelin analogs with improved affinity for the GH secretagogue receptor stimulate GH and prolactin release from human pituitary cells. Eur J Endocrinol 2004; 151: 787–795.

103 Cowley MA, Smith RG, Diano S, Tschöp M, Pronchuk N, Grove KL, Strasburger CJ, Bid-lingmaier M, Esterman M, Heiman ML, Garcia-Segura LM, Nillni EA, Mendez P, Low MJ, Sotonyi P, Friedman JM, Liu H, Pinto S, Colmers WF, Cone RD, Horvath TL: The distribution and mechanism of ac-tion of ghrelin in the CNS demonstrates a novel hypothalamic circuit regulating en-ergy homeostasis. Neuron 2003; 37: 649–661.

104 Chen HY, Trumbauer ME, Chen AS, Wein-garth DT, Adams JR, Frazier EG, Shen Z, Marsh DJ, Feighner SD, Guan XM, Ye Z, Nargund RP, Smith RG, Van der Ploeg LH, Howard AD, MacNeil DJ, Qian S: Orexi-genic action of peripheral ghrelin is medi-ated by neuropeptide Y and agouti-related protein. Endocrinology 2004; 145: 2607–2612.

105 Sun Y, Wang P, Zheng H, Smith RG: Ghre-lin stimulation of growth hormone release and appetite is mediated through the growth hormone secretagogue receptor. Proc Natl Acad Sci USA 2004; 101: 4679–4684.

106 Murphy KG, Dhillo WS, Bloom SR: Gut peptides in the regulation of food intake and energy homeostasis. Endocr Rev 2006; 27: 719–727.

107 Sibilia V, Muccioli G, Deghenghi R, Pagani F, De Luca V, Rapetti D, Locatelli V, Netti C: Evidence for a role of the GHS-R1a re-ceptors in ghrelin inhibition of gastric acid secretion in the rat. J Neuroendocrinol 2006; 18: 122–128.

108 Kawczynska-Drozdz A, Olszanecki R, Jawien J, Brzozowski T, Pawlik WW, Kor-but R, Guzik TJ: Ghrelin inhibits vascular superoxide production in spontaneously hypertensive rats. Am J Hypertens 2006; 19: 764–767.

109 Suematsu M, Katsuki A, Sumida Y, Ga-bazza EC, Murashima S, Matsumoto K, Kitagawa N, Akatsuka H, Hori Y, Nakatani K, Togashi K, Yano Y, Adachi Y: Decreased circulating levels of active ghrelin are asso-ciated with increased oxidative stress in obese subjects. Eur J Endocrinol 2005; 153: 403–407.

110 Koo GC, Huang C, Camacho R, Trainor C, Blake JT, Sirotina-Meisher A, Schleim KD, Wu TJ, Cheng K, Nargund R, McKissick G: Immune enhancing effect of a growth hor-mone secretagogue. J Immunol 2001; 166: 4195–4201.

111 Dixit VD, Schaffer EM, Pyle RS, Collins GD, Sakthivel SK, Palaniappan R, Lillard JW Jr, Taub DD: Ghrelin inhibits leptin- and activation-induced proinflammatory cytokine expression by human monocytes and T cells. J Clin Invest 2004; 114: 57–66.

112 Camina JP: Cell biology of the ghrelin re-ceptor. J Neuroendocrinol 2006; 18: 65–76.

113 Petersenn S: Structure and regulation of the growth hormone secretagogue receptor. Minerva Endocrinol 2002; 27: 243–256.

114 Bockaert J, Pin JP: Molecular tinkering of G-protein-coupled receptors: an evolution-ary success. EMBO J 1999; 18: 1723–1729.

115 McKee KK, Tan CP, Palyha OC, Liu J, Feighner SD, Hreniuk DL, Smith RG, How-ard AD, Van der Ploeg LH: Cloning and characterization of two human G-protein-coupled receptor genes (GPR38 and GPR39) related to the growth hormone secreta-gogue and neurotensin receptors. Genom-ics 1997; 46: 426–434.

116 Tan CP, McKee KK, Liu Q, Palyha OC, Feighner SD, Hreniuk DL, Smith RG, How-ard AD: Cloning and characterization of a human and murine T-cell orphan G-pro-tein-coupled receptor similar to the growth hormone secretagogue and neurotensin re-ceptors. Genomics 1998; 52: 223–229.

117 Palyha OC, Feighner SD, Tan CP, et al: Li-gand activation domain of human orphan growth hormone secretagogue receptor conserved from pufferfish to humans. Mol Endocrinol 2000; 14: 160–169.

118 Muccioli G, Papotti M, Locatelli V, Ghigo E: Binding of 125 I-labeled ghrelin to mem-branes from human hypothalamus and pi-tuitary gland. J Endocrinol Invest 2001; 24:RC7–RC9.

119 Tullin S, Hansen BS, Ankersen M, Moller J, Von Cappelen KA, Thim L: Adenosine is an agonist of the growth hormone secreta-gogue receptor. Endocrinology 2000; 141: 3397–3402.

120 Smith RG, Leonard R, Bailey AR, Palyha O, Feighner S, Tan C, Mckee KK, Pong SS, Griffin P, Howard A: Growth hormone se-cretagogue receptor family members and ligands. Endocrine 2001; 14: 9–14.

121 Carreira MC, Camina JP, Smith RG, Casa-nueva FF: Agonist-specific coupling of growth hormone secretagogue receptor type 1a to different intracellular signaling systems. Role of adenosine. Neuroendocri-nology 2004; 79: 13–25.

122 Johansson S, Fredholm BB, Hjort C, Morein T, Kull B, Hu PS: Evidence against adeno-sine analogues being agonists at the growth hormone secretagogue receptor. Biochem Pharmacol 2005; 70: 598–605.

123 Carreira MC, Camina JP, Diaz-Rodriguez E, Alvear-Perez R, Llorens-Cortes C, Casa-nueva FF: Adenosine does not bind to the growth hormone secretagogue receptor type-1a. J Endocrinol 2006; 191: 147–157.

124 Deghenghi R, Avallone R, Torsello A, Muc-cioli G, Ghigo E, Locatelli V: Growth hor-mone-inhibiting activity of cortistatin in the rat. J Endocrinol Invest 2001; 24:RC31–RC33.

125 Deghenghi R, Papotti M, Ghigo E, Muccio-li G, Locatelli V: Somatostatin octapeptides (lanreotide, octreotide, vapreotide, and their analogs) share the growth hormone-releasing peptide receptor in the human pi-tuitary gland. Endocrine 2001; 14: 29–33.

Ghrelin/GHS Receptors Neuroendocrinology 019-T1 15

126 Luque RM, Peinado JR, Gracia-Navarro F, Broglio F, Ghigo E, Kineman RD, Malagon MM, Castano JP: Cortistatin mimics so-matostatin by inducing a dose-dependent stimulatory and inhibitory effect on growth hormone secretion in somatotrope. J Mol Endocrinol 2006; 36: 547–555.

127 Ma JN, Schiffer HH, Knapp AE, Wang J, Wong KK, Currier EA, Owens M, Nash NR, Gardell LR, Brann MR, Olsson R, Brurstein ES. Identification of the atypical L-type Ca 2+ channel blocker diltiazem and its me-tabolites as ghrelin receptor agonists; Mol Pharmacol 2007; [Epub ahead of print].

128 Broglio F, van Koetsveld P, Benso A, Got-tero C, Prodam F, Papotti M, Muccioli G, Gauna C, Hofland L, Deghenghi R, Arvat E, Van Der Lely AJ, Ghigo E: Ghrelin secre-tion is inhibited by either somatostatin or cortistatin in humans. J Clin Endocrinol Metab 2002; 87: 4829–4832.

129 Woll PJ, Rozengurt E: [ D -Arg1, D -Phe5, D -Trp7,9,Leu11]substance P, a potent bombe-sin antagonist in murine Swiss 3T3 cells, inhibits the growth of human small cell lung cancer cells in vitro. Proc Natl Acad Sci USA 1988; 85: 1859–1863.

130 Holst B, Cygankiewicz A, Jensen TH, Ankersen M, Schwartz TW: High constitu-tive signaling of the ghrelin receptor – iden-tification of a potent inverse agonist. Mol Endocrinol 2003; 17: 2201–2210.

131 Holst B, Lang M, Brandt E, Bach A, How ard A, Frimurer TM, Beck-Sickinger A, Schwartz TW: Ghrelin receptor inverse ag-onists: identification of an active peptide core and its interaction epitopes on the re-ceptor. Mol Pharmacol 2006; 70: 936–946.

132 Xin Z, Zhao H, Serby MD, Liu B, Schaefer VG, Falls DH, Kaszubska W, Colins CA, Sham HL, Liu G: Synthesis and structure-activity relationships of isoxazole carbox-amides as growth hormone secretagogue receptor antagonists. Bioorg Med Chem Lett 2005; 15: 1201–1204.

133 Serby MD, Zhao H, Szczepankiewicz BG, Kosogof C, Xin Z, Liu B, Liu M, Nelson LT, Kaszubska W, Falls HD, Schaefer V, Bush EN, Shapiro R, Droz BA, Knourek-Segel VE, Fey TA, Brune ME, Beno DW, Turner TM, Collins CA, Jacobson PB, Sham HL, Liu G: 2,4-Diaminopyrimidine derivatives as potent growth hormone secretagogue re-ceptor antagonists. J Med Chem 2006; 49: 2568–2578.

134 Demange L, Boeglin D, Moulin A, Mous-seaux D, Ryan J, Berge G, Gagne D, Heitz A, Perrissoud D, Locatelli V, Torsello A, Gal-leyrand JC, Fehrentz JA, Martinez J: Syn-thesis and pharmacological in vitro and in vivo evaluation of novel triazole derivatives as ligands of the ghrelin receptor. 1. J Med Chem 2007; 50: 1939–1957.

135 Hornby PJ: Designing Spiegelmers to an-tagonise ghrelin. Gut 2006; 55: 754–755.

136 Helmling S, Maasch C, Eulberg D, Buchner K, Schroder W, Lange C, Vonhoff S, Wlotz-ka B, Tschöp MH, Rosewicz S, Klussmann S: Inhibition of ghrelin action in vitro and in vivo by an RNA-Spiegelmer. Proc Natl Acad Sci USA 2004; 101: 13174–13179.

137 Schwartz TW, Frimurer TM, Holst B, Rosenkilde MM, Elling CE: Molecular mechanism of 7TM receptor activation – a global toggle switch model. Annu Rev Pharmacol Toxicol 2006; 46: 481–519.

138 Pedretti A, Villa M, Pallavicini M, Valoti E, Vistoli G: Construction of human ghrelin receptor (hGHS-R1a) model using a frag-mental prediction approach and validation through docking analysis. J Med Chem 2006; 49: 3077–3085.

139 Feighner SD, Howard AD, Prendergast K, Palyha OC, Hreniuk DL, Nargund R, Un-derwood D, Tata JR, Dean DC, Tan CP, McKee KK, Woods JW, Patchett AA, Smith RG, Van der Ploeg LH: Structural require-ments for the activation of the human growth hormone secretagogue receptor by peptide and non-peptide secretagogues. Mol Endocrinol 1998; 12: 137–145.

140 Falls HD, Dayton BD, Fry DG, OgielaCA, Schaefer VG, Brodjian S, Reilly RM, Collins CA, Kaszubska W: Characteriza-tion of ghrelin receptor activity in a rat pi-tuitary cell line RC-4B/C. J Mol Endocrinol 2006; 37: 51–62.

141 Chen C: Growth hormone secretagogue ac-tions on the pituitary gland: multiple recep-tors for multiple ligands? Clin Exp Pharma-col Physiol 2000; 27: 323–329.

142 Malagon MM, Luque RM, Ruiz-Guerrero E, Rodriguez-Pacheco F, Garcia-Navarro S, Casanueva FF, Gracia-Navarro F, Castano JP: Intracellular signaling mechanisms me-diating ghrelin-stimulated growth hor-mone release in somatotropes. Endocrinol-ogy 2003; 144: 5372–5380.

143 Glavaski-Joksimovic A, Jeftinija K, Scanes CG, Anderson LL, Jeftinija S: Stimulatory effect of ghrelin on isolated porcine so-matotropes. Neuroendocrinology 2003; 77: 367–379.

144 Rodriguez-Pacheco F, Luque RM, Garcia-Navarro S, Gracia-Navarro F, Castano JP, Malagon MM: Ghrelin induces growth hormone secretion via nitric oxide/cGMP signaling. Ann NY Acad Sci 2005; 1040: 452–453.

145 Han XF, Zhu YL, Hernandez M, Keating DJ, Chen C: Ghrelin reduces voltage-gated potassium currents in GH3 cells via cyclic GMP pathways. Endocrine 2005; 28: 217–224.

146 Kato M, Sakuma Y: The effect of GHRP-6 on the intracellular Na + concentration of rat pituitary cells in primary culture. J Neu-roendocrinol 1999; 11: 795–800.

147 Kohno D, Gao HZ, Muroya S, Kikuyama S, Yada T: Ghrelin directly interacts with neu-ropeptide-Y-containing neurons in the rat arcuate nucleus: Ca 2+ signaling via protein kinase A and N-type channel-dependent mechanisms and cross-talk with leptin and orexin. Diabetes 2003; 52: 948–956.

148 Amar S, Mazella J, Checler F, Kitabgi P, Vincent JP: Regulation of cyclic GMP levels by neurotensin in neuroblastoma clone N1E115. Biochem Biophys Res Commun 1985; 129: 117–125.

149 Bozou JC, Rochet N, Magnaldo I, Vincent JP, Kitabgi P: Neurotensin stimulates inosi-tol trisphosphate-mediated calcium mobi-lization but not protein kinase C activation in HT29 cells. Involvement of a G-protein. Biochem J 1989; 264: 871–878.

150 Bozou JC, de Nadai F, Vincent JP, Kitabgi P: Neurotensin, bradykinin and somatostatin inhibit cAMP production in neuroblasto-ma N1E115 cells via both pertussis toxin sensitive and insensitive mechanisms. Bio-chem Biophys Res Commun 1989; 161: 1144–1150.

151 Watson MA, Yamada M, Yamada M, Cu-sack B, Veverka K, Bolden-Watson C, Richelson E: The rat neurotensin receptor expressed in Chinese hamster ovary cells mediates the release of inositol phosphates. J Neurochem 1992; 59: 1967–1970.

152 Herrington J, Hille B: Growth hormone-re-leasing hexapeptide elevates intracellular calcium in rat somatotropes by two mecha-nisms. Endocrinology 1994; 135: 1100–1108.

153 Adams EF, Lei T, Buchfelder M, Bowers CY, Fahlbusch R: Protein kinase C-dependent growth hormone-releasing peptides stimu-late cyclic adenosine 3,5-monophosphate production by human pituitary somatotro-pinomas expressing gsp oncogenes: evidence for cross-talk between transduction path-ways. Mol Endocrinol 1996; 10: 432–438.

154 Maudsley S, Martin B, Luttrell LM: The or-igins of diversity and specificity in G-pro-tein-coupled receptor signaling. J Pharma-col Exp Ther 2005; 314: 485–494.

155 Mousseaux D, Le Gallic L, Ryan J, Oiry C, Gagne D, Fehrentz JA, Galleyrand JC, Mar-tinez J: Regulation of ERK1/2 activity by ghrelin-activated growth hormone secreta-gogue receptor 1A involves a PLC/PKCva-repsilon pathway. Br J Pharmacol 2006; 148: 350–365.

156 Holst B, Holliday ND, Bach A, Elling CE, Cox HM, Schwartz TW: Common struc-tural basis for constitutive activity of the ghrelin receptor family. J Biol Chem 2004; 279: 53806–53817.

157 Nanzer AM, Khalaf S, Mozid AM, Fowkes RC, Patel MV, Burrin JM, Grossman AB, Korbonits M: Ghrelin exerts a proliferative effect on a rat pituitary somatotroph cell line via the mitogen-activated protein ki-nase pathway. Eur J Endocrinol 2004; 151: 233–240.

Muccioli /Baragli /Granata /Papotti /Ghigo

Neuroendocrinology 019-T1 16

158 Granata R, Settanni F, Trovato L, Destefa-nis S, Gallo D, Martinetti M, Ghigo E, Muc-cioli G: Unacylated as well as acylated ghre-lin promotes cell survival and inhibit apoptosis in HIT-T15 pancreatic � -cells. J Endocrinol Invest 2006; 29:RC19–RC22.

159 Bond RA, Ijzerman AP: Recent develop-ments in constitutive receptor activity and inverse agonism, and their potential for GPCR drug discovery. Trends Pharmacol Sci 2006; 27: 92–96.

160 Wang HJ, Geller F, Dempfle A, Schauble N, Friedel S, Lichtner P, Fontenla-Horro F, Wudy S, Hagemann S, Gortner L, Huse K, Remschmidt H, Bettecken T, Meitinger T, Schafer H, Hebebrand J, Hinney A: Ghrelin receptor gene: identification of several se-quence variants in extremely obese chil-dren and adolescents, healthy normal-weight and underweight students, and children with short normal stature. J Clin Endocrinol Metab 2004; 89: 157–162.

161 Pantel J, Legendre M, Cabrol S, Hilal L, Ha-jaji Y, Morisset S, Nivot S, Vie-Luton MP, Grouselle D, de Kerdanet M, Kadiri A, Epelbaum J, Le Bouc Y, Amselem S: Loss of constitutive activity of the growth hor-mone secretagogue receptor in familial short stature. J Clin Invest 2006; 116: 760–768.

162 Holst B, Schwartz TW: Ghrelin receptor mutations – too little height and too much hunger. J Clin Invest 2006; 116: 637–641.

163 Holst B, Brandt E, Bach A, Heding A, Schwartz TW: Non-peptide and peptide growth hormone secretagogues act both as ghrelin receptor agonist and as positive or negative allosteric modulators of ghrelin signaling. Mol Endocrinol 2005; 19: 2400–2411.

164 Binet V, Brajon C, Le Corre L, Acher F, Pin JP, Prezeau L: The heptahelical domain of GABA B2 is activated directly by CGP7930, a positive allosteric modulator of the GABA B receptor. J Biol Chem 2004; 279: 29085–29091.

165 Holst B, Elling CE, Schwartz TW: Metal ion-mediated agonism and agonist en-hancement in melanocortin MC1 and MC4 receptors. J Biol Chem 2002; 277: 47662–47670.

166 Schwartz TW, Holst B: Ago-allosteric mod-ulation and other types of allostery in di-meric 7TM receptors. J Recept Signal Transduct Res 2006; 26: 107–128.

167 Hataya Y, Akamizu T, Takaya K, Kanamoto N, Ariyasu H, Saijo M, Moriyama K, Shi-matsu A, Kojima M, Kangawa K, Nakao K: A low dose of ghrelin stimulates growth hormone (GH) release synergistically with GH-releasing hormone in humans. J Clin Endocrinol Metab 2001; 86: 4552–4255.

168 Bowers CY, Sartor AO, Reynolds GA, Bad-ger TM: On the actions of the growth hor-mone-releasing hexapeptide, GHRP. Endo-crinology 1991; 128: 2027–2035.

169 Wu D, Chen C, Katoh K, Zhang J, Clarke IJ: The effect of GH-releasing peptide-2 (GHRP-2 or KP-102) on GH secretion from primary cultured ovine pituitary cells can be abolished by a specific GH-releasing fac-tor receptor antagonist. J Endocrinol 1994; 140:R9–R13.

170 Pandya N, DeMott-Friberg R, Bowers CY, Barkan AL, Jaffe CA: Growth hormone (GH)-releasing peptide-6 requires endoge-nous hypothalamic GH-releasing hormone for maximal GH stimulation. J Clin Endo-crinol Metab 1998; 83: 1186–1189.

171 Bennett PA, Thomas GB, Howard AD, Feighner SD, van der Ploeg LH, Smith RG, Robinson IC: Hypothalamic growth hor-mone secretagogue-receptor expression is regulated by growth hormone in the rat. Endocrinology 1997; 138: 4552–4557.

172 Kamegai J, Wakabayashi I, Miyamoto K, Unterman TG, Kineman RD, Frohman LA: Growth hormone-dependent regulation of pituitary GH secretagogue receptor mRNA levels in the spontaneous dwarf Rat. Neu-roendocrinology 1998; 68: 312–318.

173 Kineman RD, Kamegai J, Frohman LA: Growth hormone (GH)-releasing hormone (GHRH) and the GH secretagogue (GHS), L-692,585, differentially modulate rat pitu-itary GHS receptor and GHRH receptor messenger ribonucleic acid levels. Endocri-nology 1999; 140: 3581–3586.

174 Lall S, Balthasar N, Carmignac D, Magou-las C, Sesay A, Houston P, Mathers K, Rob-inson I: Physiological studies of transgenic mice overexpressing growth hormone (GH) secretagogue receptor 1A in GH-re-leasing hormone neurons. Endocrinology 2004; 145: 1602–1611.

175 Mano-Otagiri A, Nemoto T, Sekino A, Yamauchi N, Shuto Y, Sugihara H, Oikawa S, Shibasaki T: Growth hormone-releasing hormone (GHRH) neurons in the arcuate nucleus (Arc) of the hypothalamus are de-creased in transgenic rats whose expression of ghrelin receptor is attenuated: evidence that ghrelin receptor is involved in the up-regulation of GHRH expression in the Arc. Endocrinology 2006; 147: 4093–4103.

176 Maggio R, Novi F, Scarselli M, Corsini GU: The impact of G-protein-coupled receptor hetero-oligomerization on function and pharmacology. FEBS J 2005; 272: 2939–2946.

177 Cunha SR, Mayo KE: Ghrelin and growth hormone (GH) secretagogues potentiate GH-releasing hormone (GHRH)-induced cyclic adenosine 3 � ,5 � -monophosphate pro-duction in cells expressing transfected GHRH and GH secretagogue receptors. Endocrinology 2002; 143: 4570–4582.

178 Jiang H, Betancourt L, Smith RG: Ghrelin amplifies dopamine signaling by cross-talk involving formation of growth hormone secretagogue receptor/dopamine receptor subtype-1 heterodimers. Endocrinology 2006; 20: 1772–1785.

179 Ferguson SS: Evolving concepts in G-pro-tein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol Rev 2001; 53: 1–24.

180 Camina JP, Carreira MC, El Messari S, Llo-rens-Cortes C, Smith RG, Casanueva FF: Desensitization and endocytosis mecha-nisms of ghrelin-activated growth hor-mone secretagogue receptor 1a. Endocri-nology 2004; 145: 930–940.

181 Parnot C, Miserey-Lenkei S, Bardin S, Cor-vol P, Clauser E: Lessons from constitutive-ly active mutants of G-protein-coupled re-ceptors. Trends Endocrinol Metab 2002; 13: 336–343.

182 Wilbanks AM, Laporte SA, Bohn LM, Barak LS, Caron MG: Apparent loss-of-function mutant GPCRs revealed as consti-tutively desensitized receptors. Biochemis-try 2002; 41: 11981–11989.

183 Miserey-Lenkei S, Parnot C, Bardin S, Cor-vol P, Clauser E: Constitutive internaliza-tion of constitutively active agiotensin II AT(1A) receptor mutants is blocked by in-verse agonists. J Biol Chem 2002; 277: 5891–5901.

184 Leterrier C, Bonnard D, Carrel D, Rossier J, Lenkei Z: Constitutive endocytic cycle of the CB1 cannabinoid receptor. J Biol Chem 2004; 279: 36013–36021.

185 Kim MS, Yoon CY, Park KH, Shin CS, Park KS, Kim SY, Cho BY, Lee HK: Changes in ghrelin and ghrelin receptor expression ac-cording to feeding status. Neuroreport 2003; 14: 1317–1320.

186 Tups A, Helwig M, Khorooshi RM, Archer ZA, Klingenspor M, Mercer JG: Circulating ghrelin levels and central ghrelin receptor expression are elevated in response to food deprivation in a seasonal mammal (Phodo-pus sungorus) . J Neuroendocrinol 2004; 16: 922–928.

187 Wu R, Zhou M, Cui X, Simms HH, Wang P: Upregulation of cardiovascular ghrelin re-ceptor occurs in the hyperdynamic phase of sepsis. Am J Physiol 2004; 287:H1296–H1302.

188 Ong H, McNicoll N, Escher E, Collu R, De-ghenghi R, Locatelli V, Ghigo E, Muccioli G, Boghen M, Nilsson M: Identification of a pituitary growth hormone-releasing pep-tide receptor subtype by photoaffinity la-beling. Endocrinology 1998; 139: 432–435.

189 Ong H, Bodart V, McNicoll N, Lamontagne D, Bouchard JF: Binding sites for growth hormone-releasing peptide. Growth Hor-mone IGF Res 1998; 8: 137–140.

190 Papotti M, Ghè C, Cassoni P, Catapano F, Deghenghi R, Ghigo E, Muccioli G: Growth hormone secretagogue binding sites in pe-ripheral human tissues. J Clin Endocrinol Metab 2000; 85: 3803–3807.

Ghrelin/GHS Receptors Neuroendocrinology 019-T1 17

191 Muccioli G, Broglio F, Valetto MR, Ghè C, Catapano F, Graziani A, Papotti M, Bisi G, Deghenghi R, Ghigo E: Growth hormone-releasing peptides and the cardiovascular system. Ann Endocrinol (Paris) 2000; 61: 27–31.

192 Bodart V, Bouchard JF, McNicoll N, Escher E, Carriere P, Ghigo E, Sejlitz T, Sirois MG, Lamontagne D, Ong H: Identification and characterization of a new growth hormone-releasing peptide receptor in the heart. Circ Res 1999; 85: 796–808.

193 Huang J, Zhou H, Mahavadi S, Sriwai W, Lyall V, Murthy KS: Signaling pathways mediating gastrointestinal smooth muscle contraction and MLC20 phosphorylation by motilin receptors. Am J Physiol 2005; 288:G23–G31.

194 Tremblay F, Perreault M, Klaman LD, To-bin JF, Smith E, Gimeno RE: Normal food intake and body weight in mice lacking the G-protein-coupled receptor GPR39. Endo-crinology 2007; 148: 501–506.

195 Lauwers E, Landuyt B, Arckens L, Schoofs L, Luyten W: Obestatin does not activate orphan G-protein-coupled receptor GPR39. Biochem Biophys Res Commun 2006; 351: 21–25.

196 Bresciani E, Rapetti D, Dona F, Bulgarelli I, Tamiazzo L, Locatelli V, Torsello A: Obestatin inhibits feeding but does not modulate GH and corticosterone secretion in the rat. J Endocrinol Invest 2006; 29:RC16–RC18.

197 Seoane LM, Al-Massadi O, Pazos Y, Pagot-to U, Casanueva FF: Central obestatin ad-ministration does not modify either spon-taneous or ghrelin-induced food intake in rats. J Endocrinol Invest 2006; 29:RC13–RC15.

198 Morello JP, Bouvier M: Palmitoylation: a post-translational modification that regu-lates signalling from G-protein-coupled re-ceptors. Biochem Cell Biol 1996; 74: 449–457.

199 Brighton PJ, Szekeres PG, Willars GB: Neu-romedin U and its receptors: structure, function, and physiological roles. Pharma-col Rev 2004; 56: 231–248.

200 Jethwa PH, Smith KL, Small CJ, Abbott CR, Darch SJ, Murphy KG, Seth A, Semjonous NM, Patel SR, Todd JF, Ghatei MA, Bloom SR: Neuromedin U partially mediates leptin-induced hypothalamo-pituitary ad-renal (HPA) stimulation and has a physio-logical role in the regulation of the HPA axis in the rat. Endocrinology 2006; 147: 2886–2892.

201 Nogueiras R, Tovar S, Mitchell SE, Barrett P, Rayner DV, Dieguez C, Williams LM: Negative energy balance and leptin regu-late neuromedin-U expression in the rat pars tuberalis. J Endocrinol 2006; 190: 545–553.

202 Alevizos I, Mahadevappa M, Zhang X, Ohyama H, Kohno Y, Posner M, Gallagher GT, Varvares M, Cohen D, Kim D, Kent R, Donoff RB, Todd R, Yung CM, Warrington JA, Wong DT: Oral cancer in vivo gene ex-pression profiling assisted by laser capture microdissection and microarray analysis. Oncogene 2001; 20: 6196–6204.

203 Yamashita K, Upadhyay S, Osada M, Hoque MO, Xiao Y, Mori M, Sato F, Meltzer SJ, Sid-ransky D: Pharmacologic unmasking of epigenetically silenced tumor suppressor genes in esophageal squamous cell carci-noma. Cancer Cell 2002; 2: 485–495.

204 Takahashi K, Furukawa C, Takano A, Ishi-kawa N, Kato T, Hayama S, Suzuki C, Yasui W, Inai K, Sone S, Ito T, Nishimura H, Tsuchiya E, Nakamura Y, Daigo Y: The neuromedin U-growth hormone secreta-gogue receptor 1b/neurotensin receptor 1 oncogenic signaling pathway as a therapeu-tic target for lung cancer. Cancer Res 2006; 66: 9408–9419.

205 Vincent JP, Mazella J, Kitabgi P: Neuroten-sin and neurotensin receptors. Trends Pharmacol Sci 1999; 20: 302–309.

206 Poinot-Chazel C, Portier M, Bouaboula M, Vita N, Pecceu F, Gully D, Monroe JG, Maf-frand JP, Le Fur G, Casellas P: Activation of mitogen-activated protein kinase couples neurotensin receptor stimulation to induc-tion of the primary response gene Krox-24. Biochem J 1996; 320: 145–151.

207 Botto JM, Guillemare E, Vincent JP, Mazel-la J: Effects of SR 48692 on neurotensin-in-duced calcium-activated chloride currents in the Xenopus oocyte expression system: agonist-like activity on the levocabastine-sensitive neurotensin receptor and absence of antagonist effect on the levocabastine in-sensitive neurotensin receptor. Neurosci Lett 1997; 223: 193–196.

208 Sarret P, Gendron L, Kilian P, Nguyen HM, Gallo-Payet N, Payet MD, Beaudet A: Phar-macology and functional properties of NTS2 neurotensin receptors in cerebellar granule cells. J Biol Chem 2002; 277: 36233–36243.

209 Gendron L, Perron A, Payet MD, Gallo-Payet N, Sarret P, Beaudet A: Low-affinity neurotensin receptor (NTS2) signaling: in-ternalization-dependent activation of ex-tracellular signal-regulated kinases 1/2. Mol Pharmacol 2004; 66: 1421–1430.

210 Perron A, Sarret P, Gendron L, Stroh T, Beaudet A: Identification and functional characterization of a 5-transmembrane do-main variant isoform of the NTS2 neuro-tensin receptor in rat central nervous sys-tem. J Biol Chem 2005; 280: 10219–10227.

211 Chu KM, Chow KB, Leung PK, Lau PN, Chan CB, Cheng CH, Wise H: Overexpres-sion of the truncated ghrelin receptor poly-peptide attenuates the constitutive activa-tion of phosphatidylinositol-specific phos-pholipase C by ghrelin receptors but has no effect on ghrelin-stimulated extracellular signal-regulated kinase 1/2 activity. Int J Biochem Cell Biol 2007; 39: 752–764.

212 Jeffery PL, Herington AC, Chopin LK: Ex-pression and action of the growth hor-mone-releasing peptide ghrelin and its re-ceptor in prostate cancer cell lines. J Endocrinol 2002; 172:R7–R11.

213 De Vriese C, Gregoire F, De Neef P, Rob-berecht P, Delporte C: Ghrelin is produced by the human erythroleukemic HEL cell line and involved in an autocrine pathway leading to cell proliferation. Endocrinology 2005; 146: 1514–1522.

214 Barzon L, Pacenti M, Masi G, Stefani AL, Fincati K, Palu G: Loss of growth hormone secretagogue receptor 1a and overexpres-sion of type 1b receptor transcripts in hu-man adrenocortical tumors. Oncology 2005; 68: 414–421.

215 Sato M, Nakahara K, Goto S, Kalya H, Mi-yazato M, Date Y, Nakazato M, Kangawa K, Murakami N: Effects of ghrelin and des-acyl ghrelin on neurogenesis of the rat fetal spinal cord. Biochem Biophys Res Com-mun 2006; 350: 598–603.

216 Gauna C, Meyler FM, Janssen JA, Delhanty PJ, Abribat T, van Koetsveld P, Hofland LJ, Broglio F, Ghigo E, van der Lely AJ: Admin-istration of acylated ghrelin reduces insulin sensitivity, whereas the combination of ac-ylated plus unacylated ghrelin strongly im-proves insulin sensitivity. J Clin Endocrinol Metab 2004; 89: 5035–5042.

217 Broglio F, Gottero C, Prodam F, Gauna C, Muccioli G, Papotti M, Abribat T, Van Der Lely AJ, Ghigo E: Non-acylated ghrelin counteracts the metabolic but not the neu-roendocrine response to acylated ghrelin in humans. J Clin Endocrinol Metab 2004; 89: 3062–3065.

218 Gauna C, Delhanty PJ, van Aken MO, Jans-sen JA, Themmen AP, Hofland LJ, Culler M, Broglio F, Ghigo E, van der Lely AJ: Un-acylated ghrelin is active on the INS-1E rat insulinoma cell line independently of the growth hormone secretagogue receptor type 1a and the corticotropin-releasing fac-tor-2 receptor. Mol Cell Endocrinol 2006; 251: 103–111.

219 Toshinai K, Yamaguchi H, Sun Y, Smith RG, Yamanaka A, Sakurai T, Date Y, Mondal MS, Shimbara T, Kawagoe T, Mu-rakami N, Miyazato M, Kangawa K, Naka-zato M: Des-acyl ghrelin induces food in-take by a mechanism independent of the growth hormone secretagogue receptor. Endocrinology 2006; 147: 2306–2314.

Muccioli /Baragli /Granata /Papotti /Ghigo

Neuroendocrinology 019-T1 18

220 Matsuda K, Miura T, Kaiya H, Maruyama K, Shimakura S, Uchiyama M, Kangawa K, Shioda S: Regulation of food intake by acyl and des-acyl ghrelins in the goldfish. Pep-tides 2006; 27: 2321–2325.

221 Ghè C, Cassoni P, Catapano F, Marrocco T, Deghenghi R, Ghigo E, Muccioli G, Papotti M: The antiproliferative effect of synthetic peptidyl GH secretagogues in human CALU-1 lung carcinoma cells. Endocrinol-ogy 2002; 143: 484–491.

222 Torsello A, Bresciani E, Rossoni G, Aval-lone R, Tulipano G, Cocchi D, Bulgarelli I, Deghenghi R, Berti F, Locatelli V: Ghrelin plays a minor role in the physiological con-trol of cardiac function in the rat. Endocri-nology 2003; 136: 1146–1152.

223 Deghenghi R: Structural requirements of growth hormone secretagogues; in Bercu BB, Walker RF (eds): Growth Hormone Se-cretagogues in Clinical Practice. New York, Dekker, 1998, pp 27–33.

224 Febbraio M, Haijar DP, Silverstein RL: CD36: A class B scavenger receptor in-volved in angiogenesis, atherosclerosis, in-flammation, and lipid metabolism. J Clin Invest 2001; 108: 785–791.

225 Bodart V, Febbraio M, Demers A, McNicoll N, Pohankova P, Perreault A, Sejlitz T, Escher E, Silverstein RL, Lamontagne D, Ong H: CD36 mediates the cardiovascular action of growth hormone-releasing pep-tides in the heart. Circ Res 2002; 90: 844–849.

226 Pettersson I, Muccioli G, Granata R, De-ghenghi R, Ghigo E, Ohlsson C, Isgaard J: Natural (ghrelin) and synthetic (hexarelin) GH secretagogues stimulate H9c2 cardio-myocyte cell proliferation. J Endocrinol 2002; 175: 201–209.

227 Frascarelli S, Ghelardoni S, Ronca-Testoni S, Zucchi R: Effect of ghrelin and synthetic growth hormone secretagogues in normal and ischemic rat heart. Basic Res Cardiol 2003; 98: 401–405.

228 Demers A, McNicoll N, Febbraio M, Ser-vant M, Marleau S, Silverstein R, Ong H: Identification of the growth hormone-re-leasing peptide binding site in CD36: a pho-toaffinity cross-linking study. Biochem J 2004; 382: 417–424.

229 Avallone R, Demers A, Rodrigue-Way A, Bujold K, Harb D, Anghel S, Wahli W, Mar-leau S, Ong H, Tremblay A: A growth hor-mone-releasing peptide that binds scaven-ger receptor CD36 and ghrelin receptor upregulates sterol transporters and choles-terol efflux in macrophages through a per-oxisome proliferator-activated receptor- � -dependent pathway. Mol Endocrinol 2006; 20: 3165–3178.

230 Marleau S, Harb D, Bujold K, Avallone R, Iken K, Wang Y, Demers A, Sirois MG, Feb-braio M, Silverstein RL, Tremblay A, Ong H: EP-80317, a ligand of the CD36 scaven-ger receptor, protects apolipoprotein E-de-ficient mice from developing atherosclerot-ic lesions. FASEB J 2005; 19: 1869–1871.

231 Cummings DE, Foster-Shubert KE, Over-duin J: Ghrelin and energy balance: focus on current controversies. Current Drug Targets 2006; 6: 153–169.

232 Wortley KE, del Rincon JP, Murray JD, Garcia K, Iida K, Thorner MO, Sleeman MW: Absence of ghrelin protects against early-onset obesity. J Clin Invest 2005; 115: 3573–3578.

233 Zigman JM, Nakano Y, Coppari R, Balthasar N, Marcus JN, Lee CE, Jones JE, Deysher AE, Waxman AR, White RD, Williams TD, Lachey JL, Seeley RJ, Lowell BB, Elmquist JK: Mice lacking ghrelin receptors resist the development of diet-induced obesity. J Clin Invest 2005; 115: 3564–3572.

234 Bowers CY: Historical milestones; in Ghigo E (ed): Ghrelin. Boston, Kluwer Academic, 2004, pp 1–13.