LOGO Disorders Associated with GPRs Members: 王王王 王王王 王王王王 王王 、、、
Jan 16, 2016
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Disorders Associated with GPRsMembers:王建博、秦涛、李中印、王宇
The structural and signal transmit mechanism of the class-
C G-protein-coupled receptors
First
What is this? One of the five classes GPCRs
How does the GPCRs classified? Based on sequence similarity. class-A:Rhodopsin( 视紫红质 )-like receptors class-B:secretin( 分泌素 )-like receptors class-C:mGlu-like receptors
What are class-C GPCRs include? neurotransmitters receptors glutamate( 谷氨酸盐 ) receptors GABA receptors the calcium-sensing receptor sweet taste receptors pheromone( 信息素 )receptors
Difference
A large extracellular domain——VFTVFT : Venus Flytrap (bilobate 双叶形 )
Can be regulared by allosteric modulator (变构调节剂)
Dimer ——homo or heter
The structural
Four parts1.VFT(Venus Flytrap): the agonist
binding site2.CRD(cysteine-rich domain)3.HD(heptahelical domain) C-term
VFT
Tow parts:Lobe-I&Lobe-II
Two conformations : Open(inactive) & Closed(active)
antagonist & agonist ( 抑制剂 ) ( 激动剂 )
Lobe-I
Lobe-II
CRD
The structure and function are unknown
absent CRD:GABA receptor
HD ( Heptahelical domain )
Heptahelical
long C-terminal tail
Activity site positive & negative allosteric modulators
independency
How is the signal transduced from one domain to the other?
Homodimeric receptors mGlu receptor
A disulfide-linked dimer Cys-residues
Class-C GPCRs are constitutive dimers
Lobe-I
Lobe-II
Heterodimeric receptors GABAB receptor
Absent disulfide bridge no covalent( 共价的 ) linkage
May have interaction between intracellular tail
ER retention signal (GABAB1)
Activation mechanism of class-C GPCRs
interaction between the VFTs
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On mGlus receptor
(Roo, resting-open-open) (Aco, active-closed-open) (Acc, active-closed-closed)
Are both Aco & Acc conformations lead to similar properties?
Mutated mGlu VFT composed of two distinct binding sites
Roo, none activity Aco, half activity Acc, full activity(Ca2+)
Why?Roo state: This interface revealed major charge
repulsion
Aco state: the interface consists of a number of
ionic interactions
Acc state: four acidic side chains are facing each
other, creating a cationbinding site
Only have two conformations A signal agonist can fully activate a
receptor
Surprisingly GABAB receptor in which GABA binds
in the GABAB1 VFT only
On GABAB receptor
But
GABAB2 is necessary for GABAB
only those possessing both the GABAB1 and GABAB2 VFTs display agonist-induced activity
Why Unknown
Allosteric coupling between the extracellular and HD within the
dimer
On GABAB receptor
HD of GABAB2 is a important part
Experiment IMutations into either the i2 or i3 loop
of GABAB2 suppressed G-protein activation.
The equivalent mutation in GABAB1 had a minor effect
Experiment II
GABAB1
VFTGABAB2
VFT
GABAB2 HD GABAB2 HD
Demonstrating that the HD of GABAB2 possesses enough of the molecular determinants required for G-protein coupling
GABAB2 HD expressed alone can be activated by CGP7930, a positive allosteric modulator of the GABAB receptor.
So.. trans-activation occurs in the GABAB
receptor
Experiment III
Another Experiment
GABAB1 VFT +GABAB2 HD =can not be activated
(GABAB1 VFT + GABAB2 HD) +(GABAB2 VFT + GABAB1 HD)
= can be activated
In this combination subunits cis-activation occurs
GABAB1
VFT
GABAB2 HD
GABAB2
VFT
GABAB1 HD
In a word, dimer conformation takes a very important part in GABAB receptors
On mGlu receptor
Both cis- and trans-activation occur in mGlu receptors
Because the homodimeric structure
Allosteric functioning of the HD of class C GPCRs
HD can exist in three states
HDg states:totally inactive state
HD states:Low active efficiency state
HD* states:High active efficiency state
HD can be regulared by positive & negative allosteric modulators ( 变构调节剂 )
Allosteric modulators are compounds able to regulate
the activity of a receptor by binding at a site distinct from that where endogenous ligands bind
Classification negative allosteric modulators inhibit constitutive activity of the receptor
positive allosteric modulators activate the receptor , can enhance either the
potency (力量) or the efficacy (效力) , or both
DifferenceNegative ——directlyPositive——indirectly(with agonist)
SO…
the action of negative allosteric modulators is less dependent on the concentration of endogenous ligand(agonist)
But positive allosteric modulators is highly dependent on agonist
Experiment
Bay 7620:negative allosteric modulators Ro01-6128: positive allosteric modulators
EC50 :value of glutamate
Why we intrested in allosteric modulators?
Both positive & negative modulators are highly receptor subtype selective.
less side effect , long effect Hydrophobic, allowing them to cross the
blood brain barrier more easily