Common Motif in GPC 7 TM Helix Receptors

8/3/2019 Common Motif in GPC 7 TM Helix Receptors

1/10

Journa l of Com puter-Aided Mo lecular Design, 7 (1993) 649 658 64 9ESCOMJ-CAMD 221

A c o m m o n m o t i f in G - p r o t e in - c o u p l e d s ev e nt r a n s m e m b r a n e h e l i x r e c e p t o r sL . O l i v e i r a a, A . C . M . P a i v a a a n d G . V r i e n d b'*

aEscota Paulista Medicina, Departamento de Biofisica, CP 20388, 04034 S6o Paulo, BrazilbEMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany

Received 28 A pril 1993Accepted 21 M ay 1993Key words G-protexn-coupled ecepto rs; Mod eling

S U M M A R YG-pro te in-coupled receptors a l l sha re the seven t ransmem brane he lix mo t i f similar to bac te r iorhodops in .This similari ty was exp loited to bu ild models fo r these recepto rs. Fr om an analysis of a mult i-sequencealignme nt of 225 G-pr otein -cou pled recep tors belonging to the rhodop sin-l ike superfamily, conclusionscould be drawn about functional residues. Seven residues in the transmembrane regions are conservedthroughout a l l a l igned receptors. These residues cluster a t the cytosolic side of the transmembrane helicesand are f or a ll rhodopsin -l ike G- pro tein-co uple d recepto rs implied in signal t ransduc tion. An analysis of

corre lated mutatio ns reveals a n um ber o f residues, bo th in the helices and in the cytosolic loops, tha t mightbe imp orta nt in the signal t ransdu ction p athw ay in subfamilies o f this rece ptor family.

I N T R O D U C T I O NG - p r o t e i n - c o u p l e d r e ce p t o rs ( G P C R s ) c o n s t it u t e a la r ge g r o u p o f m e m b r a n e - s p a n n i n g r e ce p -

t o r s o f d i v e r s e b i o l o g i c a l f u n c t i o n . I t is e s t i m a t e d t h a t a t l e a s t s e v e ra l h u n d r e d d i f f e r e n t m e m b e r so f t h e s e p r o t e i n s e x i s t [1 - 5 ] . T h e r e c e p t o r s h a v e b e e n d i v i d e d i n t o t h r e e s u p e r f a m i l i e s [ 6] :

( 1 ) r h o d o p s i n - l i k e r e c e p t o r s ,( 2 ) s e c re t i n - li k e r e c e p t o r s , a n d( 3) m e t a b o t r o p i c g l u t a m a t e r e c e p t o r s .

T h e r h o d o p s i n - l i k e r e c e p t o r s a r e c h a r a c t e r i z e d b y t h e f o l l o w i n g c o n s e r v e d re s id u e s : G N i n h e li xI , L x x x D i n h e li x II , D R Y a t t h e e n d o f h e li x I I I a n d N P i n he l ix V I I . N o t e n o u g h s e c r e ti n -l ik ea n d m e t a b o t r o p i c g l u t a m a t e s e q u e n c e s h a v e b e e n d e t e r m i n e d y e t t o d e f in e s e q u e n c e c o n s e r v a t i o np a t t e r n s i n t h es e s u b f a m il ie s . T h e s e c re t in - li k e a n d m e t a b o t r o p i c g l u t a m a t e r e c e p t o r s t h a t h a v eb e e n s e q u e n c e d s o f a r d o n o t p o s s es s a n y o f t h e r e si d u e s c h a r a c te r i s ti c f o r t h e G P C R f a m i l y [7 ,8 ].

S o m e i m p o r t a n t G P C R s o f th e r h o d o p s i n - l i k e g r o u p a r e a m i n e r e c e p t o r s (~ - a n d 1 3 -a dr en er gi c,*To wh om correspondenceshou ld be addressed.0920-654X/$10.00 1993 ESC OM SciencePublishers B.V.


2/10

650mu sca r in i c , s e ro to n in , d o p amin e an d h i s t amin e ) , p ep t i d e recep to rs (an g io t en s in I I , b rad y k in -in , en d o th e l i n s , t a ch y k in in s , n eu ro p ep t i d es , ch emo tac t i c p ep t i d es ) , i n t e r l eu k in -8 , o l fac to ryan d t ro p in recep to rs , an d o p s in s . Th e i r ac t i v a t i o n l ead s t o v a r i ed b as i c p h y s io lo g i ca l r e -sp o n ses , b u t can a l so i n d u ce co mp lex b eh av io ra l p a t t e rn s su ch a s a f fec t i o n , d ep re ss io n , ag g res -sion, etc.

Th e b as i c fu n c t i o n fo r a ll G PC R s seems t o b e s imi la r. A n ex t e rn a l s i g n a l is r ece iv ed , wh ich i sn o rm a l ly a l ig an d b u t i n th e ca se o f o p s in s a p h o to n a l te r s t h e s t ru c tu re o f t h e b o u n d re t i n a l. Th i sex t e rn a l s i gn a l is tr an sd u ced t h ro u g h t h e m em b ran e t o t h e cy to so l i c s i d e wh e re a G-p ro t e in i sa c t iv a t ed . T h e s ig n a l re c e p t i o n a n d t r a n s d u c t i o n is p e r f o r m e d m o s t l y b y th e m e m b r a n e - e m b e d -d ed p a r t o f t h e recep to rs . Th e sp ec if i ci ty fo r G -p ro t e in s i s m o s t l i k e ly d e t e rm in ed b y t h e cy to so l i cloops [9] .

N o d i r e c t 3 D s t r u c t u r e d e t e r m i n a t i o n o f a n y G P C R h a s b e e n c a rr i e d o u t y e t. H o w e v e r , th es t r u c tu r e o f t h e m e m b r a n e - e m b e d d e d h e li ce s o f b a c t e r io r h o d o p s i n ( B r h ) h as b e e n s o l v e d b ye l ec t ro n c ry s t a l l o g rap h y [1 0] . Ex p e r im en ta l d a t a f ro m d e l e t i o n mu ta t i o n s , a n t i b o d y t a rg e t i n g an dp r o t e o l y t ic d i g e st io n e x p e r i m e n t s p r o v i d e e v i d e n ce t h a t t h e o v e r a l l f e at u r e s o f G P C R s a n d B r hm u s t b e simil ar . B rh h as sev en t ran sm em b ran e h e l i ce s o rg an i zed i n a k id n ey -sh ap ed man n e r , w i thn ea re s t -n e ig h b o r h e l ice s ru n n in g an t i p a ra ll e l . I t h a s b een d e t e rmin ed ex p e r imen ta l l y t h a t G P C R sa l so p o ssess sev en t ran sm em b ran e h e l ice s ( fo r rev i ews see Re fs . 1 1 ,1 2 an d re fe ren ces c i t ed th e re -in ). M a n y a t t e m p t s h a v e b e e n m a d e t o d e r i v e th r e e - d im e n s i o n a l m o d e l s o f G P C R s b a s e d o n t hi ss imi l a r i t y wi th Brh [1 3 -1 5 ] . Th i s 3 D mo d e l i n g h as sh o wn th a t p o in t mu ta t i o n s an d i n h ib i t o rb i n d in g c a n b e i n a g re e m e n t w i t h t h e a s s u m p t i o n t h a t t h e 7 T M s o f G P C R s a n d B r h a r e s im i la r.

M o d e l b u i ld i n g h a s to s t a r t w i t h a se q u e n c e al ig n m e n t b e t w e e n th e G P C R s a n d B r h . A p r o b l e mi s t h a t s t an d a rd seq u en ce a l i g n men t t ech n iq u es u se s t an d a rd amin o ac id ex ch an g e ma t r i ce s .T h e s e m a t r i c es h a v e b e e n o p t i m i z e d f o r t h e a l i g n m e n t o f w a t e r -s o l u b l e p r o te i n s a n d a r e f a r f r o mi d e a l f o r m e m b r a n e - e m b e d d e d p r o t e i n s . A n o t h e r p r o b l e m i s t h a t t h e o v e r a l l s e q u e n c e i d e n t i t yb e t w e e n 7 T M s a n d B r h i s t o o l o w t o a l l o w f o r m o d e l b u i l d i n g b y h o m o l o g y u s i n g s t a n d a r dtechniques [16] .

W e u s e d a n o v e l i t e r a t i v e p r o f i l e a l i g n m e n t m e t h o d t h a t i s b a s e d o n k n o w l e d g e a b o u t t h ef u n c t i o n o f s o m e k e y r es i d ue s t o a l ig n 2 25 r h o d o p s i n -l i k e G P C R s . M a n y a d d i t i o n a l c o n s e r v e dres id u es were o b se rv ed an d , t o g e th e r w i th an an a ly s i s o f co r re l a t ed m u ta t i o n s , a se ri e s o f re s id u esp o t en t i a l l y imp o r t an t fo r s i g n a l t r an sd u c t i o n was d e t ec t ed . Th ese re s id u es a re l o ca t ed ma in ly i nth e h e li ces , b u t t en o f t h e re s id u es t h a t d i sp l ay co r re l a t ed m u ta t i o n a l b eh a v io r were fo u n d t o l iei n t h e cy to so l i c lo o p s .I TE R A T IV E S E Q U E N C E A L I G N M E N T

Man y a t t emp t s h av e b een mad e t o a l i g n GPCR seq u en ces [e .g . , 4 , 1 3 ,1 4 ,1 7 -2 5 ] . S t an d a rdseq u en ce a l i g n men t t ech n iq u es can b e u sed re l i ab ly wh en o n ly (h ig h ly h o mo lo g o u s ) seq u en ceswi th in o n e c l as s o f recep to rs a re a l i g n ed , b u t fa i l wh en a l l p re sen t l y k n o w n recep to rs n eed t o b ea l ig n ed . Th e p ro b l em i s th e o ccu r ren ce o f in se r t io n s an d d e l e t i o n s (i nd e ls ), wh ich o f co u rse a rev e ry u n l i k e ly i n th e t ran sm em b ran e h el ice s.

Th e w o rk o f Cro n e t e t a l. [1 5 ] m ad e c l ea r t h a t g en e ra l ru l e s fo r re s id u e ex ch an g es i n 7 TM s a reh a rd t o d e r i v e b ecau se re s id u e p re fe ren ces a re v e ry p o s i t i o n speci fi c. A g o o d Da y h o f f - t y p e ma t r i xcan t h e re fo re n o t b e d e r i v ed fo r t h e t ran smemb ran e h e l i ce s . S in ce we h av e a re l a t i v e ly l a rg e


3/10

6 51

n u m b e r o f se q u e n c e s t o w o r k w i t h ( 2 25 ) , w e c h o s e t o u s e a p r o f il e a l i g n m e n t m e t h o d [ 26 ,2 7 ]u t i li z i n g a u n i t a r y e x c h a n g e m a t r i x , o r in o t h e r w o r d s , t o s e t t h e p r o f il e v a l u e s e q u a l t o th ef r e q u e n c y o f o c c u r r e n c e o f t h e 2 0 a m i n o a c id s a t e a c h p o s i t i o n i n th e m u l t i - s e q u e n c e a l i g n m e n t .

T h e i n it ia l a li g n m e n t o f t h e 7 T M s e q u e n c e s o f ~ 1 00 k n o w n G P C R s , e x t r a c t ed f r o m t h e S W I S S -P R O T [2 8] a n d P I R [ 29 ] d a t a b a s e s, w a s m a d e b y v i su a l i n sp e c t io n . I n t h is a l i g n m e n t , t h e v e r yl a r g e i n s e r t i o n s t h a t o f t e n o c c u r b e t w e e n h e l i ce s V a n d V I w e r e le f t o u t . T h e p r o f i l e o b t a i n e d f r o mt h e in i ti a l m a n u a l a l i g n m e n t w a s u s e d t o r e a l i g n t h e se q u e n c e s . T h i s p r o c e s s o f r e b u i l d i n g t h ep r o fi le f r o m t h e m u l t i - s e q u e n c e a l i g n m e n t a n d a l ig n i n g a ll s eq u e n c e s o n e b y o n e a g a i n s t t h ep r o fi le w a s i t e r a te d u n t i l n o c h a n g e s o c c u r r e d b e t w e e n t w o s u b s e q u e n t a l i g n m e n t s . T h e g a p - o p e np e n a l t y w a s k e p t a t s i x t im e s t h e m a x i m a l p r o f i le v a l u e f o r r e si d u es k n o w n f r o m b i o c h e m i c a le x p e r i m e n t s t o l ie i n t h e t r a n s m e m b r a n e h e l ic e s a n d a t o n e ti m e t h e m a x i m a l p r o f i le v a lu ee l s ew h e r e . T h i s c o m p l e t e l y p r e v e n t e d i n d e ls f r o m o c c u r r i n g in h e li c es . T h e g a p e l o n g a t i o n p e n a l t yw a s 0 . 1 t i m e s t h e m a x i m a l p r o f i le v a l u e . T o a c c o m m o d a t e t h e l a r g e d if f e r e n c e s i n l o o p l e n g t h s ,t h e s ix r e s i d u e s i n t h e m i d d l e o f t h e l o o p s b e t w e e n t h e h e li c es w e r e g i v e n a g a p e l o n g a t i o n p e n a l t yo f 0 .0 . T h e p r o g r a m M A X H O M [3 0] w a s u s ed f o r a l i g n m e n t s a n d d a t a b a s e s e a rc h e s.

W h e n t h e S W I S S - P R O T d a t a b a s e [2 8] w a s s c r e e n e d w i t h th i s p ro f il e, a ll r h o d o p s i n - l i k e r e c e p -t o r s b u t n o s e c r e t i n - li k e o r g l u t a m a t e r e c e p t o r s w e r e e x t r a c t e d , a n d z e r o f a l s e p o s i t i v e s a p p e a r e d .W h e n t h e a c c e p t a n c e t h r e s h o l d w a s l o w e r e d s u c h th a t o t h e r p r o t e i n s w e r e a ls o d e te c t e d , m o s t o ft h es e p r o t e i n s w e r e G P C R s o f o t h e r g r o u p s a n d i o n c h a n n e ls . T o c h e c k t h a t o a r p r o f il e w a s ag e n e r a li z e d d e s c r ip t i o n o f r h o d o p s i n - l i k e G P C R s a n d n o t m e r e l y a d e s c r i p ti o n o f t h e s e q u e n c esT A B L E 1L O C A T I O N O F L I G A N D B I N D I N G R E S I D U E S IN T H E 7 TM S T R U C T U R E~H e l i x I I

A sp383 (224) in lutropin receptor is important for horm one binding an d G -protein a ctivation [29].A s p 79 (224) is related to sod ium regulation of a2-adrenergic receptors [47].Ser I2 (231) is involved in antagon ist b inding in m uscarinic rece ptors [48].

H e l i x I I IA sp (323j neutralizes the positive charge of the ligand s in amine receptors [49]. Th is residue corresp onds to Asp 15 in thehum an M1 mus carinic receptor; to A sp H~ in the rat 13-2adrenergic receptor; and to Asp9~ in the rat h istamine H 2 recep tor.Phe(~-'% A la ~322J,T rp (331) an d Se r~332)(corr espo ndin g to Ph e 115, A la ~17, Tr p 126 an d Ser 127 in bov ine rhod ops in) are pro xim alto the retinal [50-52]. Glu m in bovine rhodopsin (327) is the counterion for t i le Schiff base [53-56].

H e l i x VSer 513~and Se r (516) (corresponding to Ser24 and Ser28 in the [3-2 adrenergic receptor [57]) hydrog en bo nd w ith ligandhydroxyl groups. ThrTM (509) and T h r 2 34 (512) bind agonists in the m uscarinic M1 receptor [48]. As p ~85 512) and Th r ~s9(516) form a serine proteinase-like catalytic relay with the im idazole ring o f the ligand in the r at histamine H2 receptor[58,59]. Asn 2 (509) in the rat bradykm in B2 receptor is tho ug ht to be a ssociated w ith ligand b inding [60]. His2" (516)in bovine rhodopsin m odulates the mechanism of deprotonation of the Schiff base [61].

H e l i x V IPhe29 (621) in the ~-2 adrenergic rec eptor is thou ght to bind the ligand [62]. Ty rs6 (621) in the mus carinic rece ptorsare th oug ht to bind the l igand [48]. Trp 26s (618) and T yr26a (621) are close to the retinal in bovine rhodop sin [52].

H e l i x V I ILys296 (723) binds the retinal in b ovine rhod opsin [63-66]. Ty rs29 (719 ) and Ty rs33 (723) are putatively related to ligandbinding in muscarinic receptors [48].

Num bers are from the original sequence. Num bers in parentheses refer to the nu mbering o f Fag. 1.


4/10

652

H E L I XR E C E P T O R Ib a c t e r i o r h o d o p s i n

c o n s e n s u sm u s c a r l n l c M 1b e t a - 2 a d r e n e r g l cb r a d i k i n i n B 2h i s t a m i n e H 2b o v i n e r h o d o p s i n

E X T E R N A L C Y T O S O L I. . . . . . . . . 1 2 0 . . . . . . . . . 1 3 0 . . . . .P E W I W L A L G T A L M G L G T L Y F L V K G M

* * * * * * *

M C M MQ V V L I A V L Y L V L V L V T V L G N V L V I WQ V A F I G I T T G L L S L A T V T G N L L V L IW V V G M G I V M S L I V L A I V F G N V L V I TL N A I Q A P F L W V L F L L A A L E N I F V L SL K V T I S V V L T T L I L I T I A G N V V V C LQ F S M L A A Y M F L L I M L G F P I N F L T L Y

I Ib a c t e r i o r h o d o p s i n

c o n s e n s u sm n s c a r l n l C M 1b e t a - 2 a d r e n e r g l cb r a d i k i n l n B 2h i s t a m i n e H 2b o v i n e r h o d o p s i n

2 4 0 . . . . . . . . . 2 3 0 . . . . . . . . . 2 2 0 . . . .L L M S L Y M T F A I A P V L T T I A Y F K K A D

. . * * * . .

L C L M M C ML A Y V A A F P M V L L G M L L D A L A L S V I FM L L T T Y L NM S F T G I I L DA C A L S L L FL I H A A G F P V V A L G M V L D A C A L S T I FN A I T I A W F P L G C A L I L D A G A L N G L YL Q Y I A S F P L V L L G L L L D T A A L S V I FL S T Y L T T T F G G F V M F L D A V A L N L L I

I I I . . . . . . 3 2 0 . . . . . . . . . 3 3 0 . . . . . . . . . 3 4 0ba ct er io rh od op sl n E Q N P I Y W A R Y A D W L F T T P L L L L D L A L L

c o n s e n s u sm ~ s c a r l n l c M 1b e t a - 2 a d r e n e r g l cb r a d i k i n l n B 2h i s t a m i n e H 2b o v i n e r h o d o p s l n

* * * * * * * * * * * * * * *L L L M LL M MC

L C K I W L S F D V L A S T A S I L S L T A I A V D RA C D L W L A L D Y V A S N A S V M N L L L I S F D RW C E F W T S I D V L C V T A S I E T L C V I A V D RL C R V V N T M I Y M N L Y S S I C F L M L V S I D RF C N I Y T S L D V M L C T A S I L N L F M I S L D RG C N L E G F F A T L G G E I A L W S L V V L A I E R

I Vb a c t e r l o r h o d o p s l n

c o n s e n s u sm u s c a r i n l c M 1b e t a - 2 a d r e n e r g i cb r a d l k i n l n B 2h i s t a m i n e H 2b o v i n e r h o d o p s l n

. 4 3 0 . . . . . . . . . 4 2 0 . . . . . . . . .L A G V L G T G I M I G D A G V L A L I T G

* * * * * * * *

M M C ML L P P L S L L L S L V W I L A I V V K A RL I A P A W L V F S V L W A L G I M L A A RQ I P L F S T L G S V I WV ML I I VR AKV L M P S S L L L T C S W I V L S Y L K A WH I S L F S L T I S I V W I F V L S I A V RV L P P A A C A L A M V W T F A V G M I A H

Vb a c t e r l o r h o d o p s i n

c o n s e n s u sm u s c a r i n l c M 1b e t a - 2 a d r e n e r g l cb r a d i k i n l n B 2h i s t a m i n e H 2b o v i n e r h o d o p s i n

. 5 1 0 . . . . . . . . . 5 2 0 . . . . . . . .W W A I S T A A M L Y I L Y V L F F G F T

. . . . . . . . . *

L L L LM MY V I V S F I V S F L L P L L I M L V C YI T F G T A M A A F Y L P V T V M C T L YY A I A S S I V S F Y V P L V I M V F V YT N M L L N L V G F L L P L S I I T F C TY G L V D G L V T F Y L P L L I M C V T YF V I Y M F V V H F I I P L I V I F F C Y

V Ib a c t e r l o r h o d o p s i n

c o n s e n s u sm u s c a r l n l c M 1b e t a - 2 a d r e n e r g l cb r a d i k i n z n B 2h i s t a m l n e H 2b o v l n e r h o d o p s i n

. . . . . . . 6 2 0 . . . . . . . . . 6 1 0 . . . . . . . .I L W V V P Y A S W L V V T V N R L V K F T S A V E

. . . . . . . . . . .

L C M L M ML I N V I F F P L W C I F F T G V V I A L T K A A KV L V M I N Y P T W T L I F A L L I A S L T R A A KV I N V I F F P L W C L T F T G M I I G L T K L A KL F T S I Q F P F W C L V F L G L V A L V L V T A KY V F A T F Y P F W C I I F A G M V A A L T V T A KY F A V G A Y P L W C I L F A I V M I I V M R T V E

V I Ib a c t e r i o r h o d o p s i n

c o n s e n s u sm u s c a r i n l c M 1b e t a - 2 a d r e n e r g i cb r a d i k i n i n B 2h l s t a m l n e H 2b o v i n e r h o d o p s i n

. 7 1 0 . . . . . . . . . 7 2 0 . . . . . . . . . 7 3 0 . .N I E T L L F M V L D V S A K V G F G L I L L R* * * * * * * * * .

L M L M M C C MK I I K T L F I L L V W L A Y I N S C L N P I IC V P E T L W E L G Y W L C Y V N S T I N P M CL I R K E V Y I L L N W I G Y V N S G F N P L IR A V D I V T Q I S S Y V A Y S N S C L N P L VA I N E A V E G I V L W L G Y A N S A L N P I LD F G P I F M T I P A E F A K T S A V Y N P V I


5/10

653u s e d , a j a c k - k n i f e e x p e r i m e n t w a s p e r f o r m e d . A p r o f i l e w a s c o n s t r u c t e d a s d e s c r i b e d a b o v e b a s e do n ~ 1 2 5 r e c e p t o r s ; a ll o p s in s a n d o l f a c t o r y r e c e p t o r s w e r e e x c lu d e d . W h e n t h i s p r o f il e w a s u s e dt o s c re e n t h e S W I S S - P R O T d a t a b a s e , e x a c t ly t h e s a m e s e q u e n ce s w e r e d e te c t ed a s m e n t i o n e db e f o r e . S e v e r al s u c h j a c k - k n i f e e x p e r i m e n t s w e r e p e r f o r m e d , a l w a y s w i t h t h e s a m e r e s u l t.

T h e s e q u e n c e a l i g n m e n t o f t h e 7 T M s o f t h e s e G P C R s i s s h o w n i n F ig . 1 . F o r a l i m i t ed s e t o fG P C R s e x p e r i m e n t a l d a t a a b o u t r e s i d u e s i n v o l v e d in l i g a n d b i n d i n g e x i s t (s e e T a b l e 1 ); t h e s er e s i d u e s a r e i n d i c a t e d i n F i g . 1 .C O R R E L A T E D M U T A T I O N S

I n t h e m u l t i - s e q u e n c e a l i g n m e n t , s e v e r a l c l a ss e s o f r e s i d u e s c a n b e id e n t i f i ed ( s e e a l s o T a b l e 1 ):( 1) L i g a n d b i n d i n g r e s i d u e s . T h e s e a r e m o s t l y v e r y v a r i a b l e , b u t s o m e a r e c o n s e r v e d i n o n l y o n ec l a s s o f r e c e p t o r s ( e . g ., t h e a s p a r t i c a c i d t h a t b i n d s t o t h e p o s i t i v e c h a r g e i n a l l a m i n e r e c e p t o r s ) ;( 2) H i g h l y c o n s e r v e d r e s id u e s . F o r s e v e r a l o f t h e s e a r o le i n si g n al t r a n s d u c t i o n h a s b e e n o b s e r v e de x p e r i m e n t a l l y ( s e e T a b l e 1 ) ; ( 3 ) R e s i d u e s t h a t a r e c o n s e r v e d i n s u b f a m i l i e s b u t d i f f e r b e t w e e ns u b f a m i l ie s . T h e s e r e s i d u e s h a v e c o r r e l a t e d m u t a t i o n a l b e h a v i o r w h e n t h e t o t a l m u l t i - s e q u e n c ea l i g n m e n t i s a n a l y z e d . W e u s e d a m e t h o d s i m i l a r t o t h e o n e d e s c r i b e d b y G o e b e l e t a l. [3 1] t o f in dp a i r s o f r e s id u e s t h a t s h o w a c o r r e l a t e d p a t t e r n o f c o n s e r v a t i o n a n d m u t a t i o n . P a i r s o f r e s id u e st h a t a r e b o t h 4 0 - 9 0 % c o n s e r v ed , b u t w h i c h d i sp l a y s i m u l t a n eo u s m u t a t i o n t o t h e s a m e r e si d u e inm o r e t h a n 6 7 % o f a l l c a s e s, w e r e d e t e r m i n e d . T h e s e r e s i d u e s d e t e r m i n e t h e p r i n c i p a l f u n c t i o n a ld i f f e r e n c e s b e t w e e n s e q u e n c e s u b f a m i l i e s [s e e e . g. R e f s . 3 2, 33 ] . F o r t y - t h r e e r e s i d u e p a i r s w e r ef o u n d a n d a n a l y z e d . S u r p r i s in g l y , 4 2 o f t h e s e 4 3 p a i rs f o r m a n e t w o r k o f r e s id u e s c o n n e c t e d b yc o r r e l a t e d m u t a t i o n a l b e h a v i o r ( s ee F i g . 2) . T e n o f th e s e r e si d u e s a r e l o c a t e d j u s t o u t s i d e t h eh e l i c e s i n t h e c y t o s o l i c l o o p s .3 D M O D E L B U I L D I N G

T h e a l i g n m e n t o f G - p r o t e i n - c o u p l e d r e c e p t o r 7 T M s w i t h B r h is n o t s t r a i g h t f o r w a r d . D i f f e re n tt e c h n i q u e s h a v e b e e n t r i ed t o s o l v e th i s p r o b l e m [ e.g . R e f s . 1 4 ,1 5]. W e c o m b i n e d t h e a l i g n m e n t o ft h e [~ -2 a d r e n e r g i c r e c e p t o r w i t h B r h b y C r o n e t e t a l. [ 15 ], a n d t h e a l i g n m e n t o f fi v e r e c e p t o rc l as s es w i t h B r h b y T r u m p p - K a t l m e y e r et a l. [1 4], b u t m a d e s o m e m i n o r m o d i f ic a t i o n s to m a k ea l a r g e r n u m b e r o f h i g h l y c o n s e r v e d h e l i c a l r e s i d u e s p o i n t a w a y f r o m t h e li p id . T h e r a t i o n a l e f o rt h i s is t h a t t h e r e i s v i r t u a l l y n o e v o l u t i o n a r y p r e s s u r e o n t h e r e s i d u es t h a t p o i n t t o w a r d s t h e l ip i d

, ( - -Fig. 1. Spatially aligned sequences of Brh and som e representative GPCRs. The sequences of the seven helices of theG-protein-coupled eceptorsmentioned in Table 1 are displayed n their putative alignmentwith Brh. Top: sequence of the7TM of Brh. A sterisks nd icate residues that point into the central cavity. Note the reverse order of helices II, IV and V I.The consensus sequence was taken from the multi-sequence alignment. Even-numbered sequences run from right to left,thus the extracellular side of the helices is at the left and the cy tosolic side at the right. Residues that align vertically n thisfigure are equally deep in the m embrane in the m ode l shown in Fig. 2. L, C, o r M above the residues indicate Ligandbinding, Conserved, or co rrelated M utation residue, respectively. The residues are numb ered similarly as described byHibe rt et al. [13,14]: residues are num bered such that the first digit indicates the helix numbe r. In each helix the num berof the m ost conserved residue is a multiple of 10 while the start of the h elix is at the sam e time as close as possible to 100times the num ber of the helix.


6/10

654

J\

J/

,.~ .4 ---

4 " *~ , o o "

/,,'2,

/ - ,, k .r -,,. / \ ,~'4,4 2 3

F i g . 2 . C o r r e l a t e d m u t a t i o n s . R e s i d u e s w i t h p a i r w i s e c o r r e l a t e d m u t a t i o n a l b e h a v i o r a r e c o n n e c t e d . N u m b e r i n g a s i nF ig . 1 .

b i l ay e r , ex cep t t h a t t h ey sh o u ld p re fe rab ly b e h y d ro p h o b ic , so n o re s id u es fac in g t h e l i p id a reex p ec t ed t o b e h ig h ly co n se rv ed .

T h e f in al c o r r e s p o n d e n c e b e t w e e n th e 7 T M s e q u e n ce s o f t h e G P C R s a n d t h e B r h s t ru c t u r a lf ram ew o rk i s sh o wn in F ig . 1 . In t h i s f i g ure , th e re s id u es k n o w n to p o in t i n ward s i n Brh a n d t h ere s id u es fa ll in g i n o n e o f t h e t h ree a fo rem en t io n ed c l a s se s (co n se rv ed , co r re l a t ed m u ta t i n g , l i g an db in d in g ) a re i n d i ca t ed . I t c an b e seen t h a t m o s t o f t h e se re sid u es co r re sp o n d t o p o s i t i o n s i n Brht h a t p o i n t i n w a r d s . F i g u r e 3 sc h e m a t i ca l ly s h o w s t h e a v e ra g e p u t a t iv e G - p r o t e i n - c o u p l e d 7 T Mst ru c tu re w i th t h e t h ree t y p es o f re s id u es co lo r co d ed . Th e m o d e l s o f co u rse a re i n co r rec t i n th e i rd e t a il s , b u t t h ey h av e p ro v en u se fu l in en l a rg in g o u r u n d e rs t an d in g o f th e fu n c t i o n in g o f t h eG PC R s [e .g . Re fs . 1 3 ,1 4 ,3 4 ].D I S C U S S I O N

Th e fu n c t i o n o f G PC R s i s t o d e t ec t an ex t e rn a l s ig n a l an d co n v e r t t h is t o an ac t i o n a t t h ecy to so l i c s i de o f t h e 7 TM th a t l ead s t o G -p ro t e in ac t i v a t io n . In o rd e r t o p e r fo rm su ch a ro l e , th e serecep to rs f i rs t h av e t o i n t e rac t wi th a sp ec if i c l i g an d ( l i gh t ab so rp t i o n fo r o p s in s ) a t t h e p e r i p l a s-


7/10

655

~ : . : . ; ~ : ~. , : . .: > . : ;

F i g . 3 . L o c a t i o n o f r e s id u e s i n th e 7 T M m o d e l . C y l i n d e r s r u n t h r o u g h t h e m o d e l o f t h e a v e r a g e 7 T M . T h e v e r t i c a l l o c a t i o no f t h e t h r e e c l a ss e s o f r e si d u e s ( l i g a n d b i n d i n g ( g r ee n ) , c o n s e r v e d ( b l u e ) , o r c o r r e l a t e d m u t a t i n g ( r e d )) a r e i n d i c a t e d .

mic s i d e o f t h e mem b ran e . Th i s l i g an d b in d in g sh o u ld re su l t i n a s ig n a l t h a t i s sen t t h ro u g h t h erecep to r , r e su l t in g i n ac t i v a t i o n o f t h e G-p r o t e in a t t h e cy to so l i c s i d e o f th e me mb ran e .

T h e a l i g n m e n t o f t h e G P C R s e q u e n c es r e v ea l e d t h a t t h e r e s id u e s k n o w n t o b e i n v o l v ed inl i g an d b in d in g (Tab l e 1 ) a re ma in ly fo u n d i n t h e mid d l e an d i n t h e ex t race ll u l a r h a l f o f t h e 7 TM ,wh e reas a l a rg e n u m b er o f h ig h ly co n se rv e d re s id u es a re fo u n d i n th e cy to so l i c h a l f (F ig . 3 ) . Ing lo b u l a r p ro t e in s , a v e ry h ig h l ev el o f seq u en ce co n se rv a t i o n i s a lmo s t a lway s a s so c i a t ed w i th animp o r t an t ro l e in fu n c t i o n (e.g., t h e co n se rv a t i o n o f a se r in e i n se r i n e -p ro t e in ases o r a h i s ti d in e i nh aem o g lo b in s ) ra th e r t h an w i th t h e ma in t en an ce o f s tru c tu re . I t t h e re fo re seems l ik e ly t h a t t h es e q u e n c e c o n s e r v a t io n i n t h e 7 T M s o f th e G P C R s is r e l a te d t o a c o m m o n f u n c t i o n o f a ll th e s erecep to rs , n ame ly t ran sd u c in g a s i g n a l t h ro u g h t h e memb ran e .

S e v e ra l re s id u e p o s i t io n s i n G P C R s h a v e c o r r e l a t e d m u t a t i o n a l b e h a v i o r. T h e s e r e s id u e s a r em o s t l y l o c a te d a t t h e c y t o s o li c h a l f o f t h e 7 T M s a n d i n t h e in t r ac e l lu l a r l o o p s a n d s h o w c o r r e l a te dmu ta t i o n a l b eh av io r ma in ly b e tw een th e G P C R su b fami l ie s , i.e ., t h ey a re co n se rv ed i f o n ly o n esu b fami ly i s an a ly zed . Th e fac t t h a t t h e se re s id u es a re h ig h ly co n se rv ed wi th in ev e ry su b fami lys t ro n g ly su g g es t s t h a t t h ey h av e a fu n c t i o n a l ro l e i n t h o se su b fami l ie s . Th e i r c l o se sp a t ia l p ro x im -i t y t o re s id u es k n o wn to b e i n v o lv ed i n s i g n a l t r an sd u c t i o n su g g es t s t h a t t h i s fu n c t i o n i s s i g n a lt ran sd u c t i o n t o o . Th e fa c t t h a t t h ey a re o f t en d i f fe ren t b e tw een su b fami l ie s in d i ca t e s t h a t t h ey a rein v o lv ed i n th e recep to r - sp ec if i c p a r t o f th e s i g n a l t r an sd u c t i o n p a th w ay .

Tw o fac t s co r ro b o ra t e t h i s h y p o th es i s : (1 ) Th ree re s id u es t h a t a re p a r t o f th e l a rg e co r re l a t edm u t a t i o n n e t w o r k s h o w n i n F i g . 2 lie in l o o p s t h a t a r e k n o w n t o b e n e c e s sa r y f o r b i n d in g a n dac t i v a t i o n o f G -p ro t e in s . On e re s id u e (G lu 6 ) i s lo ca t ed n ex t t o a p ep t i d e t h a t i s c ru c i a l fo rG-protein act ivat ion in rhodopsin [35,36], the o~-adrenergic [37-39], the [~-adrenergic [40-43], andth e mu sca r in i c [44 ] recep to rs . Th e o th e r tw o re s id u es a re p h en y l a l an in es , l o ca t ed i n th e mid d l e o f


8/10

656a s t re tch o f re s id u es imm ed ia t e ly a f t e r h e l ix VI I i n t h e C- t e rmin a l d o m a in , w h ich is k n o w n to b en ecessa ry fo r b in d in g a n d su b seq u en t ac t i v a t i o n o f sev e ra l G-p ro t e in s [9,37 ]; (2) Th e seq u en cem o t i f D R Y a t t h e c y to s o l i c e n d o f h el ix I I I h a s b e e n s h o w n i m p o r t a n t f o r G - p r o t e i n b i n d in g[36 ,4 5 ,4 6 ]. Tw o o f t h ese t h ree re s id u es (D an d Y ) a re p a r t o f t h e n e tw o rk o f co r re l a t ed m u ta t i o n s .

M a n y q u e s t i o n s r e m a i n a b o u t G P C R s . N o t m u c h i s k n o w n a b o u t t h e f u n c t i o n a l r o l e o fex t race l lu l a r l o o p s , an d t h e d e ta i l s o f s i g n al t r an sd u c t i o n a re fa r f ro m b e in g u n d e rs to o d . Th em o d e o f i nt e r ac t io n o f m o r e s o p h i s ti c a te d l ig a n d s s u c h a s p e p t id e s a n d p r o t e i n s w i t h G P C R s a n dth e i n te rac t i o n o f t h e recep to rs wi th G-p ro t e in s rema in t o b e e lu c id a t ed . Th ree -d im en s io n a lm o d e l s o f G P C R s c o u l d b e u s e d to b e t t e r u n d e r s t a n d t h e ir s t r u c t u r e - f u n c t i o n r e la t io n s h ip .P o w e r f u l in f o r m a t i o n f o r m o d e l i n g w o u l d b e p r o v i d e d i f m u t a t i o n a l s t ud i es c o u l d p r o v e t h a t t h eh ig h ly co n se rv ed re s id u es an d t h e re s id u e p a i r s wi th co r re l a t ed mu ta t i o n a l b eh av io r a re i n d eedin v o lv ed i n s i g n a l t r an sd u c t i o n .

Th e m o re re l iab l e recep to r s t ru c tu re mo d e l s t h a t r e su l t wil l b e o f g rea t b en e f i t fo r t h e ra t i o n a ld es ig n o f n ew med ic in es. Th e m u l t i - seq u en ce a l i g n men t o f 2 2 5 G-p ro t e in -c o u p l ed re cep to rsh a s b e e n d e p o s i t e d i n t h e T M 7 f i l e s e r v e r . S e n d H E L P t o ' T M 7 @ E M B L - H e i d e l b e r g . D E 't o o b t a i n i n f o r m a t i o n a b o u t G P C R - r e l a t e d d a t a a n d t o p i c s , o r s e n d , t o t h i s s a m e i n t e r n e ta dd re ss , th e m e s sa g e G E T A L I G N : A L L . A L I G N .A C K N O W L E D G E M E N T S

W e t h an k R . S c h n e id e r fo r h el p w i t h t h e u s a ge o f t h e M A X H O M p r o g ra m . W e t h an k R .H e n d e r s o n a n d T . C e s k a f o r h e lp f u l di sc u s si o n s. W e t h a n k A . V a l e n ci a , U . G o e b e l a n d C . S a n d e rfo r mak in g t h e co r re l a t ed mu ta t i o n me th o d av a i l ab l e t o u s an d fo r s t imu la t i n g d i scu ss io n s .

R E F E R E N C E S1 B l r d s a l l , N . J . M . , T r e n ds Ph a r m a c o l . Sc i ., 12 ( 1991 ) 9 .2 M ol l on , J . , N a t u r e , 351 (1991 ) 696 .3 P r ob s t , W . C . , Snyde r , L . A . , Sc hus t e r , D . I . , B r os i u s , J. a nd Se a l f on , S . C . , D N A C e l l B i o l . , 11 ( 1992 ) 1 .4 S t r o s be r g , A . D . , E u r . J . B i oc he m . , 196 ( 1991) 1 .5 Sa va r e s e , T . M . a nd F r a s e r , C . M . , B i oc he m . J . , 283 (1992 ) 1 .6 B a r n a r d , E . , T r e nds B i oc he m . Sc i. , 17 ( 1992) 368 .7 A t t w oo d , T . K . a nd F i nd l a y , J . B . C . , P r o t e i n E ng . , 6 ( 1993 ) 167 - 176 .8 A t t w o o d , T . K . a n d F i n d l a y , J . B . C ., 7 T M , 2 (1 9 9 3) .9 K o n i g , B . , A r e n d t , A . , M c D o w e l l , J . H . , K a h l e r t , M . , H a r g r a v e , P . A . a n d H o f m a n n , K . P . , P r o c . N a t l . A c a d . S ci . U S A ,

86 (1989) 6878.10 Hen de r son, R . , Ba ldwin, J .M. , Cesk a , T .A . , Zeml in , F . , Beckmann, E . and D own ing, K.H . , J. Mo l . Biol. , 212 (1990) 899.11 N a t ha n s , J . , B i oc he m i s t r y , 31 ( 1992 ) 4931 .1 2 D o h l m a n , H . G . , T h o r n e , J . , C a r o n , M . C . a n d L e f k o w i t z , R . J . , A n n u . R e v . B i o c h e m . , 6 0 ( 19 9 1 ) 6 5 3 .13 H i be r t , M . , T r um pp- K a l l m e y e r , S . , B r u i nve l s , A . a nd H of l a c k , J . , M o l . Pha r m a c o l . , 40 ( 1991) 8 .14 T r um pp- K a l l m e ye r , S ., H of l a c k , J. , B r u i nve l s , A . a n d H i b e r t , M . , J . M e d . C h e m . , 35 ( 1992 ) 3448 .15 C r o ne t , P . , Sa nde r , C . a nd V r i e nd , G . , P r o t e i n E n g . , 6 ( 1993) 59 .16 Sa nd e r , C . a nd Sc hne i de r , R . , P r o t e i n S t r uc t . Func t . G e ne t . , 9 ( 1991 ) 56 .1 7 L i b e r t , F . , P a r m e n t i e r, M . , L e f o r t , A . , D i n s a r t , C . , V a n S a n d e , J ., M a e n h a u t , C . , S i m o n s , M . - J ., D u m o n t , J . E . a n d

Vassa r t , G. , Sc ience , 244 (1989) 569.18 L oo s f e l t , H . , M i r a s h i , M . , A t ge r , M . , Sa l es s e, R . , T h i , M . T . V . H . - L . , J ohv e t , A . , G u i oc h on- M a n t e l , A . , Sa t , S . , J a l l a l ,

B . , G a r n i e r , J . a nd M i l g r om , E . , Sc ie nc e, 245 ( 1989 ) 525 .


9/10

657

19 Hershey, A .D. an d Kra use, J .E. , Science, 247 (1990) 958.20 Sunahara, R.K., Niznik, H.B., Wemer, D.M., Stormann, T.M., Brann, M.R., Kennedy, J .L. , Gelernter, J .E. , Roz-

mabel, R. , Yang, Y., Israel, Y. , Seeman, P. and O'Dowd, B.F. , Nature, 347 (1990) 80.21 Hira ta , M. , Hayashi , Y. , Ushikubi , F. , Yokota , Y. , Kageyama, R. , Nakanishi , S. and Narumiya, S. , Nature , 349

(199t) 617.22 Van To l, H.H .M., Bunzow , J.R. , Gu an, H.-C., Sunahara, R.K ., Seeman, P. , Niznik. H.B. and Civelli, O. , Nature, 350

(1991) 610.23 Sunah ara , R.K. , Guan , H. -C. , O'Dow d, B.F., Seeman, P., Laurier , L .G. , Ng, G. , George, S.R., Torchia , J . , Van Tol ,

H.H .M. a nd Niznik, H.B., Natu re, 350 (1991) 614.24 Gera rd, N.P. and G erard, C. , Nature , 349 (1991) 614.25 Holmes, W .E., Lee, J . , Ku ang, W .-J., Rice, G.C. an d W ood , W .I. , Science, 253 (1991) 1278.26 Grib skov , M., Luethy, R. and Eisenberg, D., Meth ods Enzym ol. , 183 (1990) 146.27 Henikoff, S . and H enikoff, J .G., Nucleic Acid s Res. , 19 (1991) 6565.28 SWISS-PROT Prote in Sequence Database , EMB L D ata Library , D-69117 Heidelberg, German y and A mo s Bairoch,

De part me nt de Biochimie Medicale, Centre M edical Universitaire, 1211 Ge nev a 4, Switzerland.29 PIR , NBR F, G eorgetown U nivers i ty M edical Center, 3900 Reservoir Ro ad N.W . , Washington. DC .30 Sander, C. and Schneider, R. , In Soum pasis, D.M . and Jovin, T.M. (Eds.) Co m puta tion of Biomolecular Structures,

Springer Verlag, Berlin, 1992, pp. 15-28.31 Go ebel, U. , Sander, C. and Valencia, A., subm itted for publication.32 Breckenndge, R. and Du fton, M .J., J . M ol. Evol. , 26 (1987) 274.33 Grego ret , L .M. and Sauer, R.T. , Proc . Nat l . Acad. S ci . US A, 90 (1993) 4246.34 Oliveira, L., Paiva, A.C .M ., Sander, C. and V riend, G., subm itted for publication.35 Fran ke, R.R. , Sakmar, T .P. , Oprian, D.D. and Kh oran a, H.G. , J . Biol. Chem. , 263 (1988)2119.36 Fran ke, R.R. , Konig, B , Sakm ar, T .P., Kh orana , H.G. and Hofmann, K .P , Science , 250 (1990) 123.37 Cotecchia, S. , Exum, S. , Caron, M .G. an d Lefkow itz, R.J. , Proc. N atl. Acad . Sci. US A, 87 (1990) 2896.38 Cotecchia, S. , Ostrowski, J . , Kjelsberg, M .A., Caro n, M .G. an d Lefkowitz, R.J. , J . Biol. Chem ., 267 (1992) 1633.39 Kjelsberg, M.A ., Cotecchia, S. , Ostrowski, J . , Caro n, M .G. an d Lefkowitz, R.J. , J . Biol. Chem ., 267 (1992) 1430.40 Palm, D., Mun ch, G ., Dees, C. and H ekm an, M., FE BS Lett. , 254 (1989) 89.41 Cheung, A.H . , Huang, R . -R.C. , Graziano, M.P. an d Strader, C.D. , FEB S Let t . , 279 (1991) 277.42 Strader, C.D ., Dixon, R.A .F. , Cheung, A .H., Candelore, M .R., Blake, A.D. and Sigal, I.S. , J . Biol. Chem ., 262 (1987)16439.43 O'Dowd, B.F. , Hnatowich, M., Regan, J .W., Leader, W.M., Caron, M.G. and Lefkowitz, R.J. , J . Biol. Chem., 263

(1988) 15985.44 Ok am oto, T. and Nishi mo to, I. , J . Biol. Chem ., 267 (1992) 8342.45 Fra ser, C.M ., Chung, F.Z . , Wang, C.D. an d Venter, J .C. , Proc. Na tl. Acad. Sci. US A, 85 (1988) 5478.46 Fraser , C.M. , W ang, C.D , Robinso n, D.A. , Gocayne, J .D. and Venter , J.C., M ol . Pharmacol ., 36 (1989) 840.47 Horstman, D.A., Brandon, S. , Wilson, A.L. , Guyer, C.A., Cragoe Jr. , E.J. and Limbird, L.E. , J . Biol. Chem., 265

(1990) 21590.48 Wess, I. , Gd ula, W.I. and Brann, M .R., EMB O J. , 10 (1991) 3729.49 Strader, C.D., S igal, I.S. , Candelore, M .R., Ra nds, E. , Hill, W.S. and D ixon. R .A.F . , J . Biol. Chem ., 263 (1988) 1026750 Nak ayam a, T .A. and Kh orana , H.G . , J. Biol . Chem. , 265 (1990) 15762.51 Nak ayam a, T .A. and K horan a, H .G. , J . Biol . Chem. , 266 (1991) 4269.52 Kh ora na, H .G., J . Biol. Chem ., 267 (1992) 1.53 Zhu kov zky , E.A. and O prian , D.D., Science, 251 (1991) 558.54 Sakmar, T .P. , Franke , R.R. and Kh orana, H .G. , Proc . Nat l . Acad. Sci . US A, 86 (1989) 8309.55 Na than s, J . , Biochemistry, 29 (1990) 937.56 Sakm ar, T .P., F ranke, R.R. and K hora na, H .G. , Proc. Nat l . Acad . Scl. US A, 88 (1991) 3079.57 Strad er, C.D., Candetore, M .R., Hill, W.S. , Sigal, I.S. and Dixon, R.A .F. , J . Biol. Chem .. 264 (1989) 13572.58 Weinstein, H., Mazucek, A.P. , Osman, R . and Top ial, S. , Mol. Pharm acol. , 29 (1986) 28.59 Timm erman, H., Trends Pharmacol. Sci.~ 13 (1991) 6.60 F reed m an, R. and Jarnagin, K ., In B6nner, G., Fritz , H. , Unger, T. , Roscher, A. and Luppertz, K. (Eds.) Recent

Progress on K inins, Blrkhauser Verlag, Basel, 1992, pp . 4874 96.


10/10

6 5 8

51 W eitz, C.J. and N atha ns, J . , Neuro n, 8 (1992) 465.52 Strader, C.D., Sigal, I.S. and D ixon, R .A.F . , FA SE B J., 3 (1989) 1825.53 Bow nds, D., Natur e, 216 (1967) 1178.54 Wang, J .K., McDowell, J .H. and Hargrave, P.A., Biochemistry, 19 (1980) 555 M ullen, E. and A kh tar, M ., FEB S Lett. , 132 (1981) 261.56 F ind lay, J .B.C., Brett, M . and Papp in, D.J.C. , N ature , 293 (1981) 314.

Common Motif in GPC 7 TM Helix Receptors

Documents