1 8 2

G PR OTEI NS A N D RE G UL ATI O N OF A DE NYLYL CYCL ASE

Nobel Lect ure, Dece mber 8, 1994 b y

A LFRE D G. G IL MA N

Depart ment of Phar macology, The University of Texas South western Me dical Ce nter, Dallas, Texas 75235, US A

I NTRODUCTIO N

E arl S ut h erl a n d, a fri e n d of m y f at h er, wr ot e t o m e i n t h e s pri n g of 1 9 6 1 wit h a pro posal to partici pate i n w hat was t he n a n e d ucatio nal a dve nt ure - a co mbine d M. D.-Ph. D. training progra m that he ha d devise d at Western Reserve U niversity ( no w Case Wester n Reserve U niversity) i n O hio. My reac- ti o n w as e ntir el y n e g ati v e. I t h a n k e d hi m, p olit el y I t hi n k, b ut t h e i d e a of s pe n di ng seve n years i n Clevela n d ha d little a p peal. Ha p pily, S ut herla n d was persiste nt. He wr ote a gai n i n t he fall of 1961 (t he be gi n ni n g of m y last year i n c oll e g e), I d e ci d e d t h e i d e a w as w ort h a visit, a n d I h a d m y first gli m ps e of cyclic A M P (for w hose discovery, i n 1957, S ut herla n d was a war de d t he Nobel Prize [in 1971]). Cyclic A M P, S utherlan d, an d the M. D.- Ph. D. Progra m all looke d rat her a p peali ng. T h us, o n my arrival i n Se pte mber, 1962, I was dis- a p pointe d to learn that S utherlan d was abo ut to de part for Van derbilt University. Ho wever, there was an attractive o p port unity to work with Theo dore Rall, S utherlan d’s yo unger collaborator, who ha d playe d a pivotal r ol e i n t h e cr u ci al e x p eri m e nts of 1 9 5 7. I e nt er e d t h e R all l a b, a n d i n o v er 3 0 s ubseq ue nt years have never esca pe d t he l ure of cyclic n ucleoti de researc h, des pite occasio nal atte m pts to try. T he most deter mi ne d of t hese efforts ca me with my choice of Marshall Nirenberg’s ne wly proclai me d ne urobiolo- gy laboratory for post doctoral training. Ho wever, in o ur first conversation after my arrival at t he Natio nal I nstit utes of Healt h i n Bet hes da, Mars hall aske d me to establis h a n assay for cyclic A M P i n his laboratory. Tra p pe d a g ai n, b ut I di d n’t fi g ht b a c k v er y vi g or o usl y. Rall a n d S ut herla n d’s discovery of cyclic A M P a n d a de nylyl cyclase, t he hor mone- sensitive enzy me that synthesizes the cyclic n ucleoti de fro m A T P, gave birt h to t he co nce pts of tra ns me mbra ne sig nali ng a n d of hor mone- reg ulate d sy nt hesis of i ntracell ular seco n d messe ngers (Fig. 1). Bot h me n were trai ne d as bioc he mists (S ut herla n d wit h Carl Cori, Rall wit h Albert Le h ni nger), a n d toget her t hey i nitiate d a classical re d uctio nistic a p proac h to deci phering hor mone action. In the 1950’s, hor mones co ul d al most be d efi- ne d as reg ulatory molec ules t hat wo ul d act o nly o n i ntact cells. S ut herla n d a n d Rall’s co u p was to asse mble a s yste m i n w hic h a c haracteristic effect of epinephrine an d glucagon (activation of phosphorylase) coul d be observe d Alfr e d G. Gil m a n 1 8 3 i n ho moge nates a n d t he n to dissect t he syste m i nto its major co m po ne nts - hor mo ne-sti m ulate d sy nt hesis of a factor, cyclic A M P, by t he partic ulate frac- tio n a n d s u bse q ue nt actio n of t he factor i n t he c ytosol to acti vate phospho- r ylase (1). A n assa y (albeit tort uro us) for a de n yl yl c yclase was i n ha n d, a n d hor mone action co ul d then be st u die d by a d ding A T P to plas ma me mbranes

THE SEC OND MESSENGER SYSTE M I NV OLVI N G ADENYL CYCLASE

EF FE CT O R CELL A T P 5’- A M P I + + + +

PHOSPHDIESTERASE

A D E NI N E ( O R

+ P Pi

0’ O H

Altho ugh the conce pt of rece ptors for en dogeno us reg ulatory molec ules an d dr ugs arose wit h t he p har macological ex peri me nts of La ngley a n d E hrlic h i n t he late ni netee nt h a n d early t we ntiet h ce nt uries, t he wor d evoke d o nly m et a p h ysi c al f e eli n gs i n m a n y at t h e ti m e of t h e dis c o v er y of c y cli c A M P. T h e ter m “rece ptor” does not a p pear i n t he i n dex of t he 1955 e ditio n of T he Phar macological Basis of Therape utics, the stan dar d textbook of Phar macology, b ut t h e f oll o wi n g s e nt e n c e is t h er e: “ Years ago, La ngley na me d t he differe n- ti ati n g s u bst a n c e t h e ‘r e c e pti v e s u bst a n c e’; t his t er m is still wi d el y e m pl o y e d, b ut it m ust be realize d t hat t he ‘rece ptor’ ma y not be a mor p holo gicall y de monstrable str uct ure.” Rall a n d S ut herla n d’s ex peri me nts provi de d test- t ube assays for rece ptors, a n d t he assays de mo nstrate d t hat t he rece ptors were a ut he ntic. T he effects of e pi ne p hri ne a n d congeners o n a d e n yl yl c y cl a- se were s ho w n to co nfor m to A hlq uist’s ne w co nce pt ualizatio n of β -a dre ner- gic rece ptors (as disti ng uis he d fro m a rece ptors), a n d t he effects were blocke d wit h t he first, ne wly discovere d β -a dre nergic a ntago nist (2). Bioc he mical a p proac hes to rece ptors were t h us bor n, a n d t he q uestio n arose 1 8 4 P hysiology or Me dici ne 1994 of t h e r el ati o ns hi p of t h e β -a drenergic rece ptor to a de nylyl cyclase. Co ul d t he e nzy me be t he rece ptor? Per ha ps, b ut t his mo del wo ul d de ma n d t he exi- ste nce of a fa mil y of a de n yl yl c yclases wit h disti nct re g ulator y sites, beca use re g ulatio n of t he e nz y me was s ho w n not to be restricte d to e pine phrine a n d glucagon; A C T H, TS H, L H, A D H, an d other sti mulators were soon in evi- de nce, as was i n hi biti o n of a de n yl yl c yclase acti vit y b y c h oli ner gic a g o nists { 3}. O ver a deca de after t he disco ver y of a de n yl yl c yclase, Marti n R o d b ell a n d colleag ues provi de d reasonably co m pelling, altho ugh in direct, evi dence that rece ptors a n d a de nylyl cyclases were disti nct molec ular e ntities. T he a de ny- l yl c yclase of a di poc ytes is sti m ulate d b y a m yria d of hor mo nes. If t here were disti nct cyclases t hat eac h also ser ve d as a rece ptor, res po nses to maxi mally effective concentrations of hor mones wo ul d be a d ditive. They were not, i m plying that distinct rece ptors co ul d interact with a co m mon pool of a de- n yl yl c y cl as e ( 4). T h e iss u e w as r es ol v e d d efi niti v el y i n t h e 1 9 7 0’s wit h t h e a d ve nt of li ga n d bi n di n g assa ys for rece ptors. Rece ptors co ul d fi nall y be exa- mi ne d b y met ho ds t hat di d not rel y o n detectio n of a f u nctio nal res po nse. It w as t h e n p ossi bl e t o s ol u bili z e a n d r es ol v e a d e n yl yl c y cl as e fr o m t h e β -a dre n- er gi c rece ptor, provi ng t hat t hey were disti nct macro molec ules (5, 6).

E ARLY TI MES F OR G PR OTEI NS

The q uestion of the mo ment th us beca me the mechanis m of interaction or “co u pli ng” bet wee n rece ptors a n d a de nylyl cyclase. T he relatively si m ple notio n t hat a n ago nist- rece ptor co m plex co ul d act as a n allosteric reg ulator of t he e nzy me was also c halle nge d by R o d b ell, who first pro m ulgate d the notion of a “trans d ucer” acting as an inter me diary bet ween rece ptors an d a de n yl yl c yclase (7). Alt ho u g h t his notio n was at first base d pre do mi na ntl y o n excelle nt i nsti nct a n d t he li pi d bilayer was na me d as a ca n di date tra ns d ucer, s u p portive data for so mething more s pecific were soon forthco ming. R o d b ell, Birnba u mer, an d their colleag ues ma de the s ur prising discovery t hat o ne reg ulatory liga n d (t he rece ptor ago nist) was not s ufficie nt to acti- vate a de nylyl cyclase. A hor mo ne co ul d not activate t he e nzy me u nless g ua- n osi n e tri p hos p hate ( G T P) was also prese nt { 8}. T his cr ucial observatio n ha d been misse d for over a deca de beca use of conta mination of both me mbrane pre paratio ns a n d s ubstrate A T P wit h s ufficie nt ( µ M) co nce ntratio ns of G T P to meet the req uire ment. It was s ubseq uently deter mine d that hor monal i n hibitio n of a de nylyl cyclase activity was si milarly de pe n de nt o n G T P { 9}. I will l e a v e it t o R o d b ell to describe t hese obser vatio ns i n more detail. Ho we ver, it s ho ul d be note d t hat t here was co nsi derable ske pticis m abo ut t he si g nifi c a n c e of t h e fi n di n gs ( Fi g. 2), i n part ge nerate d b y diffic ulties i n re pr o- d uction of the res ult; most were not working‘ with the very nice me mbrane pre paratio ns t hat c haracterize d t he R o d b ell la b orat or y. Several observations of the mi d-1970’s s poke to the un deniable i m portan- ce of G T P i n reg ulatio n of hor mo ne-se nsitive a de nylyl cyclase activity. Most sig nifica ntly, Cassel a n d Seli nger detecte d a hor mo ne-sti m ulate d G T Pase Alfr e d G. Gil m a n 1 8 5

activity t hat a p peare d to be associate d wit h activatio n of a de nylyl cyclase, a n d, des pite e nor mo us tec h nical diffic ulties, t hey correctly de d uce d (fro m ki n eti c a n al ysis) t h e si g nifi c a n c e of t h e G T Pase i n t er mi n ati n g a hor monal- ly- me diate d sig nal {10}. Consistent with these tho ughts, Lon dos an d Schra m m and co workers had noted that nonhydrolyzable analogs of GTP, s u c h as Gpp( N H)p, activate d a de nylyl cyclase dra matically a n d wit ho ut t he nee d for hor mone {1 1, 1 2 }. Mic hael Mag uire, my first post doctoral fello w, discovere d t hat G T P decrease d t he affi nity of rece ptors selectively for ago- nists, b ut n ot f or a nta g o nists (13). T he i nter pretati o n of t his c o u nter - i nt ui- ti ve o bser vati o n was n ot clear, b ut it s urel y a p peare d t o be si g nifica nt. Thro ugho ut this ti me a fe w brave so uls ha d atte m pte d to sol ubilize an d p urify co m ponents of hor mone-sensitive a denylyl cyclase syste ms. All enco untere d great diffic ulties. Hor monal res ponsiveness was q uickly lost on sol ubilizatio n wit h deterge nts, a n d a de nylyl cyclase itself a p peare d to be re markably labile. E va Neer was per ha ps t he first to treat t he e nzy me as a n a p proachable bioche mical object, with a caref ul ex ploration of its hy dro dy- na mic pro perties (14). Des pite t his, a co nve ntio nal bioc he mical a p proac h to t he syste m looke d diffic ult i n dee d. T h e t ur ni n g p oi nt, f or us, st art e d wit h t h e d es cri pti o n b y D a ni el et al. { 1 5} of t he cytoci dal effect of cyclic A M P o n clo nal S49 ly m p ho ma cells. Bo ur ne a n d associates were soo n a ble to isolate a varia nt (c yc-) of t hese cells t hat a p peare d to lack a de nylyl cyclase { 1 6}, des pite co nti n ue d ex pressio n of a nor- mal n u mber of R-a dre nergic rece ptors (17). We were able to select a not her S49 cell varia nt t hat i ntri g ue d us e ve n more - an uncoupled ( U N C) mutant t hat a p peare d to have nor mal rece ptors a n d a de nylyl cyclase b ut t hat faile d to ge nerate a cyclic A M P sig nal i n res po nse to a p pro priate hor mo nes (β - a dre nergic ago nists or prostagla n di ns) {1 8 }. T he a vaila bilit y of t hese ge netic 1 8 6 Physiology or Me dici ne 1 9 9 4 varia nts ma de t he bioc he mistry a p pear more a p proac hable, partic ularly to Elliott Ross, a n extre mely tale nte d a n d well-trai ne d me mbra ne bioc he mist w ho joi ne d my lab i n 1975. Ross so ug ht to reco nstit ute t he cyc- m uta nt i n vitro, first by extracti ng a de nylyl cyclase fro m cells t hat lacke d Ra dre nergic rece ptors a n d t he n by so me ho w coaxi ng t he protei n back i nto fr uitf ul i nter- , actio ns wit h t he rece ptors prese nt i n cyc- me mbra nes. T he ex peri me nt eve n- t ually worke d; cyc- me mbra nes were reco nstit ute d to dis play catechola mine- s e nsiti v e a d e n yl yl c y cl as e a cti vit y ( 1 9). W e w er e pl e as e d t h at w e h a d t a k e n t h e first st e p i n r es ol uti o n a n d r e c o nstit uti o n of t h e s yst e m, b ut w e h a d littl e i d e a ho w q uickly t he i nvest me nt wo ul d pay divi de n ds. T he reco nstit utio n ha d not worke d for t he a ntici pate d reaso ns. W he n we i nactivate d t he a de nylyl cycla- se i n t he deter ge nt extract use d f or t he rec o nstit uti o n, we still o bser ve d u n di- mi nis he d le vels of hor mo ne-sti m ulate d a de n yl yl c yclase acti vit y. T hat is, a d di- ti o n of a deter ge nt extract de v oi d of a de n yl yl c yclase acti vit y t o rece pt or-

ri g. 3. Ex peri me nts lea di ng to t he discovery of G s . A: Cartoo n of t he protocols. I n t h e first ex peri me nts, Elli ott M. Ross a d de d a deterge nt extract of me m bra ne protei ns to s o-calle d c y c - m e m b r a n e s (le f t, I ), w h ic h w e r e t ho ug ht to lac k a de nylyl cyclase. E pi ne p hri ne sti m ulate d cyclic A M P pro d uctio n ( 2). w hic h see me d to i n di- cate t hat a de nylyl cyclase ha d bee n i nserte d i nto t he deficie nt me m bra nes. I n t he co ntrol ex peri me nt, t he a d e n y ly l c y c la s e in th e e x tra c t w a s in a c tiv a te d (c e n te r, 1 ). E v e n w ith o u t it, e p in e p h rin e c a u s e d th e c y c - me m- bra nes to ma ke cyclic A M P. T his p uzzli ng fi n di ng le d to t he discovery t hat t he cyc‘ me m bra nes di d co ntai n a de n yl yl c yclase (ri g ht, 1) b ut lac ke d a t hir d co m po ne nt necessary to activate it - a G pr otei n t hat persiste d i n t he extract after a de nylyl cyclase ha d bee n i nactivate d. Restoratio n of t he G protei n to t he me m bra nes enabled adenylyl cyclase to synthesize cyclic A MP. Reprinted fro m Linder and Gil ma n {154}, wit h per missio n. Alfre d G. Gil ma n 1 8 7 c o n ta in in g c y c - m e m b ra n e s (w h ic h a lso h a d n o a d e n y ly l c y c la se a c tiv ity ) le d to restoratio n of t he co m plete res po nse (Fig. 3). Treat me nts wit h proteases q u ic k ly re v e a le d th a t b o th th e d e te rg e n t e x tra c t a n d th e c y c - m e m b r a n e s co ntai ne d protei ns t hat were necessary for observatio n of a n y a d e n yl yl c y cl a- s e a cti vit y - basal or t hat sti m ulate d b y hor mo nes, fl uori de, or g ua ni ne n u cl e- oti des. ( We no w k no w t hat t he s pecific isofor ms of a de n yl yl c yclase t hat pre- do mi nate i n S49 cells ha ve nota bl y lo w basal acti vit y.) T h us t wo protei ns were re q uire d - t he catal yst or a de n yl yl c yclase itself, w hic h i n fact w as prese nt i n s o-calle d c y c - m e m b ra n e s, a n d a stim u la to ry p ro te in , d e fic ie n t in c y c - m e m - bra nes, t hat ha d s ur vi ve d t he mil d co n ditio ns use d to i nacti vate a de nylyl cyclase i n t he extract use d for reco nstit utio n. We pro pose d t hat t he role of t he hor mo ne rece ptor was to reg ulate t he i nteractio n bet wee n t hese t wo co m po ne nts (20, 21). Coi nci de ntally, Pfe uffer ac hieve d partial resol utio n bet- wee n a de nylyl cyclase a n d a n activati ng protei n t hat bo u n d selectively to a g ua ni ne n ucleoti de-base d affi nity resi n (22).

1 0 0

5 1 0 1 5 3 0 ( mi n)

Fi g . 3 . Data fro m t hese ex peri me nts. Reco nstit utio n of hor mo ne-se nsitive a de nylyl cyclase by mixt ure of c yc- me mbranes with heat i nactivate d wil d t y p e me mbrane e xtr acts. Deterge nt extracts of wil d t y pe me mbranes were heate d at 3 0 o C f or t hr ti mes i n dicate d o n t hr a bscissa. c hille d, a n d mixe d wit h cy c- me mbranes. T he N a F- a n d Gpp( N H)p-sti mulated a de nylyl cyclase activities of t he i nc u bate d extracts ar e sho wn by the dashed li nes. Ali q uots of t he reco nstit ute d mixt ures, pre pare d wit h t hese incubated e xtr acts, w er e assaye d wit h G T P, iso protere nol ( a congener of e pi ne p hri ne) pl us G T P, N a F, or Gpp( N H)p, as i n dicate d. Re pri nte d fro m Ross et al. { 2 1 }, wit h per missio n.

T he no vel protei n beca me t he o bject of o ur atte ntio ns, i n part beca use of its more m ysterio us nat ure a n d i n part beca use it was not as la bile as a de n yl yl cyclase. A d ditio nal ex peri me nts by Ross i m plie d t hat t he protei n (at t he ti me te r m e d G / F , b u t e v e n tu a lly n a m e d G s) w a s th e site o f a c tio n o f g u a n in e 1 8 8 P h y si ol o g y or Me dici ne 1994 n ucleoti des (an d fl uori de). {21}, a n d t hese hy pot heses were stre ngt he ne d by hy dro dy na mic c haracterizatio n of t he activity by Ally n Ho wlett; s he detecte d G p p ( N H) p a n d fl uori de-i n d uce d alteratio ns s uggestive of s ub u nit dissocia- ti o n u p o n a cti v ati o n b y t h es e li g a n ds ( 2 3). T h e r e all y h ar d w or k f ell t o P a ul Ster n weis a n d Jo h n Nort h u p, w ho toget her u n dertook t he task of p urifica- ti o n. T h e g o o d l u c k of G s w as its r e v el ati o n b y m ut ati o n i n S 4 9 c ells a n d t h e existe nce of a n eas y assa y for t he protei n b y acti vatio n of a de n yl yl c yclase. T h e b a d l u c k, u n k n o w n at t h e ti m e, w as t h at G s is a mo ng t he least ab u n da nt of t he G protei ns. Ne vert heless, perse vera nce (by all i n vol ve d) a n d skill (by t hose doi ng t he ex peri me nts) pai d off, a n d G s, wit h its u ns us pecte d 35-k Da β s ub unit, finally e merge d as a ho mogeneo us g uanine n ucleoti de bin ding pr otei n, ca pa ble of acti vati n g a de n yl yl c yclase i n its Gpp( N H)p or fl u ori d e a cti v at e d f or ms ( Fi g. 4) { 2 4, 2 5}. A t hir d, 8- k D a (γ) s ub unit went unnotice d at t h e ti m e.

Fix. 4. A: P ol yacr gla mi de gel electr o p h oresis of p urifie d fracti o ns of G s. (1) Protei n fro m a n i nter me diate ste p of p urificati o n. (2) a n d (3) P urifie d pr otei n, 3 µ g a n d 8 µ g. res pecti vel y. B: La beli n g of p urifie d G s wit h c holera toxi n a n d [3 2 P] N A D. (1) P urifie d pr otei n srai ne d wit h Coo massie bl ue. (2) an d (3) A utora diogra ms of t he c h olera t oxi n-la bele d pr otei n, ex p ose d f or 16 a n d 48 hr, res pecti vel y. The t wo higher molec ular weight ban ds in the p urifie d pre paration, both of w hic h are labele d with cholera toxin, are alternatively s plice d for ms of G sα The lo wer molec ular weight ban d is the β s u b u nit. Re printe d fro m North u p et al . {24}, wit h per missio n.

F urther st u dies of Gs perfor me d s hortly t hereafter re veale d t hat o ne e q ui- vale nt of g ua ni ne n ucleoti de bo u n d to t he α s ub u nit of t he oligo mer, t hat a cti v ati o n b y Gpp( N H)p or fl uori de was i n fact acco m pa nie d b y s u b u nit dis- s o ci ati o n, a n d t h at t h e r es ol v e d Gpp( N H)p- bo u n d α s ub unit was necessary Alfr e d G. Gil man 1 8 9 a n d s ufficie nt for acti vatio n of a de n yl yl c yclase ( Fi g. 5) {26, 27}. A d ditio nal w o rk o n th e m e c h a n ism o f a c tiv a tio n o f G s b y flu o rid e p ro v id e d su rp rise s a n d even a m use ment. The effect of fl uori de, observable when ex peri ments were perfor me d i n glass test t ubes or i n t he prese nce of co m po ne nts of t he a de- n yl yl c yclase assa y (i.e., A T P), was lost i n t he a bse nce of A T P w he n ex peri- me nts were do ne i n plastic test t ubes {28, 29}. A not her mystery factor was skillf ully p urs ue d by Pa ul Ster n weis, w ho p urifie d t he coactivator fro m bot h A T P a n d fro m aq ueo us extracts of dis posable glass test t ubes. A metal see- me d to be i nvolve d, a n d ne utro n-activatio n a nalysis reveale d A 1 3 + as t h e c ul- prit { 2 9}. T he sig nifica nce a n d val ue of t hat observatio n has beco me partic u- larly a p pare nt a deca de later (see belo w).

3 5 Fraction Nu mber

Fi g . 5 . Res ol uti o n of t he s u b u nits of G s b y gel filtrati o n. P urifie d G s was activate d by inc ubation with [3 5 S] GTP γS. After re mo val of free n ucleoti de, t he protei n was s ubjecte d to hig h perfor ma nce gel filtratio n. The to p panel sho ws the absorbance of the el ut e d protein. The inset sho ws the silver staining pattern for S DS P A G E gels of t he poole d peaks of protei n (la ne 1 = first peak, la ne 2 = seco n d peak) a n d of t he protei n a p pli- e d (la ne 3). T he lo wer pa nel s ho ws t he acti vities assaye d. G /F (G s acti vit y ( o) is q ua ntifie d b y acti vati o n of a de n yl yl c yclase. [3 5 S] GTP γ S (.) i n dicates hig h-affi nity bi n di ng of t he ra dioactive n ucleoti de. 3 5 K Acti vit y ( ) is a meas ure of t he acti vit y of t he βγ s ub unit co m plex. N ucleoti de bin ding activity an d the ca pacity to acti- vate a de nylyl cyclase were excl usi vely associate d wit h t he resol ve d a s u b u nit of G s , which dissociate d fro m βγ o n acti vati o n wit h G T P γS. Re pri nte d fro m North u p et al . (271, wit h per missi o n. 1 9 0 P hysiology or Me dici ne 1994

A n i nteresti n g si de acti vit y at t his ti me was st u d y of t he A D P-ri bos ylatio n of Gs b y c holera toxi n. T hat t his occ urre d was ver y stro n gl y i m plie d b y t he wor k of Gill {30}, Va u g ha n {31}, a n d Bo ur ne (32) a n d was pro ve n wit h p urificatio n of t he protei n. Ho wever, as p urificatio n procee de d, t he ca pacity of c holera toxi n to A D P-ribosylate Gs was lost. T his co ul d be restore d by a d ditio n of a protei n factor {33} t hat was eve nt ually p urifie d, na me d A D P-ribosylation factor or A RF, an d fo un d to be a lo w- molec ular- weight G T P bin ding protein (34, 35). A RF is, of co urse, no w lea di ng a ha p py existe nce as a n i m porta nt reg ulator of protei n trafficki ng (36) a n d as a n acti vator of p hos p holi pase D (3 7 ). Its a sso c ia tio n w ith c h o le ra to x in a n d / o r G s re m a in s to b e e x p la in e d . W or k i n J a p a n i n t h e l at e 1 9 7 0’s a n d e arl y 1 9 8 0’s b y Mi c hi o Ui a n d his c ol- leag ues res ulte d i n c haracterizatio n of islet-acti vati ng protei n (I A P) - a t o xi n fr o m Bordetella pert ussis. Treat ment of cells or me mbranes with this toxin r es ult e d i n l oss of h or m o n al i n hi biti o n of a d e n yl yl c y cl as e ( a n d i n s o m e c as es enhance ment of hor monal sti mulation of the enzy me) (38). Coinci dentally, the toxin a p peare d to catalyze the incor poration of the A D P-ribosyl moiety of N A D into a 41-k Da me mbra ne protei n (39). T he parallel wit h c holera toxi n was re markable. Tos hiaki Kata da, Professor Ui’s st u de nt, a p plie d for a post doctoral positio n i n my lab a n d was q uickly acce pte d. As Gary Bokoch, a not her ne wly arrive d post doc, a n d Kata da bega n to work wit h Professor Ui’s t o xi n, J o h n North u p realize d t hat he ha d- freq ue ntly bee n plag ue d wit h a 4 1 -k D a c o n ta m in a n t d u rin g p u rific a tio n o f G s. H e e v e n h a d fra c tio n s in th e freezer t hat were e nric he d i n t his co nta mi na nt; moral: never t hro w a nyt hi ng a w a y! ( T his c o nt a mi n a nt c a n b e s e e n i n Fi g. 4, a b o v e, fr o m t h e ori gi n al p uri- ficatio n pa per.) T he obvio us ex peri me nt worke d bea utif ully o n t he first try; North u p’s conta minant was a s u perb s ubstrate for A D P-ribosylation by I A P. O ur relati vely tort uo us ex perie nce wit h t he p urificatio n of Gs t h e n p ai d off, a n d p urificatio n of “t he” I A P s ubstrate procee de d q uickly {40}, ai de d by t he fact t hat it is s ubsta ntially more ab u n da nt t ha n G,. T he hy pot hesis t hat t he

I A P s ubstrate re prese nte d G i, a ho mologo us G protei n res po nsible for i n hi- biti o n of a de n yl yl c yclase, was o b vi o us, a n d its vali dit y was esta blis he d a year later wit h t horo ug h c haracterizatio n of G i ( 4 1 - 44). Ne vert heless, t he act ual mec ha nis ms ( pl ural i nte n de d) of i n hibitio n of a de nylyl cyclase by G i r e m ai- n e d el usi v e. T hro ug ho ut m uc h of t his ti me, Bite nsky, w ho ha d detecte d a lig ht-activa- te d cyclic G M P p hos p ho diesterase i n t he reti na { 4. 5}, calle d atte nti o n t o paral- lels bet ween the vis ual trans d uction path way an d hor mone-sensitive a denylyl cyclases. Partic ularly notable were descri ptio ns of a lig ht-activate d G T Pase (46) a n d a g ua ni ne n ucleoti de req uire me nt for activatio n of t he phospho di- esterase. T hese o bser vati o ns le d t o p urificati o n of tra ns d uci n ( G,) {47, 48} a n d a p preciatio n t hat G s, G i, a n d G t re prese nte d a fa mily of str uct urally ho mologo us g uanine n ucleoti de bin ding proteins with relate d α s ub u nits a n d v er y si mil ar ( or i d e nti c al) β s u b u nits {49} ( Fi g. 6). T he existe nce of t he γ s ub u nit of t he tra ns d uci n heterotri mer was recog nize d early beca use of its great ab u n da nce. Poor avi dity for stai n delaye d its recog nitio n as a co m po- ne nt of G s a n d G i { 4 1, 5 0}. Alfre d G. Gil m a n 1 9 1

IAP SUBSTRATE G/ F 3 5 3 9 3 5 4 1

P L A S E - B S A- O V A L -

TI-

F or a bri ef p eri o d of ti m e i n t h e e arl y 1 9 8 0’s it s e e m e d t h at t hi n gs mi g ht c al m do wn. Guanine nucleoti de- me diate d sti mulation an d inhibition of a denylyl c y c la se w a s in th e h a n d s o f G s a n d G i, w hile tra ns d uci n ex plai ne d t he obser- v ati o ns i n t h e vis u al s yst e m. I n c oll a b or ati o n wit h R oss, w e w er e a bl e t o o bs er- ve hor mo nal sti m ulatio n of a de nylyl cyclase usi ng t hree p urifie d protei ns - t h e β -a dre nergic rece ptor, G s, a n d a de n yl yl c yclase - reco nstit ute d i nto p hos- p holi pi d vesicles {51}. W hat more was t here? Major cl ues ca me q uickly. Pa ul

Ster n weis {52}, no w i n de pe n de ntly, a n d Eva Neer { 5 3} disco vere d G o as a st art- li ngly ab u n da nt protei n i n brai n; Fai n ( 5 4) an d Go m perts { 5 5} observe d hor- mo ne- a n d GTP-dependent sti m ulatio n of i nositol tris p hos p hate s y nt hesis; a mino acid sequence ho mologies were detected behveen signal-transducing G pr ot ei ns a n d t h e p 2 1 r as ge ne pr o d ucts (56); a n d t he cl o ners arri v e d ( 5 7, 5 8). It was clearly ti me to a d d so me ne w tec h nology to t he re pertoire a n d welco- m e m a n y n e w p e o pl e t o t h e p art y.

G PR OTEI NS FR O M P HER O M O NES T O P H OT O NS

It no w see ms a p pro priate to aban don the historical, story-telling a p proach a n d atte m pt to describe t he c urre nt le vel of u n dersta n di ng of G pr otei n- me diate d trans me mbrane signaling. It has beco me ab un dantly clear, parti- c ul arl y o v er t h e p ast d e c a d e, t h at t his r el ati v el y l ar g e f a mil y of heterotri me- ric G T P-bi n di ng a n d hy drolyzi ng protei ns plays a n esse ntial tra ns d uci ng role 1 9 2 Physiology or Medicine 1994 i n li nki ng h u n dre ds of c ell s urf a c e r e c e pt ors t o effect or pr otei ns at t he plas- ma me mbra ne. T hese syste ms are wi dely utilize d i n nat ure, co ntrolli ng pro- cesses ra n gi n g fro m mati n g i n yeast to co g nitio n i n ma n. Rece ptors t hat acti- vate G protei ns are corres po n di ngly diverse a n d e nco m pass protei ns t hat interact with hor mones, ne urotrans mitters, a utacoi ds, o dorants, tastants, phero mones, an d photons. Several revie ws of this area are reco m men de d { 5 9 - 70} a n d s h o ul d als o be c o ns ulte d f or refere nces t o t he ori gi nal literat ure. Overvie w of G Protei n F u nctio n a n d Str uct ure . Alt ho ug h G protei ns are str uct ural heterotri mers, t hey f u nctio n as dissociable di mers. T he β a n d γ s ub u nits exist as tig htly associate d co m plexes t hat f u nctio n as a u nit. T he i d e ntit y of t h e α s u b u nit is c urre ntl y use d t o defi ne a gi ve n G pr otei n oli g o- mer. Alt ho ug h a n u mber of differe nt βγ s ub unit co m plexes can a p parently associate fr uitf ull y a n d pro misc uo usl y wit h a variet y of cx s u b u nits, it is u n- k no w n to w hat exte nt t his occ urs i n vi v o . Sixtee n disti nct ge nes e nco de G protei n α s ub u nits i n ma m mals; 20 or m ore pr otei ns are s y nt hesize d, i ncl u di n g t he pr o d ucts t hat arise as a res ult of alternative s plicing of m R N A. The co m monly recognize d s ubclassification of t h e α s ub u nit fa mil y is base d o n str uct ural relatio ns hi ps, b ut t his sc he me d o es r e as o n a bl e j usti c e t o f u n cti o n al r el ati o ns hi ps as w ell { 6 6} ( Fi g. 7). F o ur s u bf a mili es ar e us u all y dis c uss e d: ( 1) t h e s m all G s gr o u p ( G, a n d G olf ), b est recog nize d as acti vators of a de nylyl cyclases; (2) t he large a n d f u nctio nally di verse G i gro u p, w hose me mbers are pert ussis toxi n s ubstrates wit h o ne exce ptio n ( G z ) ; ( 3) t h e G q gro u p, acti vators of t he se veral isofor ms of p h os- p holi pase C β ; a n d (4) t he most rece ntly recog nize d G 1 2 gro u p, whose f unc- tions are unkno wn. There are five kno wn genes enco ding β s u b u nits a n d six f or γ’s, If all possible co mbi natio ns of α , β , a n d γ were allo we d, we wo ul d nee d to co nsi der at least 600 G protei n oligo mers. Alt ho ug h so me co mbi na- ti o ns of β a n d γ a p pear to be forbi d de n a n d t here are so me prefere nces of a’s f or s p e cifi c βγ di m ers, t h e n u m b er is still li k el y t o b e v er y l ar g e.

4 0 6 0 6 0 1 0 0 I I I

% A mi n o Aci d I de ntit y

Fi g. 7. S e q u e n c e r el ati o ns hi ps b et w e e n m a m m ali a n G α s u b u nits a n d fa mil y gr o u pi n gs. M o difie d, wit h per- missi o n, fr o m { 6 6 } a n d { 6 5 }. Alfr e d G. Gil man 1 9 3

Eac h G pr otei n α s ub u nit has .a si n gle hi g h-affi nit y bi n di n g site for g ua ni ne n ucleoti de. The G D P-bo un d for m of α is r el ati v el y i n a cti v e a n d h as hi g h affi- nit y f or B y. T h us, G D P- α βγ co nstit utes t he i nactive oligo mer. Rece ptor-cata- lyze d g uanine n ucleoti de exchange res ults in for mation of G T P- α , a n d r es ul- ta nt co nfor matio nal c ha nges ca use dissociatio n of α fr o m βγ (s e e Fi g. 8). T wo regulators of do wnstrea m effecters, G T P- α a n d βγ ar e t h us li b er at e d. G pr ot ei n α s ub u nits are t he mselves e nzy mes, albeit poor o nes, wit h i ntri nsic G T Pase acti vit y. After a peri o d of ti me c haracteristic of i n di vi d ual α s u b u nits, G T P is h y drol yze d to G D P, s u b u nits associate, a n d t he basal state is restore d. G protei ns t h us f u nctio n as s witc hes a n d ti mers. T he hi g h affi nit y of α ( a n d partic ularl y of t he oli go mer) for G D P hol ds t he s witc h off; n ucleoti de exc ha n ge t ur ns t he s witc h o n; h y dr ol ysis of G T P t ur ns it off a gai n wit h a c ha- r a ct eristi c d el a y (s e c o n ds t o p er h a ps mi n ut es) as a r es ult of sl o w c at al ysis; t his co nstit utes t he ti mer, w hic h is a n i m porta nt ele me nt of sig nal a m plificatio n.

G PR OTEIN ACTIVATI ON/ DEACTIVATI ON CYCLE

Basal State Receptor Activati o n

1. 2.

Subunit Dissociation

Eff e ct or Activati o n

Fi g. 8. C- pr ot ei n- m e di at e d trans me mbrane si g n alli n g. I n t h e b as al st at e ( 1) G pr ot ei ns e xist as heterotri mers wit h G D P b o u n d ti g htl y t o t he α s u b u nit; t h e hor mone receptor ( R) is unoccupied and the effect or ( E) is unregulated. Upon hor mone bi n di n g a n d rece pt or acti vati o n ( 2), t he rece pt or i nteract s wit h th e h et er otri m er t o pr o m ote a confor mational change and dissociation of G D P fr o m t h e g u a ni n e n u cl e oti d e bi n di n g sit e; at n or mal cell ular c o nce ntrati o ns of guanine n u cl e oti d es, G T P fills t h e sit e i m m e di at el y. ( U n d er e x p eri m e nt al conditions where G T P is absent, the hor mone has high affinity for the receptor and the H- R- G-protein c o m pl e x is st a bl e.) Bi n di n g of G T P t o G α ( 3) in d u c es a confor mational change with t wo consequences. First, t h e G pr otei n diss ociates fr o m t h e H- R co mplex, reducing the affi nit y of hor mone f or receptor a n d, i n t ur n, freei n g t h e receptor for- another liaison with a neighboring quiescent G protein. Second, G T P binding also re d uces t he affi nit y of G α f or G βγ , a n d s u b u nit diss o ci ati o n o c c urs. T his fr e es G α - GTP t o f ulfill its pri mar y r ol e as a re g ulat or of effect ors ( 4). At le ast i n s o m e s yst e ms, t h e fr e e G βγ co mplex als o i nteracts directl y wit h effect ors ( E l) and modulates t h e acti vit y of t he acti ve c o m ple x, or it acts i n d e p e n d e ntl y at disti n ct effect ors ( E 2 ). G α possesses an intrinsic G T P as e a cti vit y ( 5). T h e r at e of t his G T Pase d et er mi n es t h e lif eti m e of t h e a cti v e s p e ci es a n d t h e associated physiological response. The G α -catalyzed hydrolysis of G T P leaves G D P in t h e bi n di n g sit e a n d causes diss ociati o n a n d deacti vati o n of t he acti ve c o m ple x. G a - G DP has hi g h affi nit y f o r G βγ ; subsequent reassociation of G α - G DP wit h G βγ ret ur ns t h e s yst e m t o t h e b as al st at e ( 1). R e pri nt e d fr o m Hel per a n d Gil m a n { 6 8 }, wit h p er missi o n. 1 9 4 Physiology or Medicine 1994

G protei n s ub u nits are s ubjecte d to a n u mber of co vale nt mo dificatio ns, bot h physiologically an d pathologically. Li pi d covalent mo difications are partic u- larly evi de nt. Me mbers of t he G i s ubfa mily are myristoylate d, with the Cl4 fatty aci d incor porate d in a mi de linkage to a mino-ter minal glycine resi d ues ( 7 1 - 73). T his mo dificatio n is a n i m porta nt deter mi na nt of t he affi nity of t h e s e α s u b u nits f or βγ a n d of t h e affi nit y of t h e G iα ’s f or a d e n yl yl c y cl as e (s e e b el o w) ( 7 4, 7 5). All α s u b u nits wit h t he exce pti o n of G tα are pal mito ylate d, i n s o m e c as es d o u bl y s o, a n d t h e Cl 6 f att y a ci d is b o u n d i n t hi o est er li n k a g e t o c yst ei n e r esi d u es n e ar t h e a mi n o t er mi n us ( 7 6). I n t h e c as e of m e m b ers of t h e

G i f a mil y, t h e p al mit o yl at e d c yst ei n e r esi d u e is i m m e di at el y a dj a c e nt t o t h e myristoylate d glycine. While myristoylation pres u mably occ urs c otra nslati o- nall y a n d t he mo dificatio n is irre versi ble, pal mitate is i ncor porate d posttra ns- l ati o n all y a n d t h e b o u n d f att y a ci d t ur ns o v er r el ati v el y r a pi dl y. Of p arti c ul ar i nterest, t ur nover of pal mitate is a rece ptor-reg ulate d p he no me no n, wit h co n- trol a p pare ntl y exerte d at t he le vel of re mo val of pal mitate fro m t he α s u b u- nit (77, 78). T he si g nifica nce of t his p he n o me n o n is n ot yet f ull y a p preciate d, b ut it co ul d re prese nt part of a pat h wa y for atte n uatio n of tra ns me m bra ne si g- n ali n g. Fi n all y, f or t h e li pi ds, γ s ub u nits co ntai n ty pical C A A X boxes at t heir c ar b o x yl t er mi ni a n d ar e pr e n yl at e d; t h e γ 1 s u b u nit, f o u n d i n t h e r eti n a, is f ar- n es yl at e d ( 7 9 1, w hil e t h e ot h er γ’s a p pear to be gera nylgera nylate d (80, 81). Alt ho u g h pre n ylatio n is not esse ntial for t he for matio n of hi g h-affi nit y βγ di m ers, it is cr u ci al f or t h e i nt er a cti o ns of βγ wit h α a n d wit h at least certai n effect ors ( e. g., a d e n yl yl c y cl as es) ( 8 2). All of t h e li pi d m o difi c ati o ns m a y pl a y i m porta nt roles i n localizatio n of G protei n s ub u nits to me mbra nes, alt ho ug h t he mec ha nis ms t hat dictate t he s pecificities of t hese protei n- me mbra ne i nter- actio ns re mai n to be disco vere d. We s us pect t hat G sα c o ntai ns a n as yet u ni- de ntifie d covale nt mo dificatio n. T he nat ural protei n ( p urifie d fro m liver or brai n) has a s u bsta ntiall y hi g her affi nit y f or a de n yl yl c yclase t ha n d oes rec o m- bi n a nt G sα s y nt hesize d i n bacteria (83). A D P-ribosylatio n of G protei n α s u b u nits b y bacterial t oxi ns is a partic u- larl y i nteresti n g, irre versible co vale nt mo dificatio n of pat holo gical si g nifi- cance. The diarrheagenic enterotoxin pro d uce d by Vi brio c holer ae a n d t he heat la bile t oxi n s y nt hesize d b y certai n strai ns of E. c oli ar e A D P-ribosyltra ns- ferases wit h great s pecificity for G,,. N A D is t he do nor of t he A D P-ribosyl moiet y, w hic h is attac he d to a n acti ve site ar gi ni ne resi d ue of t he s u bstrate

{ 8 4}. T h e r es ult a nt i n hi biti o n of t h e G T Pase a cti vit y of G sα ca uses persiste nt acti vati o n of b ot h G sα a n d a de nylyl cyclase. Diarr hea is t he do mi na nt sig n of disease beca use of t he local, e nteric nat ure of t he i nfectio n. A toxi n (isl et- activati ng protei n) pro d uce d by Bor detell a pert ussis catalyzes A D P-ribosylation of a cystei ne resi d ue near t he carboxyl-ter mi n us of me mbers of t he G i f a mi- l y of α s u b u nits {85}. T his res ults i n i n hi biti o n of i nteracti o ns bet wee n G pr o- tei ns a n d rece ptors, effectively blocki ng t he affecte d pat h ways, i ncl u di ng t h ose t hat ca use i n hi biti o n of a de n yl yl c yclase. As a si d eli g ht, it i s i nt er e sti n g to note t hat ot her microorga nis ms have develo pe d differe nt strategies for ele vatio n of host cell co nce ntratio ns of c yclic A M P. A toxi n elaborate d b y Alfr e d G. Gil man 1 9 5

B acill us a nt h arcis a n d a di sti n ct Bor detell a pert ussis t o xi n ar e t h e ms el v es c al- mo d ulin-activate d a denylyl cyclases that per meate ma m malian cells.

Hi g h-resol utio n cr ystal str uct ures of t wo G protei n α s u b u nits, G t a a n d G iα l, i n differe nt liga n de d states ha ve bee n describe d rece ntly { 8 6 - 88}. We have bee n please d to collaborate wit h Ste p he n S pra ng i n t hese efforts. T he ge neral arc hitect ure of t hese closely relate d protei ns is esse ntially i de ntical (Fig. 9). Eac h is co nstr ucte d of t wo very disti nct do mai ns: a p2l r as -li k e α β d o m ai n t h at is fl a n k e d b y a u ni q u e (t o G pr ot ei ns) α h eli c al d o m ai n. T h e t w o str uct ures are c o n necte d b y a pair of li n ker stra n ds. Alt h o u g h all of t he direct co ntacts bet wee n t he protei n a n d g ua ni ne n ucleoti de are for me d wit h eit her t h e p 2 1 r as -li k e d o m ai n or t h e li n k er 2 p e pti d e, t h e n u cl e oti d e is virt u all y b uri- e d i n t h e cl eft b et w e e n t h e t w o m aj or d o m ai ns. It is h y p ot h esi z e d t h at r e c e p- tor- me diate d confor mational changes s ufficient to per mit g uanine n u cl e oti- de exc ha n ge res ult i n s u bsta ntial se paratio n of t he helical a n d p 2 1 r as -li k e

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r a s Fi g. 9. Ri b bo n a n d coil sc he matic of G iα l s u b u nit. T he helical do mai n is colore d yello w, a n d t he p 2 1 -li k e do mai n is gree n a n d cya n. Li n ker 1 a n d li n ker 2 stra n ds are colore d re d. T he G T P γS is s h o w n as a ball a n d stick model, and the magnesiu m ion is depicted as a magenta sphere. Secondary structure ele ments are labe- le d. T he re d N a n d C mar k t he positio ns of t he first or dere d resi d ues at t he a mi no a n d car boxy ter mi ni of t he molec ule. Re pri nte d fro m Cole ma n et al. { 8 8}, wit h per missio n. Des pite t he existe nce of se veral s u perb cr ystal str uct ures of p 2 1 r as pr otei ns a n d of G T Pase- deficie nt m uta nts of p21 r as , it h as b e e n diffi c ult t o d e d u c e t h e mec ha nis m of G T P hy drolysis, per ha ps i n large part beca use t he protei ns are s uc h p o or catal ysts i n t he a bse nce of acti vat ors ( GTPase acti vati n g pr otei ns or G A PS). T he sa me is tr ue of t he str uct ures of t he G T P γS-bound f or ms of - G tα a n d G iα l H a p pil y, h o w e v er, t h e Al F 4 -bound co nfor matio ns of t hese pr o- 1 9 6 Physiology or Medicine 1994 t ei ns ar e m or e ill u mi n ati n g. As n ot e d a b o v e, Al 3 + was unex pecte dly fo un d to be a cofactor necessar y for acti vatio n of G protei ns b y F-, a n d it ha d bee n - de d uce d that AlF 4 probably bo u n d to G α protei ns i n proxi mity to G D P, mi mi c ki n g t h e γ-phosphoryl m oiet y of G T P (29, 89, 90). T he X-ra y str uct ure re veale d t his hy pot hesis to be nearly correct {88}. Ho we ver, rat her t ha n si m- - pl y mi mi c ki n g G T P, G D P- Al 4 a p pears t o be acti n g as a tra nsiti o n-state a na- l o g, r e v e ali n g criti c al r ol es pl a y e d b y a cti v e-sit e a mi n o a ci d r esi d u es. 1 7 8 2 0 4 T w o r esi d u es, Ar g ( G iα 1 n u mbering) an d Gl n , ha d bee n i m plicate d i n catalysis as t he res ult of isolatio n or co nstr uctio n of G T Pase- deficie nt pro- tei ns wit h m utatio ns at t hese sites (91 - 9 4). I n a d diti o n, t his Ar g r esi d u e

corres po n ds to t he Ar g i n G sα t hat is A D P-ribosylate d by c holera toxi n, a n d r as t he Gl n corres po n ds to Gln 6 1 i n p 2l , a r esi d u e k n o w n t o b e criti c al f or catal ysis. ( T here is no ho molo g of Ar g 1 7 8 i n p 2l r as Ar g 1 7 8 i s i n t h e li n k er 2 p e pti d e.)

1 7 8 Fi g . 1 0 . A sc he matic of t he active site i n t he G T P γS- G i al co m plex, s ho wi ng t he dis positio n of Ar g a n d Gl n 2 0 4 ; t hese resi d ues are not wit hi n hy droge n bo n di ng dista nce of t he n ucleoti de. T he p utative w at er n u cl e- o p hile is p ositi o ne d 3.85 Å fr o m t h e γ p hos p hor us tra ns axial t o t he βγ , bridging phosphate oxygen ato m. The β 1 - α 1 loo p is colore d gree n, t he β 2 - α 3 s wit c h pe pti de yello w, a n d t he li n ker 2 stra n d is bl u e.

- Rearra n ge me nt of t he positio ns of t hese t wo resi d ues i n t he G DP- AIF 4 str uc- t ur e (r el ati v e t o t h eir p ositi o ns i n t h e G T P γ-Sbound pr otei n) re veals t heir r ol es i n c at al ysis ( Fi g. 1 0). Gl n 2 0 4 a p pears to be stabilizi n g a n d orie nti n g t he hy drolytic water molec ule i n t he trigo nal-bi pyra mi dal tra nsitio n state, w hile Ar g 1 7 8 stabilizes t he negati ve c harge at t he eq uatorial oxyge n ato ms of t he pentacoor dinate phos phate inter me diate. Since this Ar g r esi d u e is u ni q u e t o G α pr otei ns, its prese nce ma y ex plai n t he hi g her h y dr ol ytic acti vit y of G α pr o- t ei ns r el ati v e t o t h os e of m e m b ers of t h e p 2 1 r as f a mil y. Alfre d G. Gil man

- c o ntact t he AlF 4 cl u st er, a n d t h e n ucleo p hilic w at er h a s m o v e d i nt o t h e li g a n d fi el d of t h e al u mi n u m i o n.

H y dr ol ysis of G T P b y G iα 1 is acco m pa nie d by relaxatio n of bot h t he li nker 2 stra n d a n d a t we nty-resi d ue seg me nt t hat co ntai ns Gl n 2 0 4 . T he loss of a ny or dere d confor mation in these resi d ues ( which are invisible in the electron de nsity ma p) acco u nts for alteratio ns i n pro perties k no w n to be c haracteris- ti c of t h e G D P- b o u n d f or m of t h e pr ot ei n: l oss of t h e M g 2 + bi n di n g sit e, a so me what re duce d affinity for guanine nucleoti de, enhance d susceptibility to proteolysis in this region, an d q uenching of try pto phan fl uorescence.

N H I

Fi g. 1 0. C: M o del of t he acti ve site of G iα 1 at t he tra nsiti o n state of t he p h os p h or ol ysis reacti o n, base d o n t he - str uct ure of t he G D P. AlF 4 co m plex. Re pri nte d fro m Cole ma n et al. { 8 8}, wit h per missio n. 1 9 8 P h ysi ol og y o r Medicine 1994

A s ur prisi n g c o ns e q u e n c e of G T P h y dr ol ysis is t h e ass e m bl y of t h e a mi n o a n d carboxyl ter mi ni i nto a disti nct, orga nize d α - h eli c al d o m ai n. T his str u ct ur al c ha nge occ urs nearly 30 Å fr o m t h e c at al yti c sit e, a n d it is diffi c ult t o dis c er n a n i ntra molec ular pat h way of co nfor matio nal tra nsitio n bet wee n t hese sites. Even more surprising is the discovery that the ne wly for me d do main for ms an exceedingly co mpli mentary and extensive packing interface with the α - helical do main an d the linker 2 stran d of the neighboring molec ule in the cr ystal lattice ( Fi g. 11). T h us, t he tra ns missio n of str uct ural c ha n ges bet wee n the G TP bin ding site an d the a mino an d carboxyl ter minii ( which for m part of t he pres u m ptive bi n di ng s urface for t he βγ subunit co mplex) might be by mea ns of i nter molec ular co ntacts { 9 5}. I nteresti ngly, t hese observatio ns co ul d be perti ne nt to rece nt s pec ulatio ns by R o d b ell o n t h e p ossi bilit y of G

F a g. 1 1 Sy m metry-related molecules of G DP- G iα 1 f or m a h elic al arr a y i n t h e cr ystalli ne state. T he car boxyl- a n d a mino-ter minal a m mo aci d resi d ues of G iα 1 are disor dere d i n t he G T P co m plex b ut fol d i nt o a discrete pr otei n microdo main co mprising nearl y 40 resi d ues upon hydrolysis of the nucleoside tnphosphate. A p h os- p hate i o n, w hic h co ntacts t hree argi ni ne residues fro m the a m mo ter minus and lysine 180 fro m the li n ker-2 stra n d, ma y act as a n ucleatio n ce nter f or t he microdo ma m. ( A s ulfate i o n ser ves t his r ol e i n crystals of G D P-

G iα 1 .) T his microdo main f or m s a n exte nsive and co mple mentary interface with the a-helical do main a n d t he h n ker-2 stra n d of t he neig h bori ng molec ule T he co ntact b uries more t ha n 1800 Å2 of solve nt-accessi ble s ur- face, a n area e q uivale nt to that enco mpassed by many pr otei n antigen-i m munoglobulin co mplexes.

S u m mary of t he F u nctio ns of I n divi d ual G Protei n S ub u nits. α S ub u nits ( T a bl e l). Al m ost all kno wn Gprotein asubunitsand manydistinct βγ s ub unit co m plexes have been p urifie d to ho mogeneity fro m tiss ue so urces or p urifie d after ex pressi o n i n heter ol o g o us s yste ms (eit her E. c oli or Sf 9 c ells). Pro perties of several s ub u nits have also bee n i nferre d by a p plicatio n of ne w wave bioche mistry - experi ments perfor med “i n tra nsfecto”. Each syste m has advantages and disadvantages. While the E. c oli- derive d proteins may be mis- sing certain covalent mo difications (altho ugh myristoylation can be acco m- Alfr e d G. Gil m a n 1 9 9

plishe d by coex pression of protein N- myristoyl transferase), they have the disti nct a dva ntage of bei ng u na mbig uo usly free of ot her G protei n s ub u nits. T his ca n be diffic ult to pro ve wit h G protei ns isolate d fro m ot her so urces.

Table 1. Pro perties of Ma m malia n G Protei n α S ub u nits

Fa mily / M (k Da % A. A. Toxi n b Li pi d c Tiss ue Re prese ntative Effect or / r S u b u nit x 1 0 - 3 ) I de ntity” Distri b utio n Rece ptors d R ol e

4 4. 2 C T P U bi q uit o u s c y cl a s e 4 5. 7 P U bi q uit o u s c ha n nels T S H, ot hers c ha n nels 4 4. 7 8 8 C T P ? Olf act ory A dc nylyl

1 0 0 P T Adc nylyl cyclase

P T c ha n nels 9 4 P T Ne arl y

P T M, Brai n, ot hers Met- E n k, c ha n nels P T Br ai n, ot hers c y cl a s e ot hers C T, P T M r o d s Rhodopsin Reti nal co nes C T h uds

ot hers ot h er,

4 2 1 0 0 P

4 2 P

P li v e r B’ s, ot h er s 4 3 P ? ot hers (?)

4 3. 5 P T- c ell s, ot h er s ( ?) c ell s

4 4 1 0 0 U bi q uit o u s ? 4 4 6 7 P

Foot notes: Ta ble 1. a. % A mi no acid ide ntity: co mpariso n is wit h t he first-listed me mber of eac h fa mily b. C holer a toxi n ( C T) a n d pert ussis t o xi n ( P T) c at alyze t he A DP-ribosylatio n of a n ar gi ni n e resid ue ( CT) a nd a cystei ne r e si- d ue ( P T). res pectively, of t he i ndicated a s u b u nits.

c. Lipid modificatio ns: T he i ndicated G α s ub u nits ar e covale ntly mo difie d at or near the a mino ter min us on cystei ne resi d ues by S-pal mitoylatio n (P) a nd/or glyci ne resi d ues by N- myrisroylatio n ( M). d. Rece ptor a b breviatio ns: B A R, β -a dre nergic; M 2 c h o, M 2 - m uscari nic c h oli nergic; α 2 A R, α 2 -a dre nergic; met-e n k, met-e nkep ha- li n; M 1 C h o, M 1 - m uscari nic c h oli nergic; α 1 A R, α 1 -a dre nergic. e. S pli c e v ari a nts. α s( s) =s hort for ms of a α s a n d α s( 1) =lo ng for ms of α s . 2 0 0 Physiology or Medicine 1994

T he me m bers of t he G s u bf a mil y ( α , α ) acti vate vario us a de n yl yl c yclases, sα s olf a n d t hey do so by direct i nteractio ns wit h t hese protei ns. All k no w n isofor ms of me mbrane- bo un d ma m malian a denylyl cyclase are activate d by G sα . G sα i s ex presse d as fo ur disti nct poly pe pti des ( + /- resi d ues e nco de d i n exo n 3; + /- a seri ne resi d ue at t he s plice j u ncti o n) as a res ult of alter nati ve s plici n g of a single prec ursor m R N A, b ut t hese varia nts have not bee n well disti ng uis he d f u nctio nall y { 9 7 - 9 9}. T h e α s u b u nit of G olf is expresse d pre do minantly in olfactory ne uroe pitheli u m, where it pres u mably co u ples o dora nt rece pt ors wit h a lar gel y olfactor y-s pecific isofor m of a de n yl yl c yclase (t y pe III) {l 0 0}. 2 + P urifi e d G s α also activates di hy dro pyri di ne-se nsitive, voltage-gate d C a c ha n- nels i n patc hes excise d fro m skeletal a n d car diac m uscle (101) a n d i n hibits car diac Na + c ha n nels (102). T he p hysiological sig nifica nce of t hese last t wo eff e cts is diffi c ult t o j u d g e. G s is acti vate d b y rece ptors t hat sti m ulate a de n y- lyl cyclase acti vity; B-a dre nergic rece ptors are prototy pical.

M e m b ers of t h e G i s ubfa mily were first e nco u ntere d as reti nal tra ns d uci ns a n d t he n as s u bstrates for islet-acti vati n g protei n. T he t wo isofor ms of tra ns- d uci n are selecti vel y ex presse d i n reti nal ro ds a n d co nes { 1 0 3}. T he y are acti- vate d by p hotolyze d r ho do psi n or t he co ne o psi ns, a n d eac h sti m ulates a cyclic G MP-specific phospho diesterase, res ulti ng i n lo were d i ntracell ular concentrations of cyclic G M P on ill u mination. A trans d ucin-like G protein, g us d uci n, is ex presse d selecti vel y i n taste b u ds (104). T he relati o ns hi p of g us- d uci n to t he tra ns d uci ns is s ufficie ntly close t hat a cyclic n ucleoti de p hos- p ho diesterase is hy pot hesize d to be t he effect or i n a pat h way me diati ng res po nse to certai n tasta nts.

T hree closely relate d ge nes e nco de G iα l,2, a n d 3 . T hese pr otei ns are f u nc- ti o n all y v er y si mil ar i n vitr o, alt h o u g h t h e y diff er i n b ot h t h eir c ell ul ar a n d s ubcell ular distrib utio n. De mo nstratio n of t he direct i nvolve me nt of t hese α s ub u nits as i n hibitors of a de n yl yl c yclases was lo n g dela ye d for se veral tec h- nical reaso ns, i ncl u di ng a req uire me nt for co m paratively hig h co nce ntra- ti o ns, t h e n e e d f or m yrist o yl ati o n of t h e α s ub u nit, a n d differe ntial res po n- ses of differe nt isofor ms of a de n yl yl c yclase (75, 105). T his role is no w well establishe d. Altho ugh the G iα protei ns were ori gi nall y t ho u g ht to acti vate K + c ha n nels i n car diac myocytes a n d ne ural tiss ue { 1 0 6}, t h eir r ol e i n t his p at h- wa y is no w more co ntro versial a n d ma y be seco n dar y to t hat pla ye d b y t he βγ s ub unit co m plex { 1 0 7}. E vi de nce has also acc u m ulate d for a role of at least certai n G i α protei ns i n me mbra ne trafficki ng {108 - 110}. Partici pati o n of t hese protei ns i n s uc h a p pare ntly disti nct cell ular pat h ways is co nf usi ng.

G z α differs s u bsta ntiall y fr o m t he G i α pr ot ei ns, b ut it als o i n hi bits a d e n yl yl c y cl as e a cti vit y i n tra nsfecte d c ells ( 1 1 1) or i n vitr o ( 1 1 2) N ot a bl y, G z α is n ot a s u bstrate f or pert ussis t oxi n a n d has a ver y sl o w rate of G T P h y dr ol ysis { 1 1 3}.

As me ntio ne d abo ve, t he disco very of G o was a n e ye-o pe ner beca use of its ab u n da nce i n brai n (1 - 2 % of brai n me mbra ne protei n) a n d a p pare nt lack of involve ment with kno wn guanine nucleotide-regulated syste ms. Although 2 + G o α a p p e ars t o pl a y a m aj or r ol e as a n i n hi bit or of v olt a g e-s e nsiti v e C a c ha n- nels {114}, I ass u me it has ot her extre mel y i m p orta nt r oles. Hi nts are s u p pli- Alfr e d G. Gil m a n 2 0 1 e d b y its hi g h co nce ntratio n i n ne ural gro wt h co nes {115} a n d a p pare nt i nter- actio ns wit h G A P-43, a Ca 2 + - bi n di n g protei n t hat is also co nce ntrate d i n t hese str uct ures (116). Co m pelling evi dence for reg ulation of phos phoinositi de-s pecific phos- p holi pase C activities was i n ha n d well before t he releva nt G protei ns co ul d be i de ntifie d (54, 55). T his is a pert ussis t oxi n-i nse nsiti ve pr ocess i n m ost cells, a n d t he P C R-base d clo ni ng of me mbers of t he G q α s ubfa mily provi de d c a n di d at es f or t his r ol e { 1 1 7}. Nearly si m ultaneo usly, G proteins that serve t his f u nctio n were i de ntifie d by classical reco nstit uti ve tec h niq ues (118, 119) a n d b y p urificati o n of n o vel α s ub u nits usi ng cle ver s ub u nit affi nity a n d exchange techniq ues { 1 2 0}. All t hree pat hs mer ge d wit h t he i de ntificatio n of

G a n d t he n G ,G as acti vat ors of t he vari o us is of or ms of q α 1 1 α 1 4 α, a n d G 1 5/ 1 6 α p hos p holi pase C B. P urificatio n of t he releva nt protei ns prove d t hat i nterac- ti o ns of G q α fa mily me mbers wit h t he p hos p holi pases are direct, a n d t hese a p pear to occ ur at carboxyl-ter mi nal do mai ns of t he e nzy mes. Partic ularly i nteresti ng is t he observatio n t hat t he p hos p holi pase C β ’s a ct as G APS or

G T Pase-activating proteins to war ds G q α (121). I n t he a bse nce of t he effect or, t h e k c at f or h y dr ol ysis of G T P b y t h es e pr ot ei ns is v er y sl o w, b ut it is i n cr e as e d o ver 50-f ol d b y t he effect or. T he si m plest i nter pretati o n of t his effect is t hat phos pholi pase C β s ho ul d block its o w n activatio n. Ho wever, ki netic a nalyses of t hese i nteractio ns s uggest t hat rece ptor, G q ; an d the phos pholi pase asso- ciate i n a co m plex t hat bi n ds a n d h y drol yzes G T P ra pi dl y, s uc h t hat t here is s ubsta ntial stea dy-state activatio n of p hos p holi pase C associate d wit h parti- c ul arl y r a pi d r es p o ns es.

T h e r ol es of G 1 2 α a n d G 1 3 α are u nk no w n. Bot h of t hese protei ns are str uc- t ur all y r el at e d t o t h e pr o d u ct of t h e Drosop hila co ncerti na ge ne, w hic h a p p e ars t o pl a y a r ol e i n g astr ul ati o n { 1 2 2}. Tra nsfectio n of NI H 3 T 3 c ells wit h G 1 2 α c D N A r es ults i n c ell ul ar tr a nsf or m ati o n { 1 2 3, 1 2 4}.

Table 2. Pro perties of Ma m malia n G Protei n β a n d γ S ub u nits

S u b u nit Mr % A. A. Tiss ue Distri b utio n EfFector/ Role (k Daxl0 3 ) I de ntit y a

β 3 7. 3 1 0 0 U bi q uito us

3 7. 3 9 0 Nearly u bi q uito us Re q uire d for G a -rece ptor i nteractio n 3 7. 2 8 3 Nearly u bi q uito us

3 7. 2 8 9 Nearly u bi q uito us I n hi bitio n of G a act vati o n 3 8. 7 5 2 Brai n S u p port of ago nist-i n d uce d rece ptor phosphorylation and desensitization

Y or (isofor m s pecific res po nses) 8. 4 1 0 0 Reti nal ro ds Phosphohpase 8, 7. 9 3 8 a dre nal 8. 5 3 6 Brai n, testis channels ( 3 4) ( Ki d ney, reti na 7. 3 2 5 U bi q uito us Phospholipase A’ 7. 5 3 5 a. A mi no aci d i de ntity: co m pariso n is wit h t he first-liste d me m ber of eac h fa mily. 2 0 2 Physiology or Medicine 1994

βγ S u b u nits ( Ta ble 2). General acce ptance of do wnstrea m reg ulation of effect ors b y t h e βγ s ub u nit co m plex is relati vely rece nt {69, 70}. T hese s ub u- nits were first assig ne d less gla moro us roles. T he bi n di ng of G D P a n d βγ t o α is p ositi vel y c o o perati ve. βγ t h us stabilizes t he i nactive G D P-bo u n d for m of α b y mar ke dl y re d uci n g t he rate of dissociatio n of n ucleoti de (125). As a r e s ult, βγ acts as a noise s u p pressor (126). By co ntrast, i nteractio ns of G T P a n d B y wit h α are negatively coo perative, a n d it was hy pot hesize d t hat βγ co ul d s pee d deacti vatio n of α a n d t hereby ca use i n hibitio n of releva nt do w n- strea m res po nses { 1 2 7}. T h e si g nifi c a n c e of t his p ossi bilit y r e m ai ns ill d efi- ne d, b ut t he e ve nt ual o bser vati o n of i n hi bit or y effects of G iα pr otei ns o n a de- nylyl cyclase has obviate d t he “ nee d” for t his hy pot hesis. Rece ptor-catalyze d exchange of G D P for G T P on G α r e q uir es βγ { 1 2 8}, a n d βγ ca n act catal yti- c all y i n t his r ol e. T h e G pr ot ei n h et er otri m er is t h us t h e f or m t h at is r e c o g- nize d b y rece pt or, a n d reass ociati o n of s u b u nits is a re q uisite f or acti vati o n. T he first stro n g e vi de nce for i nteractio n of B y wit h effect ors ca me fro m L o g ot hetis et al. { 1 0 7}, w ho detecte d acti vatio n of K + c h a n n el s i n c ar di a c atri- al m yoc ytes wit h βγ b ut n ot wit h G iα . Co ntroversy abo ut t he i nter pretatio n of t hese observatio ns ke pt βγ at l e ast p arti all y i n t h e cl os et f or a f e w y e ars, d es- pite ge netic evi de nce t hat By was t he pri mary me diator of do w nstrea m sig- naling in the phero mone res ponse path way of b u d ding yeast { 1 2 9}. I nteresti ng a n d direct i nteractio ns bet wee n βγ a n d effect ors s uc h as a de n yl yl c yclases {130 - 132} a n d p hos p holi pases {133, 134} have no w been observe d usi ng si m ple bioc he mical assays t hat have bee n wi dely re pro d uce d; t he iss ue t h us n o w see ms t o be settle d. Effects of βγ o n differe nt a de n yl yl c yclases will be disc usse d belo w. T he iss ue of s pecificit y a m o n g differe nt s pecies of βγ s ub u nits re mai ns vex- i ng. Ot her t ha n observatio ns t hat no n-reti nal α s u b u nits a n d effect ors a p pe- ar t o di s cri mi n at e a g ai n st r eti n al βγ (β 1 γ1), littl e s p e cifi cit y is o bs er v e d i n e x a- mi nati o n of t he i nteracti o ns of a n u m ber of βγ s ub u nit co m plexes wit h a vari- et y of α s ub u nits a n d effect ors { 8 2, 1 3 5, 1 3 6}. T hese observatio ns, ma de i n vitr o, fl y i n t he face of stri ki n g o bser vati o ns of s pecificit y ma de i n i ntact cells b y KIe uss a n d ass ociates {82, 135 - 1 3 9}. Voltage-se nsiti ve C a 2 + c ha n nels i n G H 3 cells are i n hi bite d b y b ot h M4- m uscarinic a n d so matostati n rece ptors.

Selecti ve s u p pressi o n of eit her of t he t w o s plice varia nts of G o α wit h a ntise n- se oligonucleoti des de monstrates that the muscarinic response depen ds on t he ex pressio n of G ol α b ut n ot G o 2 α w hile t he res po nse to so matostati n is selectively de pe n de nt o n G o 2 α Si milar s u p pressio n of i n di vi d ual β or γ s u b u nits also yiel de d striki ng res ults, co nsiste nt wit h m uscari nic sig nali ng via

α ol β 3 γ 4 a n d so matostati n sig nali ng t hro ug h α o 2 β 1 γ 3 . T he best c urre nt g uess is t hat t his s pecificit y is exerte d at t he le vel of rece pt or- G pr otei n i nteracti o ns, b ut de mo nstratio ns of s uc h by reco nstit utio n of p urifie d co m po ne nts i n vitr o re mains less than convincing. Alfre d G. Gil man 2 0 3

A DE NYLYL CYCL ASES

We have mai ntai ne d o ur i nterest i n a de nylyl cyclases t hro ug ho ut t he “ diver- sio n” i nto G protei ns, alt ho ug h “t he job” of a de nylyl cyclase was for so me ti me t he do mai n of o nly o ne i n divi d ualistic lab me mber. T his sit uatio n c ha n- ge d a n d i m prove d s ubsta ntially i n 1989. Ma m malia n a de nylyl cyclases are activate d by forskoli n, a diter pe ne fo u n d i n t h e r o ots of t h e pl a nt C ole us f ors k olii . T he de vel o p me nt of a f ors k oli n-affi- nity matrix by Pfe uffer a n d Metzger {140} ma de p urificatio n of t he e nzy me p ossi bl e, b ut n ot si m pl e. S mi g el w as t h e first i n o ur l a b or at or y t o p urif y a c al- mo d ulin-sensitive for m of a denylyl cyclase fro m bovine brain by a da pting Pfe uffer’s tec h niq ues {141}, a n d Kr u pi nski a n d co workers (142) fi nally p urifi- e d a s ufficie nt a mo u nt of protei n to obtai n a mi no aci d seq ue nce. Wit h t he inval uable hel p of Ran dall Ree d at Johns Ho pkins, whose collaborative efforts we so ug ht beca use of t he ab u n da nce of a de nylyl cyclase i n olfactory ne uroe pit heli u m, c D N A’s e nco di ng ty pe I (by defi nitio n) a de nylyl cyclase were obtai ne d fro m a bo vi ne brai n library. Se veral labs ha ve no w co ntrib ute d t o t h e is ol ati o n of si x a d diti o n al f ull-l e n gt h cl o n es (t y p es II - VI a n d VIII) b y a p plicatio n of lo w-stri nge ncy hybri dizatio n a n d P C R tec h niq ues; all of t hese protei ns ha ve bee n ex presse d, a n d t heir reg ulatory pro perties are bei ng defi- n e d ( 1 4 3 - 1 4 5). Ma m malian, me mbrane-bound adenylyl cyclases have a co mplex (dedu- ce d) str uct ure t hat is re mi nisce nt of a variety of tra ns porters a n d c ha n nels ( Fi g. 12). T heir to po gra p hical relatio ns hi p to t he P gl yco protei n a n d t he c ys- tic fibrosis trans me mbrane con d uctance reg ulator is striking, altho ugh they s hare no a mi no aci d seq ue nce ho mologies wit h t hese protei ns. A s hort cyto- plas mic a mi no ter mi n us is follo we d by six p utative tra ns me mbra ne s pa ns

( designate d M 1 ) an d a ro ughly 40-k Da cyto plas mic do main ( C 1 ) . T his a p pa- re nt str uct ural u nit is t he n re peate d: a seco n d set of six tra ns me mbra ne s p a n s ( M 2 ) is again follo we d by a secon d large cyto plas mic do main ( C 2 ) . Alt ho ug h t his str uct ure is u ni q ue for a “si m ple” e nzy me, its sig nifica nce is el usi v e. I fi n d it f as ci n ati n g t h at t h e r e g ul at or y m otif f or a d e n yl yl c y cl as es - acti vatio n by a G T P bi n di ng protei n - is a p p ar e nt i n Saccharo myces cereviseae. Altho ugh this mo de of reg ulation is conserve d fro m yeast to ma m mals, the m olec ular pla yers are n ot. T he a de n yl yl c yclase of Succharo myces is a v er y lar g e peripheral me mbrane protein with little rese mblance to its ma m malian co unter part (146). The G T P-bin ding protein in yeast res ponsible for sti m u- latio n of cyclic A M P sy nt hesis is t he resi de nt ho molog of ma m malia n p 2 1 r as {147}, eve n t ho ug h yeast have heterotri meric G protei ns. Evol utio n works i n strange ways. 2 0 4 Physiology or Medicine 1994

M 1 M 2 \ \

Fi g . 1 2. Predicted topology of me mbrane-bound adenylyl cyclases. Cylinders represent me mbrane-spanning regio ns, w hile bol d li nes i n dicate regio ns of hig h a mi no aci d si milarity a mo ng all me mbers of t h e f a mil y.

No menclat ure is as follo ws: N, a minoter minal do main; M 1 , first set of me mbrane-s panning regions; C l a a n d

C l b , t h e first large i ntracell ular cyto plas mic do mai n; M 2 , sec o n d set of trans me mbrane s panning regions; an d

C 2 a a n d C 2 b , t he seco n d large i ntracell ular do mai n. Re pri nte d fro m Ta ussi g a n d Gil m a n {145}, wit h per- missi o n.

T wo regio ns of ro ug hly 200 a mi no aci d resi d ues eac h ( C l a , a n d C 2 a ) are hi g h- ly conserve d a mong the ma m malian a denylyl cyclases, an d this relationshi p also exte n ds to t he to pogra p hically si milar e nzy mes of Droso p hil a a n d

Dictyosteli u m. T h e C 1 a a n d C 2 a do mai ns are also q uite si milar to eac h ot her an d to the catalytic do mains of both me mbrane-bo un d an d sol uble g uanylyl cyclases. T hese relatio ns hi ps i n dicate t hat o ne or bot h of t hese do mai ns is a sit e of c at al ysis. U nfort u natel y, it has not bee n possible to detect si g nifica nt a de n yl yl c y cl a- se acti vity follo wi ng ex pressio n of eit her of t hese p utati ve catalytic do mai ns as discrete pr otei ns; t he sa me is tr ue if i n di vi d ual hal ves of t he m olec ule are

ex presse d i n Sf 9 cells. Ne vert heless, co nc urre nt ex pressio n of M 1 C 1 a n d

M 2 C 2 res ults i n a p peara nce of a s u bsta ntial le vel of a de n yl yl c yclase acti vit y t hat ca n be re g ulate d c haracteristicall y b y G pr otei n s u b u nits a n d, i n t he case of t h e t y p e I e n z y m e, c al m o d uli n { 1 3 0}. We te ntatively ass u me t hat i nterac-

tio n bet wee n t he C l a n d C 2 d o mai ns is necessar y f or catal ysis. T his is c o nsis- te nt wit h t he facts t hat bot h s ub u nits of hetero di meric, sol uble g ua nylyl cyclases are req uire d for catalysis (eac h s ub u nit co ntai ns seq ue nces ho molo- g o us t o C 1 a a n d C 2 a ) {148 } an d that the me mbrane-bo un d g uanylyl cyclases are ho mo-oligo mers { 1 4 9 }. It is als o i nt er esti n g t h at p oi nt m ut ati o ns i n eit h er

C 1 a or C 2 a ca n i m pair a de n yl yl c yclase acti vit y se verel y a n d t hat m utatio ns i n

eit her d o mai n ca n ele vate t he K m for A T P. Bot h do mai ns may bi n d A T P; bot h mig ht also catalyze cyclic A M P sy nt hesis, or o ne may be t he do mi na nt cata- l yst w hile t he ot her ser ves a re g ulat or y r ole. Reg ulatio n of A de nylyl Cyclases by G Protei n S u b u nits. All s e v e n of t h e

isofor ms of a de n yl yl c yclase i de ntifie d to date are acti vate d b y G sα ( a n d f or- skoli n). S ur prisi ngly, t hese feat ures (a n d so-calle d P-site i n hibitio n by a de- n osi n e a nalo gs) are t he o nl y s hare d re g ulator y motifs. T he t y pe I isofor m is als o a cti v at e d b y c al m o d uli n, w hil e it is str o n gl y i n hi bit e d b y t h e G pr ot ei n βγ s ub u nit co m plex. Alt ho ug h t his effect was origi nally attrib ute d to seq uestra- Alfr e d G. Gil man 2 0 5 ti o n of c al m o d uli n b y βγ , p urificatio n of t he ex presse d cyclase has per mitte d de m o nstrati o n of its direct i nteracti o n wit h βγ (132). T he t hree isofor ms of

G iα a n d G o α c a n als o i n hi bit t y p e I a d e n yl yl c y cl as e, b ut t h e eff e ct is m u c h l ess pro mi ne nt t ha n t hat of βγ w he n cal mo d uli n is t he activator of t he e nzy me a n d it nearl y disa p pears w he n t he c yclase is acti vate d b y G sα { 1 0 5}. T y p e I a d e n yl yl c y cl as e is t h e o nl y is of or m f o u n d t o d at e t h at is i n hi bite d- b y βγ . W he n we looke d for s uc h i nteractio ns wit h ot her isofor ms, we were v er y s ur pris e d t o fi n d str o n g sti m ul ati o n of e n z y m ati c a cti vit y wit h t h e t y p e II a n d t y pe I V protei ns (131, 150). Partic ularl y i nteresti n g, t hese sti m ulator y eff ects of βγ are hi g hl y co n ditio nal. T he s u b u nits ha ve little or no effect o n a de nylyl cyclase activity w he n a d de d alo ne, b ut t he co m plex sti m ulates e nzy- m ati c a cti vit y 5- t o 1 0-f ol d w h e n G sα is als o pr es e nt. Sti m ul ati o n of t y p e II a n d I V a de nylyl cyclases by βγ re q uires s ubsta ntially hig her co nce ntratio ns of βγ t h a n of G sα a n d we pres u me t hat effective co nce ntratio ns of bot h activators ca n not arise b y dissociatio n of oli go meric G,. T he so urce of βγ is b eli e v e d t o b e t h e G i or G o oligo mers, bot h of w hic h are prese nt i n hig h co nce ntratio ns i n br ai n. W e t h us e n visi o n t y p e II a n d I V a d e n yl yl c y cl as es as m ol e c ul es d esi g- ne d to detect coi nci de ntal activatio n of t wo reg ulatory pat h ways - marking s uc h e ve nts wit h a disti ncti ve si g nal. T he bioc he mical pro perties of t hese a de- nylyl cyclases provi de an excellent ex planation for pheno mena describe d in t he 1970’s b y Rall a n d associates {151} w ho obser ve d hig hly sy nergistic sti- m ulatio n of c yclic A M P acc u m ulatio n i n brai n slices after a p plicatio n of pairs of ne urotrans mitters no w kno wn to work thro ugh G S - a n d G i-reg ulate d pat h- w a ys. Gi v e n a cti v ati o n of t y p e II a d e n yl yl c y cl as e b y βγ , w hic h pres u mably ari- s es fr o m G i, it w o ul d b e pr o bl e m ati c if G iα were t o i n hi bit t he e nz y me; grati- f yi n gl y, it d o es n ot. T he first reall y belie vable de mo nstratio ns of i n hibitio n of a de n yl yl c yclases b y G iα were obser ve d wit h t he ty pe V a n d ty pe VI isofor ms, w here t he effect is pro mi ne nt (75, 105). As note d abo ve, it is de pe n de nt o n myristoylatio n of t hese a s ub u nits a n d req uires fairly hig h, b ut we believe q uite reaso nable, co nce ntratio ns of t he protei ns ( hig h n M - µ M). T y pe V a n d VI a de n yl yl c y cl a- s es ar e t h us r e g ul at e d i n t h e r el ati v el y si m pl e w a y t h at w as e n visi o n e d t o b e t h e g e n er al r ul e - acti vati o n b y G sα a n d i n hi biti o n b y G iα - b ut e v e n t h es e is o- for ms pro vi de d s ur prises, i n t hat t hey are i n hibite d by lo w ( µ M) c o nce ntra- ti o ns of C a 2 + . T hree disti nct patter ns of reg ulatio n of ma m malia n a de nylyl cyclases are t h us e vi de nt ( Fi g. 13). All isofor ms are acti vate d b y G s a , a n d t wo ot her s ub classes of G pr otei ns, G i a n d G q ar e i m pli c at e d as w ell, eit h er dir e ctl y or i n di- 2 + , rectl y. T he effects of G q fa mily me mbers are exerte d thro ugh G a eit her a cti n g al o n e, wit h c al m o d uli n, or wit h pr ot ei n ki n as e C. G i- a n d G q - me dia- te d pat h ways ca n bot h acti vate a n a de nylyl cyclase (ty pe II a n d probably I V) i n c o ncert wit h G sα or t he y ca n b ot h o p p ose s uc h acti vati o n (t y pes V a n d VI).

T he effects of G i a n d G q are a nta g o nistic t o eac h ot her wit h t he t y pe I e nz y- me. E ve n at t his relati vel y earl y sta ge of i n vesti gati o n of t he re g ulat or y c o m- plexities of a de n yl yl c yclases, it is clear t hat t hese e nz y mes ha ve e v ol ve d t o 2 0 6 Physiology or Medicine 1994 per mit extensive integration an d cross-talk bet ween signaling path ways. The a de n yl yl c yclases are f ocal p oi nts f or t he c o n ver ge nce of a great deal of re g u- latory i nfor matio n.

Fi g. 1 3. Patter ns of re g ulati o n of a de n yl yl c yclase acti vit y. P K C = pr ot ei n ki n as e C; C A M = c al m o d uli n; A C = a de n yl yl c yclase. S e e t e xt f or dis c ussi o n. R e pri nt e d fr o m Ta ussi g a n d Gil m a n { 1 4 5 }, wit h p er missi o n.

F ut ure Directio ns for A de nylyl Cyclase. A d e n yl yl c y cl as es ar e l a bil e i ntri nsi c m e m br a n e pr ot ei ns; t h eir l e v el of e x pr essi o n is l o w, e v e n u n d er artifi ci al c o n- ditions. Ne w tools are nee de d to probe their str uct ures an d mechanis ms of re g u la tio n . W ith th e se th o u g h ts in m in d , W e i-Je n T a n g h a s a tte m p te d to c o n - str uct a sol uble a de nylyl cyclase t hat wo ul d retai n c haracteristic reg ulatory pro perties, be s y nt hesize d i n lar ge q ua ntities, a n d be a me nable to ge netic a nalysis. He has rece ntly s uccee de d i n desig ni ng a n d sy nt hesizi ng a molec u- le t hat ma y ha ve all of t hese pr o perties {152}. T he c urre nt pr o d uct is a c hi- m er a of t h e C 1 a d o m ai n of t y p e I a d e n yl yl c y cl as e, j oi n e d b y a li n k er t o t h e C 2 do mai n of ty pe II a de nylyl cyclase. T he molec ule is sy nt hesize d by E. c oli, w here it acc u m ulates i n t he cyto plas m. It is acti vate d dra matically (fro m a n extre mel y l o w basal acti vit y) b y G sα a n d, s ur prisi n gl y, f ors k oli n. C yclase- defi- ci e nt str ai ns of E. c oli are de pe n de nt o n ex pressio n a n d acti vatio n of t he a de- nylyl cyclase for gro wt h o n maltose. Ge netic selectio n of m uta nts wit h i nfor- mative phenoty pes th us see ms possible, as does p urification, detaile d cha- racterizatio n, a n d, ho pef ully, str uct ural a nalysis. We ho pe t hat t his a p proac h will o pe n t he door for tr ue u n dersta n di ng of t hese i m porta nt protei ns.

W HY G PR OTEI NS?

O ne mig ht well ask w hy G protei ns are i ncl u de d i n sig nali ng pat h ways a n d w hy t he syste ms are so co m plex str uct urally. Tra ns me mbra ne sig nali ng is cle- arly acco mplishe d with si mpler (altho ugh us ually oligo meric) molec ular asse mblages, s uc h as tyrosi ne ki nases, liga n d-gate d io n c ha n nels, a n d rece p- Alfr e d G. Gil m a n 2 0 7

tor g ua n yl yl c yclases. I belie ve t here are se veral reaso ns for t he e vol utio n of co m plex si g nali n g s yste ms. At a relati vel y si m ple le vel, t he existe nce of t hese molec ular s witches an d ti mers per mits enor mo us a m plification in the signa- li ng process. A si ngle ago nist-rece ptor co m plex ca n catalyze t he activatio n of ma n y G protei ns d uri n g t he ti me t hat a si n gle G protei n α s ub u nit re mai ns, a cti v e { 1 5 3}; dela ye d deacti vati o n of t he α s ub u nit per mits f urt her a m plifica- ti o n at t h e l e v el of c at al yti c effect or m ol e c ul es. T h er e is als o t h e p ossi bilit y of s ubstantial reg ulatory co m plexity, with o p port unities to mo d ulate both the q ua ntitati ve a n d q ualitati ve as pects of si g nali n g b y alterati o ns i n rates of s y n- t hesis a n d degra datio n of ma ny ge ne pro d ucts, as well as more ac ute reg ula- tio n by covale nt mo dificatio n of t hese molec ules. Most i m porta ntly, per ha ps, t he tri partite nat ure of t hese sig nali ng syste ms per mits e nor mo us di versity of o ut p uts. G protein-reg ulate d signaling path ways are characterize d by both convergence an d divergence at each ste p. Many different kin ds of rece ptors ca n co n verge to acti vate a si ngle ty pe of G protei n, w hile a si ngle ty pe of rece pt or ca n i nteract wit h m ore t ha n o ne s pecies of G pr otei n t o i nitiate se ve- ral e ve nts. Si milarl y, differe nt G protei ns ma y co n ver ge o n a si n gle effect or t o alt er its a cti vit y, eit h er a d diti v el y, s y n er gisti c all y, or a nt a g o nisti c all y, w hil e a si ngle G protei n may also i nteract wit h more t ha n o ne effector. G protei ns ca n als o e xert effects via eit her t heir α or βγ s ub u nits. T he co m plexity of t he cell ular s witc hboar d t h us a p pears s ufficie ntly vast to per mit eac h cell to desig n a hig hly c usto mize d sig nali ng re pertoire by ex pressio n of a relati vely mo dest n u mber of mo d ular co m ponents. I dentification of all of these co m- po ne nts see ms certai n i n t he next deca de or so. Wit h t his i nfor matio n i n ha n d, we will be able to co m plete o ur u n dersta n di ng of t he wiri ng diagra m of t he sig nali ng s witc hboar d i n eac h ty pe of cell. S uc h k no wle dge, co u ple d wit h bot h i ncreasi ng so p histicatio n i n ratio nal dr ug desig n a n d i ncreasi ngly clever a p proac hes to scree n h uge c he mical libraries, will revol utio nize bot h phar macology and therapeutics.

ACKNO WLEDG MENTS There are many who have earned my most profound gratitude. My father e nco ura ge d a lo ve of scie nce b y exa m ple, a n d it was caref ull y n urt ure d b y m y doctoral an d post doctoral mentors - Theo dore W. Rall, who perha ps sho ul d ha ve wo n a Nobel Prize, a n d Mars hall W. Nire nber g, w ho di d. All of t he st u- dents an d post doctoral fello ws who have worke d in o ur laboratory have ma de s ubsta ntial co ntrib utio ns. T hese i n divi d uals are (i n al p habetical order): David Ber man, Gary Bokoch, La wrence Br u nto n, Patrick Casey, Fra ncoise Coussen, Car men Dessauer, Alex Duncan, Kenneth Ferguson, Michael Freiss m uth, Boning Gao, Michael Graziano, Tats uya Haga, E man uel Hanski, Br uce Harris, Jo h n He pler, Ts uto m u Higas hiji ma, Ally n Ho wlett, J or g e Itii g u e z- Ll u hi, Hir os hi It o h, Richar d Kahn, Toshiaki Kata da, Christiane Kle uss, Tohr u Kozasa, John Kr u pinski, Ethan Lee, Hsin Chieh ( Calvin) Lin, Ma uri ne Lin der, Michael Maguire, Davi d Manning, Pa mela Mi d dleton, S usanne Mu mby, John Nort h u p, Bruce Posner, Lynn Quar mby, Andre Ra w, 2 0 8 Physiology or Medicine 1994

Janet Robisha w, Elliott Ross, Leonar d Schleifer, Jose ph Sch warz meier, M urray S migel, Pa ul Ster n weis, Roger S u na hara, Wei-Jen Ta ng, Ro nal d Ta ussig, an d Nats uo Ue da. Ma ny me mbers of o ur staff have also playe d cr ucial roles, b ut t wo lo ng- ter m e m ployees tr uly sta n d o ut. Pa mela Ster n weis, w ho joi ne d t he lab i n 1973, still pr o vi des s killf ul assista nce a n d a n u plifti n g s pirit t o t h ose f ort u na- te e no ug h to work wit h her. We n dy Dea ner, w ho bega n to work wit h me i n 1976 as a n A d mi nistrati ve Assista nt, no w kee ps m uc h of my life a n d t hat of the De part ment of Phar macology in goo d working or der. It is notable that o ur r el ati o ns hi p h as als o s ur vi v e d t hr e e e diti o ns of Good man and Gil m a n’s T he P har macological Basis of T herape utics. Collaboratio ns wit h ot hers o n o ur fac ulty a n d else w here have bee n i nva- l uable. Partic ularly notable roles have been playe d by S usanne Mu mby, Elliott Ross, Ste p he n S pra ng, a n d Pa ul Ster n weis. O ur research has been s u p porte d contin uo usly since 1972 by grants fro m t he Natio nal I nstit utes of Healt h (first t he Natio nal I nstit ute of Ne urological Disor ders a n d Stroke a n d t he n t he Natio nal I nstit ute of Ge neral Me dical Scie nces) a n d, si nce 1977, by t he A merica n Ca ncer Society. Ot her i nval uable so urces of s u p port have i ncl u de d Mr. a n d Mrs. Peter O’ Do n nell, t he L ucille P. Markey C harita ble Tr ust, t he Ra y mo n d a n d Elle n Willie C hair of Molecular Neurophar macology, the Perot Fa mily Foundation, the Meado ws Foun dation, an d the Robert A. Welch Foun dation.

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