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Intramolecular disulfide bridges: avenues to receptor activation?
172
TIBS 12 - May 1987
Open Question Intramolecular disulfide bridges: avenues to receptor activation? Craig C. Malbon, Shaji T. George and Cary P. Moxharn Evidence suggests that many cell surface receptors that are coupled to their effector systems via G proteins possess mtramolecular disulfide bridges At least m the case o f the [I. adrenergw receptors, cleavage o f disulfides by thlol compounds appears to act,ate the receptor m a manner slmdar to agontst bmdmg A role ts proposed for mtrcanolecular &sulfide bndges and suifhvdD,ls m the structure and acmanon o f G protem-coupled ret eptors
The ability of cells to transduce mformalion across b~ologlcal membranes has captivated the interest of researchers from a wide range of disciplines A vanely of strategies has been employed to tackle the problem of understanding how cells manage to respond to extracellular signals and propagate this reformation across the cell membrane to the intracellular compartment Both for visual excitation and for many hormones that bind to cell surface receptors, the basic composition of the membrane elements involved in signal transduction includes a receptor (or photoplgment). an cffector molecule that generates the intracellular second messenger, and a guanine nucleottde binding (G) protein that propagates the signal from the receptor to the effector molecule(s) I Each of these elements is btfuncttonal, each must be capable of receiving input and then communicating the signal to the next element (protem) in the system as output Ligand recognmon (or photoexcitation) and signal propagation to a G protein are functions of the receptor Thus recognmon of the input signal must "activate' the recemor,~'he signal is to be propagated to the G pr3tem Although our knowledge of the hgand binding properties of many cell surface hormone receptors is impressive, little ~s known about what constitutes the functional domains t,f the receptor and even less is C C Malbon. S T George mrd C P Moxham are members of the Dtabete~ and Metabohsm Research Group and are at the Department o] Pharmacologt cal Scwnces State Umversav of New York at Stony Brook. Stony Brook, NY 11794--8651 USA, C P M t~ currently at the Department of Molecular Biology The Welltome Research Laboratories Research Trmngle Park NC27709 USA It/~TFl~e~ltrPubll~dhon~
C imhntlgL
11"t76-Ch1~7~7/~1121111
known o f the process by which hgand binding yields receptor 'activation' Catecholamme modulauon of intracellular cyclic AMP (cAMP) levels provides a useful model system for analysis of signal transduetion across biological membranes ~-Adrenerglc receptors bind catecholammes and stimulate intracellular accumulation of cAMP by increasing the activity of the effector molecule adenylate cyclase, via the activauon of the G~ proteml Structurally, mammalian I~-adrenergic receptors are single-chat polypeptlde glycoproteins of M~ 64 I)00-67 fl00 in sodium dodecyl sulfate polyacrylamlde gel electrophoresis (SDS-PAGE) 2-~ Successful large-scale purification of these Iow~,bundance membrane proteins provided amounts of receptor required to apply several powerful strategies to the study of receptor structure and function Chemical modification of 13-adrenerglc receptors by group-specihc and site-specific reagents, molecular cloning of these molecules, and analysis of receptor function by defined reconstltution of receptor and G~ in phosphohpld vesicles have been applied to the questions of what constitutes functional domains of the receptor and how does agomst bmdmg 'activate' these receptors Our interest in the contnbutlon of disulfide bridges and free sulfhydryl groups of the receptor to both the snucture and the function of this molecule has been reinforced by recent results obtamed from studies m which these three distinct approaches have been employed in this article we develop the hypothesis that disulfide bndges and free sulfhydDI groups play an integral role m the structure of cell surface receptors that are coupled to G
proteins A role for mtramolecular disulfide bridges and free sulfhydryl groups in the process of receptor activation by agomsts is proposed Role in receptor structure
The effects of agents that chemically modify disulfide bndges and free sulfhydryl groups of proteins on the hormone-sensztzve adenylate cyclase system have been described in numerous reports (see Ref 5) Defining the precise targets of these chemical modifications was a difficult task due not only to the mtnnsm complexity of the individual elements of the system, but also to that of the biological membrane m which the system was probed l'he first structural evidence supporting the presence of intramolecular disulfide bridges in the receptor was the demonstration that chemical reduction of these bridges decreased the electrophoretm mobility of the molecule s In SDS-PAGE, pure 13adrenergic receptor migrates with an M, of 54 000, this changes to 65 000 after chenucal reduction with reagents that cleave disulfide bridges of proteins Possible explanations for this behavior are that the receptor exists m a 'compact' form maintained by intramolecular disulfide bndges under non-reducmg conditlons and that cleavage of these bonds results in an 'unfolding' of the molecule increasing the apparent M r of the molecule s Analym ot partially purified receptor preparations replete with other membrane proteins clearly suggested that the mobility shift observed for the receptor after treatment with reagents that cleave disulfide bonds was not a property observed for most of the other membrane protems ~ But is this behavior unique to 13adrenergm receptors 'J Do other membrane receptor proteins display an increased M r when sublected to SDSPAGE under reducing as compared to non-reducmg condmons '~ If the answer Is yes, what ff anything, do these proteins have m common '~ The list of proteins that are reported to display a mobility shift after reduction mcludes, in addition to the mammalian 131and ~z-adrenergm receptors s 6, the hepatic glucagon receptor 7, the opiate receptor purified from bovine strtatum 8, the receptors for interleukm-2 ~ and interleukm-31", and the LH/hCG receptorit Chemical reduction and alkylation of the photopigment
TIBS 12-May
173
1987
rhodopsln and the turkey 13]-adrenerglc receptor also i n c r e a s e s the M r of these proteins m S D S - P A G E (C Moxham, E M Ross and C C Malbon, unpubhshed) Most, if not all, of these cell surface receptors appear to share the common feature of bemg coupled to their effector molecules via G protems Immunological analysis of the [~adrenerglc receptor suggests that the molecule IS the M r 55 000 rather than the 65 000 species m s u u t 2 Thus, the 55 000 species is not formed as a result of the solubfl~zatlon of the receptor from the membrane ~2 Furthermore, structural evidence has been provided suggesting that the integnty of intramolecular d~sulfide bndge(s) is essentml for hgand binding 5 Chemtcal cleavage of these bonds destabilizes the ablhty of the receptor to b i d radlohgands ~ These data suggest that mtramolecular disulfide bridges may play a critical role in the structure of G protein-coupled receptors Molecular clonmg of the hamster lung l~2-adrenergtc receptor ~ as well as the turkey erythrocyte I~radrenerg~c receptor ~4 has been reported recently The mammalian [12-adrenerg~e receptor possesses 15 cystemyi restdues (cysteme content = 3 5 mol % ) and the avmn ~ receptor contains 17 (cy~eme content = 3 6 mol %) The proposed onentatlon of the mammalian [~-adrenergm receptor with respect to the cell membrane ~s dis-
EXTRACELLULAR
played in Fng 1 F o u r of the cysteLnyl residues are predicted to be w]thLn transmembrane-spanning regmns (TMSR) of the protenn, and two additional cysteunyl resndues have been assngned at or near the hpud bnlayer/aqueous interphase The remannnng nine cystennyl residues are predicted to be nn regions outsnde of the lnptd bulayer three mn the extracellular loops, one mn cytoplasmic loop 5-6, and five mn the C-terminal portion of the molecule Companson of pnmary sequences of the avian and the mammahan I~-receptors reveals smct conservation (mdentmty) of nine cystemne residues, includmg those predicted to lie within the lipid bflayer (Table I) Primary sequence information ts avamlable for the muscanmc acetylchohne receptor I~ and bovmne opsm t6, two cell surface receptors that are also coupled to G proteins ) Smmllantles among the muscanmc receptor and [3-adrenerglc receptors are stnkmng TMSRs I and 4 as well as loops I-2 and 3-4 are devoid of c)stemyl residues, TMSRs 2 and 7 contain at least one cystemyl residue, and the Ctermini contam two to six res,dues All three receptors lack cystemnyl residues in the N-terrnlnus, T M S R i, and loop 1-2 (Table I), but opsm displays cystelnyl residues mnTMSRs 4, but not 7, a feature not observed in the fl-adrenerglc or muscannle receptors Elucidation of the membrane elements w t h whmch other
Table l Predwted asslgmnou of c~ ~temt/ resMues m t ell surfaca membrane receptor~
Receptor ' Regmn N-terminal TMSR I
Loop I-2 TMSR2 Loop 2-3 'l%lSn 3 Loop 3-4 TMSR4 Loop 4-5 TMSR 5 Loop 5-6 TMSR6 Loop6-7 TMSR7 C-termmnal Total Mol % Ref
131-Ar"
p~Ar
mAchr
I
--
--
. .
. .
. .
Ops m --
. .
I 2 ---
I 1 2 ---
l
3
-I
-I I -1 0
-I I -i 'i
I 4 -2 ~ 2
-3
17 3~; 14
15 36 13
15 ~'~ I'i
l0 29 16
I
i
-
I
--
---
I
I i
2 I -I
~Abbrev=atlons used are as Iollows 13rAr 13=adren,~r_'~creceptor of turkey eDthrocyle [~:-Ar [1.- adrenerg~e receptor or ha'-.~ter lung mAchr muscanmc chohnerg~c receptor ol porcme cerebrum Ops m bo~me reunal opsm and TMSR transmembr.ne-sp~,mmgregmn putative membrane receptors xx+thseven predicted TMSRs and far less cysteme content (hke the m a s oncogene product IT) mteract, may assist m the cntlcal review of our hypothesis that cystelnyl re+ldues o f cell surface receptors coupled
,8 2- AdrenergJc receptor
W CYTOPLASMIC
HOOC(
Fig I Pr~p~sed~rten~att~n~f~hemammahan[~-radrenergwrecepl~rLi~threrpeUt~theplasmamembrane Transnlembrane-spanmngreglon~ctreuumber,'dsequen ttally from the N- to C termtm, from omt to se~en (lefl to nghO The asslgnmem of teraarv smlcmre ts arberar) The pnmart sequence n as taken from Daon el al i, based upon a model o f Yarden et al i.Imade by analogy to dw known onentanon o f opsm -'7 The c~stem~l re.dues are m blacA
T I B S 12
-
M a y 1987
174 p
m i m m m l m
/%
to G proteins part~opate i n intramolecular disulfide bridges that are important to receptor structure and function
~ I I
II
N
H
........
.~,-\
2
~ 1
~--~'-.'~
---
.I L - - ~ ' J
t,~ ~
/
.--'"
,,'
'activation' Although receptor 'actwatlon' ~s a term frequently apphed in the literature to the description of receptor sequellae that follow agomst hgand binding, we know little of the molecular details of this m i I #/' , ~1 Ik o o . V _qA-I phenomenon Two advances provide the capability to probe features of receptor activation by agomst directly the ablhty to isolate essentially pure receptors and i m ",,,,',,.,, k \~'~JJJ ! % %~-.J)'J I ~ v . ~ , ~ j G proteins, and the ability to reconstitute membrane orotems into defined, "-..:,-"7 r " T ', . k unmlamellar phosphohpld vesicles Pedersen and Ross used this approach I I ~ ~ e,^~H with ~-adrenerp0c receptor msolated from 1I II t ~ ! , buvn turkey er)throcytes and G~ from rabbit I I ",, ",,,_ . - " / ,"-7 liver, and demonstrated that treatment I \ e e ~ " .... " ~ _s of ~-adre ~erg~c receptor ~ t h dithmo- • ""~'" ~" . . . . . . . . I ....... • s" thremtol or other tfuol agents capable of cleawng disulfide bridges of proteins caused the functional activation of the receptor u Furthermore. treatment with 0 )EXTRACELLULAR dlthlothreltol also markedly potentiated activation of G, (as measured by GTPyS binding) by agomst hgand, enhancing Fig 2 proposed model ofthe [badrenerglcreceptoras seenfrom the extracellularude of the cell membrane hpM the initial rate by nearly tenfold Thlol bdayer Predictedtrammembrane-spanmng regions are depmted as seven cods. numbered m sequence stamng activation of the receptor was sho~m to from dle N termmus C) stemyl resMuesare mdrcated by '--S' Assfgnmenl of tertmry strucfltre ~ arbgrary occur in the absence of agomst and mnthe presence of antagonist hgands TreatWhether or not the M r 65 000, fully- in the M, 65 000 rather than the 55 000 ment of Gs with thlol compounds was speoes 21 One possible explanation for w~thout effect, defining the receptor pro- reduced form of the receptor m the 'acuthese data Is that in sgu, prolonged expovated' species is an open question. On tern itself as the target of the functional sure of receptors to agomst sumulatlon the basis of the membrane organization activ.qmon by thmolsI~ results m a disulfide-exchange reaction 19 of the j~-adrener ,,c receptor proposed in Recent studies in which mammalian that favours the Mr 65 000 receptor F,g, 2, it Is possible that there are more rather than avian D-adrenergmcreceptors species This hypothesis, though were reconstituted with G s in defined than one intramolecular disulfide bndge speculative tn nature, Is consistent with m the molecule The observation that hposomes demonstrate that mammahan the data discussed above and deserves D-receptors are functionally activated by treating these receptors vmh ,ncreasmg cntlcal evaluation concentrations of reductant yields receptreatment voth thlol compounds, and tor species ~ t h intermediate Mr to the that it is the M r 55 00~ receptor species Disulfides and membrane organization of that binds agomst hgand as well as acts as 55 000 and 65 000 formss, supports this receptors possibility In wew of the effects of the substrate for thlol activation (C Finally, can this mformaUon on the exhaustive chemical reductmn on the Moxham, E M Ross, S T George, H effects of treatment with thlol comhgand binding capability of the recepJ Brandwem and C C Malbon, unpubpounds on the apparent structure and hshed) These studies provide addmonal tor 5, ,t is hkely that the 'activated' form function of I]-adrenergtc receptor be evidence that cleavage of disulfide of the receptor may be analogous to utilized to develop a first-approximation receptor that has been only partially bridges by th,ols generates a species of model for the orgamzation of this recepreceptor capable of activating G~ in a reduced or undergone disulfide bndge tor in the lipid bilayer ° Just such a model rearrangement Only detailed structural manner essentially mndlstingmshable from the agomst-bound form of the analysis of both the agomst- and thiol- is'proposed in Fig 2 This model was designed only to emphasize one possible receptor Agomst binding, like treat- actwated species of the receptor wdl orgamzation of the transmembraneanswer this question ment wffh thmolcompounds, may promPhotoaffinRy labeling studies with spanning reglc,ns predicted from the ote disulfide exchange reactions I~and/or cleavage of intramolecular disulfide [12Sl]iodoazldobenzylplndolol and mem- molecular cloning data 13.[4 Although bndges and transform the receptor into branes prepared from $49 mouse lym- other arrangements of the transmema structurally-altered state capable of phoma void-type cells revealed labeling brane-spannmg regions are equally tenactivating G s The "activated" species of of both M r 55 000 and 65 000 peptides 21 able, we offer the rendition shown in this the receptor may act also as the substrate When the photolabehng was performed figure on the basis of several considerafor receptor sequestration or desensitiza- w~th membranes from cells hrst incu- tions The symmetry of this model is tion processes which have been observed bated wRh Isoproterenol ('desensmzed'), obvious TMSRs 2, 3, 6 and 7 which each the predominant labeling was observed possess cystemyl residues are organized after agomst bmndmgz°
Role in r e c e p t o r
,, )J
s"%
"-b,
0-----3
175
T I B S 1 2 - M a y 1987
in close proximity O n e would expect that disulfide bridges within the hpld bllayer would be r a t h e r resistant to cleavage by low concentrations of hydrophihc thiol c o m p o u n d s like dithlothreftol Conversion o f the receptor from the M r 55 000 to the 65 000 species does reqmre, in fact. s t n n g e n t conditions vath relatively high concentrations of reductant 5 Thus, we would propose that halfcysttnyl groups of disulfide bridges are hkely to exist within T M S R s 2, 3, 6 and 7 T h e model also highhghts the positioning of T M S R s 4 a n d 5, p r o w d m g their isolation from the T M S R s core via loops 3 - 4 (which are devoid of cysteinyl residues) and 5--6. thus creating a possible 'flexpolnt" In the molecule Cysteinyl residues are relatively abundant m o t h e r cell surface proteins, in addition to the G protein-coupled receptors described above T h e E G F receptor 22, the L D L receptor 2a, the N G F receptor 24, the n e u o n c o g e n e product 25, a n d the D r o s o p h i l a N o t c h protein 26 are good examples In these m e m b r a n e proreins, most of the cysteane is found in cystelne-nch repeats of extracellular de .'auras O n the basis o f the p n m a r y sequence reformation, the model proposed for the m e m b r a n e orgamzatlon of [I-adrenerglc receptors contrasts sharply with that of these o t h e r cell surface protelns The presence of cysteinyl residues m T M S R s and intracellular domains of [I-adrenerg~c receptors as well as the observation that thiols can activate the receptor suggest a possible role for miramolecular disulfide b n d g e s within the lipid bilayer both m maintaining the organization of the receptor in the m e m b r a n e and in participating in the process of agomst-mediated receptor activation T h e future direction of receptor biology will certainly include the analysis of the roles of cysteinyl residues and lntramolecular disulfide bridges m receptor structure and activation Attacking the model presented herein as well as o t h e r speculative models w~th the same wgor that has brought us to this threshold will certamly answer the question of w h e t h e r receptor chsulfides are avenues to receptor activation o r merely a n unavoidable but provocative ' d e a d - e n d ' Acknowledgements This work was s u p p o r t e d by N I H grants DK25410, AM30111, and a C a r e e r D e v e l o p m e n t A w a r d from the N I H to C C M We thank Beth Maynard (University of Massachusetts Medical Center) for t h e illustrations References I Gdman, A G (1984)Ce1136,577-579 2 Cubero A and Malbon, C C (1984)J Bfol
Chem 259, 1344-1350 3 Benowc J L Shorr R G L,Caron M G and Lelko~tz R J (1984)Biochemw~3,~-~, 4510-.4518 4 Graz]ano, M P, Moxham, C P and Malbon C ? (1985)J Btol Chem 260,7665-7674 5 Moxham. C P and Malbon, C C (1985) Biochenustry 24, 6072--6077 6 Moxham, C P George, S T , Graz, ano M P , Brandwem, H and Malbon, C C (1986)J Btol Chem 261, [4562-14570 7 Iwant). V and Hut, K C (1985)Proc Nail Acad Sa USA 82,325-329 8 Gioannlm, T L , Howard, A D . Hdler, J B and Simon E J (1985)J Biol Chem 260 I."~17-15121 9 Leonard, W J , Depper, J M, Kronke, M Robb. R J , Waldmann, T A and Greene W C (1985)J Biol (?hem 260 1872-1880 10 Sorensen, P Farber, N M and Krystal G (1986)J Biol {?hem 261 9094-9097 I I Wimalasena, J Abel, J A . Jr, W]ebe. J P and Chen, T T (1986)J Biol Chem 261 9416-9420 12 Moxham, C P, George, S T Brandwem. H J and Malbon, C C 0986)Fed Proc 45 1569 (Abstr 516) 13 Dixon, R A F, Kobllka B K Sirader, D J , Benovic, J L, Dohlman, H G , Fnelle, T , Bolanoswskl, M A
Bennett. C D .
Rands, E , Dlehl, R E , Mum[ord R A Slater, E E Sigal, 1 S, Caron, M G Lefkow]tz, R J and Stradar, C D (1986) Nature 321,75-79 14 Yarden, Y, Rodnguez, H, Wong. S K-F Brandt, D R , May, D C Burmer J Harlons, R N,Chen E Y, Ramachundran, J , Ullnch, A and R.3ss. E M 0986) Proc Natl Acad Scl USA 83. 679~¢~-6799 15 Kubo, T , Fukuda. K, M]kann, A , Maeda, A , Takahashl, H M]shma, M, Haga, T . Haga K, Icluyama. A , Kangawa. K, Kopma, M. Matsuo H Htrose, T and Numa, S (1986) Nature 323.41 I.-416 16 Hargrave. P A (1982)Prog RetmaIRes I 151
17 Young D Wa]tches G Birchmeler C Fasano O and Wigler M (198b)Cel145 711719 18 Pedersen, S E andRoss E M (1985)J Biol Chem 260 14150.-14157 19 Creighton. T E 0984) Methods End,tool 107, 305-329 20 LefkowKz R J Benovlc, J L Kobdka B and Caron. M G (198b) Trends Pharmacol Scl 7. "!!~. A.!S
21 Rashldbalg], A , Ruoho. A E. Green D A and Clark. R B (1983) Proc NatlAead Sct USA 80, 2849-285~
22 Ullnch. A . Coussens L. Hayihck .I S Dull T J.Gray, A.Tam. A W.Lee J Yarden Y, Laberman, T A , Schlesslnger J Downward J Mayes, E L, Whltlle N Waterfield, M D and Secburg. P H 0984) Nature309 418-425 23 Yamamoto, T , Davis. C G , Brown M S Schneider W J Casey M L Goldstem J L and Russell D W (1984)Ce1139 27-38 24 Johnson. D, Lanahan, A . Buck. C R, Sehgal, A Morgan C, Mercer. E , Bothwell M andChao, M 0986) Cell47 545-554 Bargman. C I , Hung, M-C and Wemberg, R A 0986) Nature319, 226-230 26 Wharton. K A,Johansen K M Xu T and Artavams-Tsakonas.S (1985)Cell43 ~67-581 27 Dratz E A and Hargrave, P A (1984) Trends Btochem Sa 8 128-131 Note added in proof The recent molecular cloning of the human ~zadrenerDc receptor (Kobilka el a/ Proc Nail Acad Sct USA 84, 46-50, 1987) and Ihe porcine cardmc muscanmc acetylchohne receptor (Kubo el al FEBS Left 209, 367-372 1980) reveals strict
consarvallon o[ the c)steinyl residues that are predicted to be posmoned within the lipid balayer These reports provide additional support to our hypothesisthat cystemylresidues play a criticalrole m the membrane orgamzatton of G prolem-hnked receptors and perhaps in the activation of the receplors by asomsI hgand
John Innes Institute Plant Cell Biology Summer School 7-18 September1987 This course of pract]cals a n d lectures for advanced research workers vail cover the isolation a n d characterization of macromolecules, in sire hybridization. protoplast techniques, the subcellular localization of antigens by tmmunofluorescence and immunogold methods, electron microscopy including cryotechmques, deep etching, negative staining and image analysis T h e C o u r s e O r g a m s e r s are K Roberts, D R Hills, N Brewfn and A Maule I n v i t e d Lecturers include D H Northcote, A Selvendran, D Bowles. G Galffe, J Beezley, and M A k a m T h e C o u r s e Fee, wluch will include full board 2-week course fee, IS £500 Enrollment 15 places
Davies, C W Lloyd, G J Trewavas, H Dickinson, R J Ellis, R Perham, P Shaw, and accommodation and the
F u r t h e r Information and apphcatlon forms from Dr K Roberts, Dept of Cell Biology, J o h n Innes Institute, Colney Lane, Norwich, NR4 7UH (Deadline for a p p h c a u o n l J u n e 1987) (This school is m o u n t e d vath support from the C o m p a n y of Biologists and from the Nuffield Foundation )
TIBS 12 - May 1987
Open Question Intramolecular disulfide bridges: avenues to receptor activation? Craig C. Malbon, Shaji T. George and Cary P. Moxharn Evidence suggests that many cell surface receptors that are coupled to their effector systems via G proteins possess mtramolecular disulfide bridges At least m the case o f the [I. adrenergw receptors, cleavage o f disulfides by thlol compounds appears to act,ate the receptor m a manner slmdar to agontst bmdmg A role ts proposed for mtrcanolecular &sulfide bndges and suifhvdD,ls m the structure and acmanon o f G protem-coupled ret eptors
The ability of cells to transduce mformalion across b~ologlcal membranes has captivated the interest of researchers from a wide range of disciplines A vanely of strategies has been employed to tackle the problem of understanding how cells manage to respond to extracellular signals and propagate this reformation across the cell membrane to the intracellular compartment Both for visual excitation and for many hormones that bind to cell surface receptors, the basic composition of the membrane elements involved in signal transduction includes a receptor (or photoplgment). an cffector molecule that generates the intracellular second messenger, and a guanine nucleottde binding (G) protein that propagates the signal from the receptor to the effector molecule(s) I Each of these elements is btfuncttonal, each must be capable of receiving input and then communicating the signal to the next element (protem) in the system as output Ligand recognmon (or photoexcitation) and signal propagation to a G protein are functions of the receptor Thus recognmon of the input signal must "activate' the recemor,~'he signal is to be propagated to the G pr3tem Although our knowledge of the hgand binding properties of many cell surface hormone receptors is impressive, little ~s known about what constitutes the functional domains t,f the receptor and even less is C C Malbon. S T George mrd C P Moxham are members of the Dtabete~ and Metabohsm Research Group and are at the Department o] Pharmacologt cal Scwnces State Umversav of New York at Stony Brook. Stony Brook, NY 11794--8651 USA, C P M t~ currently at the Department of Molecular Biology The Welltome Research Laboratories Research Trmngle Park NC27709 USA It/~TFl~e~ltrPubll~dhon~
C imhntlgL
11"t76-Ch1~7~7/~1121111
known o f the process by which hgand binding yields receptor 'activation' Catecholamme modulauon of intracellular cyclic AMP (cAMP) levels provides a useful model system for analysis of signal transduetion across biological membranes ~-Adrenerglc receptors bind catecholammes and stimulate intracellular accumulation of cAMP by increasing the activity of the effector molecule adenylate cyclase, via the activauon of the G~ proteml Structurally, mammalian I~-adrenergic receptors are single-chat polypeptlde glycoproteins of M~ 64 I)00-67 fl00 in sodium dodecyl sulfate polyacrylamlde gel electrophoresis (SDS-PAGE) 2-~ Successful large-scale purification of these Iow~,bundance membrane proteins provided amounts of receptor required to apply several powerful strategies to the study of receptor structure and function Chemical modification of 13-adrenerglc receptors by group-specihc and site-specific reagents, molecular cloning of these molecules, and analysis of receptor function by defined reconstltution of receptor and G~ in phosphohpld vesicles have been applied to the questions of what constitutes functional domains of the receptor and how does agomst bmdmg 'activate' these receptors Our interest in the contnbutlon of disulfide bridges and free sulfhydryl groups of the receptor to both the snucture and the function of this molecule has been reinforced by recent results obtamed from studies m which these three distinct approaches have been employed in this article we develop the hypothesis that disulfide bndges and free sulfhydDI groups play an integral role m the structure of cell surface receptors that are coupled to G
proteins A role for mtramolecular disulfide bridges and free sulfhydryl groups in the process of receptor activation by agomsts is proposed Role in receptor structure
The effects of agents that chemically modify disulfide bndges and free sulfhydryl groups of proteins on the hormone-sensztzve adenylate cyclase system have been described in numerous reports (see Ref 5) Defining the precise targets of these chemical modifications was a difficult task due not only to the mtnnsm complexity of the individual elements of the system, but also to that of the biological membrane m which the system was probed l'he first structural evidence supporting the presence of intramolecular disulfide bridges in the receptor was the demonstration that chemical reduction of these bridges decreased the electrophoretm mobility of the molecule s In SDS-PAGE, pure 13adrenergic receptor migrates with an M, of 54 000, this changes to 65 000 after chenucal reduction with reagents that cleave disulfide bridges of proteins Possible explanations for this behavior are that the receptor exists m a 'compact' form maintained by intramolecular disulfide bndges under non-reducmg conditlons and that cleavage of these bonds results in an 'unfolding' of the molecule increasing the apparent M r of the molecule s Analym ot partially purified receptor preparations replete with other membrane proteins clearly suggested that the mobility shift observed for the receptor after treatment with reagents that cleave disulfide bonds was not a property observed for most of the other membrane protems ~ But is this behavior unique to 13adrenergm receptors 'J Do other membrane receptor proteins display an increased M r when sublected to SDSPAGE under reducing as compared to non-reducmg condmons '~ If the answer Is yes, what ff anything, do these proteins have m common '~ The list of proteins that are reported to display a mobility shift after reduction mcludes, in addition to the mammalian 131and ~z-adrenergm receptors s 6, the hepatic glucagon receptor 7, the opiate receptor purified from bovine strtatum 8, the receptors for interleukm-2 ~ and interleukm-31", and the LH/hCG receptorit Chemical reduction and alkylation of the photopigment
TIBS 12-May
173
1987
rhodopsln and the turkey 13]-adrenerglc receptor also i n c r e a s e s the M r of these proteins m S D S - P A G E (C Moxham, E M Ross and C C Malbon, unpubhshed) Most, if not all, of these cell surface receptors appear to share the common feature of bemg coupled to their effector molecules via G protems Immunological analysis of the [~adrenerglc receptor suggests that the molecule IS the M r 55 000 rather than the 65 000 species m s u u t 2 Thus, the 55 000 species is not formed as a result of the solubfl~zatlon of the receptor from the membrane ~2 Furthermore, structural evidence has been provided suggesting that the integnty of intramolecular d~sulfide bndge(s) is essentml for hgand binding 5 Chemtcal cleavage of these bonds destabilizes the ablhty of the receptor to b i d radlohgands ~ These data suggest that mtramolecular disulfide bridges may play a critical role in the structure of G protein-coupled receptors Molecular clonmg of the hamster lung l~2-adrenergtc receptor ~ as well as the turkey erythrocyte I~radrenerg~c receptor ~4 has been reported recently The mammalian [12-adrenerg~e receptor possesses 15 cystemyi restdues (cysteme content = 3 5 mol % ) and the avmn ~ receptor contains 17 (cy~eme content = 3 6 mol %) The proposed onentatlon of the mammalian [~-adrenergm receptor with respect to the cell membrane ~s dis-
EXTRACELLULAR
played in Fng 1 F o u r of the cysteLnyl residues are predicted to be w]thLn transmembrane-spanning regmns (TMSR) of the protenn, and two additional cysteunyl resndues have been assngned at or near the hpud bnlayer/aqueous interphase The remannnng nine cystennyl residues are predicted to be nn regions outsnde of the lnptd bulayer three mn the extracellular loops, one mn cytoplasmic loop 5-6, and five mn the C-terminal portion of the molecule Companson of pnmary sequences of the avian and the mammahan I~-receptors reveals smct conservation (mdentmty) of nine cystemne residues, includmg those predicted to lie within the lipid bflayer (Table I) Primary sequence information ts avamlable for the muscanmc acetylchohne receptor I~ and bovmne opsm t6, two cell surface receptors that are also coupled to G proteins ) Smmllantles among the muscanmc receptor and [3-adrenerglc receptors are stnkmng TMSRs I and 4 as well as loops I-2 and 3-4 are devoid of c)stemyl residues, TMSRs 2 and 7 contain at least one cystemyl residue, and the Ctermini contam two to six res,dues All three receptors lack cystemnyl residues in the N-terrnlnus, T M S R i, and loop 1-2 (Table I), but opsm displays cystelnyl residues mnTMSRs 4, but not 7, a feature not observed in the fl-adrenerglc or muscannle receptors Elucidation of the membrane elements w t h whmch other
Table l Predwted asslgmnou of c~ ~temt/ resMues m t ell surfaca membrane receptor~
Receptor ' Regmn N-terminal TMSR I
Loop I-2 TMSR2 Loop 2-3 'l%lSn 3 Loop 3-4 TMSR4 Loop 4-5 TMSR 5 Loop 5-6 TMSR6 Loop6-7 TMSR7 C-termmnal Total Mol % Ref
131-Ar"
p~Ar
mAchr
I
--
--
. .
. .
. .
Ops m --
. .
I 2 ---
I 1 2 ---
l
3
-I
-I I -1 0
-I I -i 'i
I 4 -2 ~ 2
-3
17 3~; 14
15 36 13
15 ~'~ I'i
l0 29 16
I
i
-
I
--
---
I
I i
2 I -I
~Abbrev=atlons used are as Iollows 13rAr 13=adren,~r_'~creceptor of turkey eDthrocyle [~:-Ar [1.- adrenerg~e receptor or ha'-.~ter lung mAchr muscanmc chohnerg~c receptor ol porcme cerebrum Ops m bo~me reunal opsm and TMSR transmembr.ne-sp~,mmgregmn putative membrane receptors xx+thseven predicted TMSRs and far less cysteme content (hke the m a s oncogene product IT) mteract, may assist m the cntlcal review of our hypothesis that cystelnyl re+ldues o f cell surface receptors coupled
,8 2- AdrenergJc receptor
W CYTOPLASMIC
HOOC(
Fig I Pr~p~sed~rten~att~n~f~hemammahan[~-radrenergwrecepl~rLi~threrpeUt~theplasmamembrane Transnlembrane-spanmngreglon~ctreuumber,'dsequen ttally from the N- to C termtm, from omt to se~en (lefl to nghO The asslgnmem of teraarv smlcmre ts arberar) The pnmart sequence n as taken from Daon el al i, based upon a model o f Yarden et al i.Imade by analogy to dw known onentanon o f opsm -'7 The c~stem~l re.dues are m blacA
T I B S 12
-
M a y 1987
174 p
m i m m m l m
/%
to G proteins part~opate i n intramolecular disulfide bridges that are important to receptor structure and function
~ I I
II
N
H
........
.~,-\
2
~ 1
~--~'-.'~
---
.I L - - ~ ' J
t,~ ~
/
.--'"
,,'
'activation' Although receptor 'actwatlon' ~s a term frequently apphed in the literature to the description of receptor sequellae that follow agomst hgand binding, we know little of the molecular details of this m i I #/' , ~1 Ik o o . V _qA-I phenomenon Two advances provide the capability to probe features of receptor activation by agomst directly the ablhty to isolate essentially pure receptors and i m ",,,,',,.,, k \~'~JJJ ! % %~-.J)'J I ~ v . ~ , ~ j G proteins, and the ability to reconstitute membrane orotems into defined, "-..:,-"7 r " T ', . k unmlamellar phosphohpld vesicles Pedersen and Ross used this approach I I ~ ~ e,^~H with ~-adrenerp0c receptor msolated from 1I II t ~ ! , buvn turkey er)throcytes and G~ from rabbit I I ",, ",,,_ . - " / ,"-7 liver, and demonstrated that treatment I \ e e ~ " .... " ~ _s of ~-adre ~erg~c receptor ~ t h dithmo- • ""~'" ~" . . . . . . . . I ....... • s" thremtol or other tfuol agents capable of cleawng disulfide bridges of proteins caused the functional activation of the receptor u Furthermore. treatment with 0 )EXTRACELLULAR dlthlothreltol also markedly potentiated activation of G, (as measured by GTPyS binding) by agomst hgand, enhancing Fig 2 proposed model ofthe [badrenerglcreceptoras seenfrom the extracellularude of the cell membrane hpM the initial rate by nearly tenfold Thlol bdayer Predictedtrammembrane-spanmng regions are depmted as seven cods. numbered m sequence stamng activation of the receptor was sho~m to from dle N termmus C) stemyl resMuesare mdrcated by '--S' Assfgnmenl of tertmry strucfltre ~ arbgrary occur in the absence of agomst and mnthe presence of antagonist hgands TreatWhether or not the M r 65 000, fully- in the M, 65 000 rather than the 55 000 ment of Gs with thlol compounds was speoes 21 One possible explanation for w~thout effect, defining the receptor pro- reduced form of the receptor m the 'acuthese data Is that in sgu, prolonged expovated' species is an open question. On tern itself as the target of the functional sure of receptors to agomst sumulatlon the basis of the membrane organization activ.qmon by thmolsI~ results m a disulfide-exchange reaction 19 of the j~-adrener ,,c receptor proposed in Recent studies in which mammalian that favours the Mr 65 000 receptor F,g, 2, it Is possible that there are more rather than avian D-adrenergmcreceptors species This hypothesis, though were reconstituted with G s in defined than one intramolecular disulfide bndge speculative tn nature, Is consistent with m the molecule The observation that hposomes demonstrate that mammahan the data discussed above and deserves D-receptors are functionally activated by treating these receptors vmh ,ncreasmg cntlcal evaluation concentrations of reductant yields receptreatment voth thlol compounds, and tor species ~ t h intermediate Mr to the that it is the M r 55 00~ receptor species Disulfides and membrane organization of that binds agomst hgand as well as acts as 55 000 and 65 000 formss, supports this receptors possibility In wew of the effects of the substrate for thlol activation (C Finally, can this mformaUon on the exhaustive chemical reductmn on the Moxham, E M Ross, S T George, H effects of treatment with thlol comhgand binding capability of the recepJ Brandwem and C C Malbon, unpubpounds on the apparent structure and hshed) These studies provide addmonal tor 5, ,t is hkely that the 'activated' form function of I]-adrenergtc receptor be evidence that cleavage of disulfide of the receptor may be analogous to utilized to develop a first-approximation receptor that has been only partially bridges by th,ols generates a species of model for the orgamzation of this recepreceptor capable of activating G~ in a reduced or undergone disulfide bndge tor in the lipid bilayer ° Just such a model rearrangement Only detailed structural manner essentially mndlstingmshable from the agomst-bound form of the analysis of both the agomst- and thiol- is'proposed in Fig 2 This model was designed only to emphasize one possible receptor Agomst binding, like treat- actwated species of the receptor wdl orgamzation of the transmembraneanswer this question ment wffh thmolcompounds, may promPhotoaffinRy labeling studies with spanning reglc,ns predicted from the ote disulfide exchange reactions I~and/or cleavage of intramolecular disulfide [12Sl]iodoazldobenzylplndolol and mem- molecular cloning data 13.[4 Although bndges and transform the receptor into branes prepared from $49 mouse lym- other arrangements of the transmema structurally-altered state capable of phoma void-type cells revealed labeling brane-spannmg regions are equally tenactivating G s The "activated" species of of both M r 55 000 and 65 000 peptides 21 able, we offer the rendition shown in this the receptor may act also as the substrate When the photolabehng was performed figure on the basis of several considerafor receptor sequestration or desensitiza- w~th membranes from cells hrst incu- tions The symmetry of this model is tion processes which have been observed bated wRh Isoproterenol ('desensmzed'), obvious TMSRs 2, 3, 6 and 7 which each the predominant labeling was observed possess cystemyl residues are organized after agomst bmndmgz°
Role in r e c e p t o r
,, )J
s"%
"-b,
0-----3
175
T I B S 1 2 - M a y 1987
in close proximity O n e would expect that disulfide bridges within the hpld bllayer would be r a t h e r resistant to cleavage by low concentrations of hydrophihc thiol c o m p o u n d s like dithlothreftol Conversion o f the receptor from the M r 55 000 to the 65 000 species does reqmre, in fact. s t n n g e n t conditions vath relatively high concentrations of reductant 5 Thus, we would propose that halfcysttnyl groups of disulfide bridges are hkely to exist within T M S R s 2, 3, 6 and 7 T h e model also highhghts the positioning of T M S R s 4 a n d 5, p r o w d m g their isolation from the T M S R s core via loops 3 - 4 (which are devoid of cysteinyl residues) and 5--6. thus creating a possible 'flexpolnt" In the molecule Cysteinyl residues are relatively abundant m o t h e r cell surface proteins, in addition to the G protein-coupled receptors described above T h e E G F receptor 22, the L D L receptor 2a, the N G F receptor 24, the n e u o n c o g e n e product 25, a n d the D r o s o p h i l a N o t c h protein 26 are good examples In these m e m b r a n e proreins, most of the cysteane is found in cystelne-nch repeats of extracellular de .'auras O n the basis o f the p n m a r y sequence reformation, the model proposed for the m e m b r a n e orgamzatlon of [I-adrenerglc receptors contrasts sharply with that of these o t h e r cell surface protelns The presence of cysteinyl residues m T M S R s and intracellular domains of [I-adrenerg~c receptors as well as the observation that thiols can activate the receptor suggest a possible role for miramolecular disulfide b n d g e s within the lipid bilayer both m maintaining the organization of the receptor in the m e m b r a n e and in participating in the process of agomst-mediated receptor activation T h e future direction of receptor biology will certainly include the analysis of the roles of cysteinyl residues and lntramolecular disulfide bridges m receptor structure and activation Attacking the model presented herein as well as o t h e r speculative models w~th the same wgor that has brought us to this threshold will certamly answer the question of w h e t h e r receptor chsulfides are avenues to receptor activation o r merely a n unavoidable but provocative ' d e a d - e n d ' Acknowledgements This work was s u p p o r t e d by N I H grants DK25410, AM30111, and a C a r e e r D e v e l o p m e n t A w a r d from the N I H to C C M We thank Beth Maynard (University of Massachusetts Medical Center) for t h e illustrations References I Gdman, A G (1984)Ce1136,577-579 2 Cubero A and Malbon, C C (1984)J Bfol
Chem 259, 1344-1350 3 Benowc J L Shorr R G L,Caron M G and Lelko~tz R J (1984)Biochemw~3,~-~, 4510-.4518 4 Graz]ano, M P, Moxham, C P and Malbon C ? (1985)J Btol Chem 260,7665-7674 5 Moxham. C P and Malbon, C C (1985) Biochenustry 24, 6072--6077 6 Moxham, C P George, S T , Graz, ano M P , Brandwem, H and Malbon, C C (1986)J Btol Chem 261, [4562-14570 7 Iwant). V and Hut, K C (1985)Proc Nail Acad Sa USA 82,325-329 8 Gioannlm, T L , Howard, A D . Hdler, J B and Simon E J (1985)J Biol Chem 260 I."~17-15121 9 Leonard, W J , Depper, J M, Kronke, M Robb. R J , Waldmann, T A and Greene W C (1985)J Biol (?hem 260 1872-1880 10 Sorensen, P Farber, N M and Krystal G (1986)J Biol {?hem 261 9094-9097 I I Wimalasena, J Abel, J A . Jr, W]ebe. J P and Chen, T T (1986)J Biol Chem 261 9416-9420 12 Moxham, C P, George, S T Brandwem. H J and Malbon, C C 0986)Fed Proc 45 1569 (Abstr 516) 13 Dixon, R A F, Kobllka B K Sirader, D J , Benovic, J L, Dohlman, H G , Fnelle, T , Bolanoswskl, M A
Bennett. C D .
Rands, E , Dlehl, R E , Mum[ord R A Slater, E E Sigal, 1 S, Caron, M G Lefkow]tz, R J and Stradar, C D (1986) Nature 321,75-79 14 Yarden, Y, Rodnguez, H, Wong. S K-F Brandt, D R , May, D C Burmer J Harlons, R N,Chen E Y, Ramachundran, J , Ullnch, A and R.3ss. E M 0986) Proc Natl Acad Scl USA 83. 679~¢~-6799 15 Kubo, T , Fukuda. K, M]kann, A , Maeda, A , Takahashl, H M]shma, M, Haga, T . Haga K, Icluyama. A , Kangawa. K, Kopma, M. Matsuo H Htrose, T and Numa, S (1986) Nature 323.41 I.-416 16 Hargrave. P A (1982)Prog RetmaIRes I 151
17 Young D Wa]tches G Birchmeler C Fasano O and Wigler M (198b)Cel145 711719 18 Pedersen, S E andRoss E M (1985)J Biol Chem 260 14150.-14157 19 Creighton. T E 0984) Methods End,tool 107, 305-329 20 LefkowKz R J Benovlc, J L Kobdka B and Caron. M G (198b) Trends Pharmacol Scl 7. "!!~. A.!S
21 Rashldbalg], A , Ruoho. A E. Green D A and Clark. R B (1983) Proc NatlAead Sct USA 80, 2849-285~
22 Ullnch. A . Coussens L. Hayihck .I S Dull T J.Gray, A.Tam. A W.Lee J Yarden Y, Laberman, T A , Schlesslnger J Downward J Mayes, E L, Whltlle N Waterfield, M D and Secburg. P H 0984) Nature309 418-425 23 Yamamoto, T , Davis. C G , Brown M S Schneider W J Casey M L Goldstem J L and Russell D W (1984)Ce1139 27-38 24 Johnson. D, Lanahan, A . Buck. C R, Sehgal, A Morgan C, Mercer. E , Bothwell M andChao, M 0986) Cell47 545-554 Bargman. C I , Hung, M-C and Wemberg, R A 0986) Nature319, 226-230 26 Wharton. K A,Johansen K M Xu T and Artavams-Tsakonas.S (1985)Cell43 ~67-581 27 Dratz E A and Hargrave, P A (1984) Trends Btochem Sa 8 128-131 Note added in proof The recent molecular cloning of the human ~zadrenerDc receptor (Kobilka el a/ Proc Nail Acad Sct USA 84, 46-50, 1987) and Ihe porcine cardmc muscanmc acetylchohne receptor (Kubo el al FEBS Left 209, 367-372 1980) reveals strict
consarvallon o[ the c)steinyl residues that are predicted to be posmoned within the lipid balayer These reports provide additional support to our hypothesisthat cystemylresidues play a criticalrole m the membrane orgamzatton of G prolem-hnked receptors and perhaps in the activation of the receplors by asomsI hgand
John Innes Institute Plant Cell Biology Summer School 7-18 September1987 This course of pract]cals a n d lectures for advanced research workers vail cover the isolation a n d characterization of macromolecules, in sire hybridization. protoplast techniques, the subcellular localization of antigens by tmmunofluorescence and immunogold methods, electron microscopy including cryotechmques, deep etching, negative staining and image analysis T h e C o u r s e O r g a m s e r s are K Roberts, D R Hills, N Brewfn and A Maule I n v i t e d Lecturers include D H Northcote, A Selvendran, D Bowles. G Galffe, J Beezley, and M A k a m T h e C o u r s e Fee, wluch will include full board 2-week course fee, IS £500 Enrollment 15 places
Davies, C W Lloyd, G J Trewavas, H Dickinson, R J Ellis, R Perham, P Shaw, and accommodation and the
F u r t h e r Information and apphcatlon forms from Dr K Roberts, Dept of Cell Biology, J o h n Innes Institute, Colney Lane, Norwich, NR4 7UH (Deadline for a p p h c a u o n l J u n e 1987) (This school is m o u n t e d vath support from the C o m p a n y of Biologists and from the Nuffield Foundation )