Guanine nucleotides stimulate car☐yl methylation of kidney cytosolic proteins

Biochimica ct Biopl5"sica Acta. 1136(1992) 151)-154 z: 1992_Elsexier Science Publishers B.V. All rights rcscr.'¢d 0167-4889/92/S05.00

1.50

Guanine nucleotides stimulate carboxyl methylation of kidney

cytosolic proteins Denis Gingras and Richard

B61iveau

Lahoratoire de Membranologic .ttoldculaire. I ~ r t t T n e n t dr" chinue-biochimie. Unit c~iL.: du Qta~occ A ,*.fon/r~a/Momr~al. Q u ~ c ICanada~ and Groupe de Recht'rche en Transport Mt~zbranaire. Unire~ti de Montreal. Montrial Quf,bec ¢Cana~a)

(Recei~.cd4 [X-cember 1991) (Revis4.'dmanuscript reoeixetl20 March Iq92_1

K~y words: Protein cztrbo:q;tnlcll'q,'lllalasferas¢:Guanine nudeotid¢

We studied the effect of guanine nucleotides on the carbo~l methyla:ion catabzed by class ii protein carbox~/lmethyltransferases tPCMT). Additioo of guanosine 5°-O4y-thiohriphosphate (GTPyS) promoted a time- and concentration-dependent enhancemcnt of protein mctbylatio.~ ia tnc cytosolic fraction isolated from kidney cortex. GTPyS affected the kinetics of the methylation reaction, as reflected by alterations of both apparent K~ and V ~ of the metl~'ltransfetase. This effect was specific for guanine nucleotides and was completely abolished by addition of S-adenosyl-t_-homocysteine, a well-known inh~itor of methyltransferasc--catalyzed reactions. No GTP~,S stimulation of melh~,°.~t~ was found in q,losolic extracts from any of the other tissues studied, including brain, testis, spleen, and liver, nor in brush-border membranes isolated from the kidney cortex. The metbylated proteins were highly sensitive to moderately alkaline conditions, suggesting that the methyl esters were formed on t.-isoaspartyl residues and thus mcthylated by a cla.gs II PCMT. Th.csc results suggest that class-II-associated protein mcthylation activity from the soluble iraction of the kidney can be regulated by g,,anine nuclcotid,~.

h droduO.iou Protein carboxylmethyhransferases (PCMT) represent a broad group of enzymes that transfer a methyl group from S-adenosyI-L-methionine to a carboxyl group in proteins [1]. In eucar~'otes, two major classes of PCMT have been studied to date. C!ass II PCMT (E.C. 2.1.1.77) methylate a wide variety of proteins and peptides to form extremely base-labile atethyl esters and possess a high :eactivity towards aspartic residues covalently altered by isomerization reactions [2.3]. These unusual properties have led to the hypothesis that these methyltransferases may be involved in the metabolism of d a m a g e d proteins [4.5]. Another class of ir, ethyltransferases (class l i d catalyze the metbylation Cortespondenc~ to: R. I~liveau. D~partement de chimie-biochimie. Uni','ersit6 dy Qu6bec ~ MontrdaL C.P. 8888. Succursalc "A". Mow tribal. Out,bee IDC 3P8. Canada. Abbreviations: AdoMet. S-adenos¥l-i.-mcthionine: AdoHcy. Sadcnosyl-t-homo~'st¢in~: ApplNHh~.adenyl-5'-yl imidodiphnsphatc; BBM. brush-border mcrqbrant:s; G-protein. guanine-nucleotidebinding prolein; GTPyS. guanosin¢ 5"-O4y-thioRriphosphate: GDP~OS. guannsine 5°-O4/~-thiokliphosphate: PCMT. protein carbox~'lmethyltransfe~ase; 16-BAC. benz~'ldimethyl-n-hexadeq,'lammonium chloride.

of the a-cart~'~.~.jl group of C-terminal residues located in a consens::s sequence ( C A A X ) of yeast and mammalian pro:~.ins. The pro!ein substrates of this class include t~c small GTP-binding protein ras [6-8], the nuc:-_ar |amin B [9], the -~- subonit of some trimeric G - p r m e i n s [10L and c G M P phnsphodieslerase [11]. The data obtained so far with these substrates have suggested that metbylation occurs after isoprenylation of a cysteine residue 4 amino acids from the C-terminns and proteolytic removal of the last 3 amino acids [ 12,13]. The nature of the a-carboxyl-terminal-metbylated proteins and the fact that this methylation is in some cases enhanced by guanine nucleotides [14,15], suggest an involvement of this methylation reaction in hormonal transduction. However, no studies have focnsed so far on a similar regulatory role of d a s s - l l catalyzed protein metbylation. In the kidney, the cells lining the proximal tubule are the site of reabsorption of important metabolites filtered by the glomerolns. These cells are characterized by a high degree of asymmetry with segregation of sodium-coupled active-transport systems in the brush-border membrane [16], while hormone receptors and bormone-sensitive adenylyl t3'clases are concentrated at the basolateral pole, in close contact with the circulation [17]. W e have recently reported the pres-

encc of two distinct class !1 PCMT in the kidney cortex, a soluble and a brush-border-membrane-associated PCMT [18]. In the present work, we examined the effects of guanine nucieotides on protein methylation occurring in the cytosolic fraction and in brush-border membranes of the kidney cortex. We demonstrate that guanine nucleotides enhance the activity of the soluble class !1 PCMT.

60 ~

,--r---

Materials and Meflmds All experiments were performed on adult SpragueDawley male raLs weighing 300-350 g (Charles River). Tissues were homogenized in 5 vols. of 250 mM sucrose-5 mM Tris-HCI (pH 7.5) using a Potter-Elvehjem tissue grinder. The homogenates were centrifuged at l l 0 0 0 0 x g for 60 min and the resulting supernatants used as cytosols. Brush-border membranes from the kidney cortex were isolated by a magnesium precipitation method [19]. The medium used for metbylation reactions contained, in a final volume of 30/zl : 100 mM Hepes-Tris buffer (pH 7.5), 2 p.M (2/zCi) [methyl-3H]S-adenosyi L-rnethionine (69-73 Ci/mmol, Dupont-New England Nuclear) and 100 p.g of proteins. The reaction mixture was incubated at 3TC for 60 rain or the times indicated in the figures. For GTP-dcpcndent methylation, GTP,/S (10 to 200 p.M) was added to the medium. Total incorporation of radioactivity into protein methyl esters was measured with a methanol vapor diffusion assay [20]. Kinetic parameters were determined by non-linear regression analysis of the data using a computer graphics program (Kaleidagraph). Methylated proteins were separated by 16-BAC acid electrophoresis using 7.5% acrylamide gels followed by fluorograpby with i M sodium salicylat¢, as described previously [18]. Control experiments have previously shown that the total amount of radioactivity present in the acidic gel is in the form of basc-scusith'c methyl esters. No significant radioactivity is detected following SDS-PAGE at pH 8.8, the methyl estcrs being hydrolyzed at this alkaline pH [18]~

10

t~~" ~

8 0

P

~

Incubationtime(min) Fig. I. Time dependence of endogenouscarboxyl mcthylation of kidney c~o.-,olicproteins.Cytosolicproteins(100 pg) were incubated in 100 mM Hepes/Tris(pH 7.5) with 2 pM [3HlAdoMct(2 ~.Ci) in the absence (Q) or presence of 100 pM App[NH.]p (11). GDP~S (o), GTP',/S(o) or GTP-tS+ 500 pM AdoHcy( A). Methylationwas quantified as described elsewhere[20]. Values are means+_S.D,of three experimentsdone in triplicate. Inset. Initial velocitiesof protein carboxyl mcthylation for contrd (1:3) and GTPTS-trcatcd (e) cytosols. (100 pM) also resulted in a stimulation of the endogenous methylation, but to a smaller extent, while App[NH]p, a non-hydrolysable analog of ATP, was without effect (Fig. 1). Both basal and GTPyS-stimulated methylation activities were almost completely abolished by the addition of 500/zM AdoHcy, a wellknown inhibitor of methyltrausferase-catalyzed reactions. The effect of GTPTS was strongly concentration-dependent. As shown in Fig. 2, addition of GTP~/S to the incubation medium resulted in a progressive stimula-

~0o

:~'~

is0.

Results

Time-, concentration-, and AdoMet-dependence GTPyS-stimulated protein methflation

of

Incubation of the soluble fraction isolated from kidney cortex with the radioactive methyl donor [3H]AdoMet resulted in a time-dependent methylation of endogenous proteins (Fig. i). The addition of 100 lzM GTPyS, a non-hydrolysable analog of GTP, increased markedly both the rate and extent of methylation, with an average 3-fold stimulation of the initial rate of incorporation of labelled methyl groups (33 vs. 10 pmol/mg per h) (Fig. i, inset). Addition of GDP/3S

o'

. ~ . , , ~. 50

i00

. , tso

2oo

Nucleotides (I.tM) Fig Z Concentration depcndcocc and specificity of nuclontides on carboxyl methylation ol kidney-corlex cylosolie proteins. Cytosolic proteins (I00 p.g) were incubated in I00 mM Hepes/Tris (~'H 7.5) with 2 p M [~H]AdoMet (2 pCi) in the presence of increasing concentrations of App[Nll]p ([3), GDP~S (o) or GTPTS (o) for 60 rain at 37*(::. Control value refers to methyhtion measured in the absence of nucleotides (9.8 pmol/mg per h). Means _+S.D. of three experiments done in triplicate are shown.

152 TABLE I Effect of GIPyS on rite etulogou,ui u#uhle PC.'tfT activity of t~ti~nL~ tusues

.

i

Preparatton of the soluble e~lraCts rfld determination of I ~ , l T act~.'ilywere I~rformed as ~k'-~'ribet:in "Materials and Methods'. A reprc~cnZatk¢ cxperlmenl :~ilh lrip.t-~:alcdeterminations is sh~rwn.

20b 150 100

Seluble cslracl

O

I~.?,fF a,_-lk-il) Ipmol/mg per h~

control

5O 0

. 0

5

10

15 20 AdoMet (pM)

25

,

30

Fig. 3. Effca of GTI'?S on the AdoMet dependence ot kidno'
tion o f the e n d o g e n o u s p r o t e i n methylation acth,ity. which r e a c h e d a m a x i m u m a r o u n d 150 p M G T P y S . H e r e a g a i n . G D P ~ S was m u c h less effective, with a maximal stimulation half o f t h a t p r o d u c e d by GTP3,S, while A p p [ N H ] p w a s ineffective. T h u s . g u a n i n e nucleotides, but not a d e n i n e nucleotides, specifically enh a n c c J the m e t h y l a t i o n o f e n d o g e n o u s soluble p r o t e i n s f r o m k i d n e y cortex. T h e stimulation o f the soluble protein m c t h y l a t i o n by G T P - y S w a s also studied at different c o n c e n t r a t i o n s o f A d o M e t (Fig. 3). T h e p r e s e n c e of G T P T S e n h a n c e d b o t h the maximal velocity o f the reaction (267 vs. 156 p m o l / m g p e r h) a n d the a p p a r e n t K , , o f the P C M T for A d o M e t (4 vs. 1 5 / t M ) , as d e t e r m i n e d by n o n - l i n e a r regression analysis a s s u m i n g single-site MichaelisM e n t e n kiEetics. It t h u s a p p e a r s that the a d d i t i o n o f G T P 3 , S to the kidney cytosol p r o m o t e s a n increase in the velocity o f the carboxyl m c t h y l a t i o n catalyzed by the P C M T .

Effect o f G T P T S o,~ endogenous protein tnethylation o f i'a~o~G rat tLr$:zes W e next e x a m i n e d the possible presence o f this GTP3,S-stimt:la:ed soluble carboxyl methylation activity in o t h e r t i ~ u e s . A s s h o w n in T a b l e i, a d d i t i o n o f G T P y S to the incubation m e d i u m resulted in a 1.8a n d 2.8-fold stimulation o f e n d o g e n o u s p r o t e i n m e t h ylation in whole kidney a n d kidney cortex cytosols, respectively. However, G T P y S w a s not s t i m u l a t o r y in the cytosol o f a n y o f the o t h e r lissues tested (Table 1). T h e i n c o r p o r a t i o n o f radioactivity into specific protein s u b s t r a t e s w a s also d e t e r m i n e d a f t e r s e p a r a t i o n o f the p r o t e i n s f r o m various eytosolic extracts by acid gel electrophoresis. A s s h o w n in Fig. 4. the addition o f G T P T S greatly e n h a n c e d the labelling o f v, hole-kidney

"*Vholekidne3Kidne3-eerier Brain .t|eaEl 5~¢en

t~cr Inlestine Testes

+ tOO#M GTPyS ~_2.5+ 42 --~.t "_.-4.t 39.3_+.3.7 fi.S_+l.t t 12_+ t.5 ~_.7_+0.5

t2.6+~0 10.5+_1.5 3~.3-_ 2.5 6~ +0.9 12.J _~1.9 ?~-+03 8.6-+0.4 ?,5.9+ 1.fi

8.8_+0.3

24.4 -+4,5

x-fold stimulation t.8 2.8 l.I t.O 0.9 t.| 1.0 0.o4

a n d kidney-cortex oitosolie p r o t e i n s (lanes 9 - 1 2 L but w a s without effect in b,-ain, h e a r t , liver, a n d spleen (lanes ! - 8 ) . A n u m b e r o f methyl a c c e p t i n g p r o t e i n s w e r e particularly evident in the cortex cytosol (lane 12), w h e r e at least five distinct p o l y p c p t i d e s :','ere me*.hylated in a G T P 3 , S M e p e n d e n t m a n n e r . W e have previously s h o w n t h a t t h e radiolabelling o f the k i d n e y cytosolic p r o t e i n s w a s completely abolishcd, except for a 2 1 - k D a protein, w h e n s t a n d a r d alkaline S D S - P A G E was u s e d for s e p a r a t i o n [18]. T h e hydrolysis o f these methyl e s t e r s at m o d e r a t e l y alkaline p H u s e d in SDS-

'1 2

3

4-5

•~ ~

qmIllP-'-~.

-6

7

a

9

IOTI

: l / l l l l k

112

1~

-6IG

_2,

-i,¢ Fig. 4. Elf.ca of GTP?S on the methylation of the mctl~l-acccpting proteins in the C)lOSOlsof different rat IL.%~Ues.T'he ~l~,~Is(100pg protcim,) of brain (I. 2). hcarl (3. 4). Iher (5. 6). spleen (7. 8). whole kidney (9. t0) and kidney codex (I I. 12) were incubated for 60 rain al 3TC ",~ith ~i ~ M ['H]AdoMel (5 pCi) in lint mM Hepes~Fris buffer (pit 7.5) ~,~itl, (lanes 2.. 4. 6. 8. 10. 12) or without (lanes i, 3. 5. 7. 9. I I ) 100 ~ M G'TP.yS. Mctl~,:latcd proteins ~ere fraclionatc:d by tfi-BAC gel eteclzophoresis at pH "~0. to a~x~l bydmh'sis of the

ba~:-Ia~':,lc methyl estcr~ and the incorporated radioactivity was detected by flumography. Molecular-weight rnarkcm arc pho~phoO""la-~ h (97 kDa), bovine serum albumin (66 kDa). o~'albumin (45 kDa). carbonic anh)drasc (31 kDa), soybean-t~qy~in inhibitor (21 kDa) and b~o~'mc ( 14.4 kDaL

153

GTP~S

Cytosol _ +

-

BBM +

~'~

~

-97

-31

--21

I:i~ 5. Efi'cctof GTP~,Son the metl~'lationof kidn¢~cones c~aosolic enc BBM proteins. Cslosolic and BBM proteins (100 #g) were incubateJ for 60 mm at 37"-C*ith 5/~M I~HJAdoMcI(5 ~aCi)in 1O0 mM Hc~°s/l"r/sbuffer(pit 7.5) with or vdthout I00 #M GTP~,S. Mclh~lated proteins~'¢r¢ fraclionated 1~' I6-BACgel elcctrophorcsis at pH ZO and det~qed by fluorogmphy,as described in the legend to Fig.4.

PAGE is thus consistent with the formation of L-isoaspartyl methyl esters 121. We have previously shown that the kidney cortex also contains a brnsh-border-membrane-associated PCMT, distinct from the soluble PCMT [18]. The endogenous substrates are different for both enzymes (Fig. 5). In addition, GTPyS had no effect on the methylation of the membrane-bound metbyl-accepting proteins by the membrane-bound PCMT (Fig. 5). Discussion In this study, we have examined the modulation by guanine nuclcotides of class il PCMT activity located in the kidney cortex. Our results show that the addition of GTP'yS, an activating ligand of GTP-binding proteins, causes a marked enhancement of class-ll-associated protein carboxyl methylation in the renal cytosol. This phenomenon is time-, concentration- and AdoMet--dependent, can be inhibited by AdoHey and is specific for guanine nucleotides. GDP,OS was also stimulatory, but to a lesser extent. Although this result may be surprising, GDP/3S has also been shown to increase the metbylation of low-molecular-weight Gproteins by another class of methyltransferase (class-! I I

PCMT) [14,15]. However, the guanine-nucleotide-dependent mcthylat.ion of these proteins results in the formation of base-stable C-terminal methyl esters [14,15]. These results differ significantly from ours. since we have previously shown that the kidney-cortexsoluble PCMT belongs to another class of methyltransferases (class il), based on the alkaline-lability of the metbylated cytosolic proteins on SDS-PAGE [18]. a widely used criterion to distinguish th:: class-ll-catalyzed, base-labile protein methyl esters, from class-illassociated base-resistant C-terminal methyl esters [6]. It thus appears that the GTPyS dependence of protein metbylation described here is related to class-ll PCMT activity and is distinct from that observed for the methylation of low-molecular-weight G-proteins. An intriguing feature of the cymsolic GTPyS-dependent protein methylation described here is its apparent predominance in the kidney, particularl7 in the kidney cortex. Cytosolic extracts from tissues such as brain and testes, which possess a strong dass-ll PCMT activity, were not affected by the presence of guanine nucleotides. Interestingly, the BBM-associated endogenous PCMT activity previously reported [18] remained unaffected by GTPyS, suggesting a compartmentalization of this modulation within tht: same cell. It is not clear at this point whether this restriction of GTPySstimulated class ll PCMT to the kidney cytosol is related to the presence of a PCMT with distinct properties, to a specific GTP-binding protein, or to specific endogenous substrates. The nature of the interaction of GTPyS with the carbo~,l methylation pathway described here is unclear, it is unlikely that GTPyS interacts directly with the enzyme since PCMT sequenced so far possess no consensus sequence for a GTP-binding site [21]. Interaction of GTPyS with the substrates is also unlikely since it would require that each endogenous substrate of the PCMT be GTP-binding proteins. Non-hydrolysable analogs of GTP, such as GTPyS, interact with high affinity with GTP-binding sites and are widely used to assess the involvement of a GTP-binding protein in the modulation of enzymatic activity [22]. Two major groups of G-proteins have been described to date: high-molecular-weight trimeric G-proteins involved in receptor-eflector coupling, and low-molecular-weight G-proteins such as ras, for which no clear function is known yet. it is not clear at this point which group may he involved in the stimulation of the cytosolie methylation activity reported here. Both groups of G-proteins are assumed t.o be active only in a membrane-associated form [22,23]. However, although it is generally assumed that beterotrimeric G-proteins are associated exclusively with plasma membranes [22]. a number of reports have recently described the presence of a subunits in a soluble form, including oc~ [24,9-5] and some substrates of pertussis toxin [26,27].

154 G i v e n t h e u b i q u i t o u s role o f g u a n i n e n u c l e o t i d e s in t h e a c t i v a t i o n o f p r o t e i n s i n v o l v e d in s i g n a l t r a n s d u c lion. i n t e r a c t i o n s bep~'cen carboxyl m c t h y l t r a n s f e r a s e s and such proteins may reflect an important functkn of t h i s m e t h y l a t i o n r e a c t i o n . T h e m e t h y l a t i o n c a t a l y z e d %, c l a s s I! P C M T h a s b e e n a s s o c i a t e d w i t h m c t h y l a t i o n o f a b n o r m a l a s p a r t y l r e s i d u e s a n d w i t h a role in ~he m e t a b o l i s m o f a g e d p r o t e i n s [ I ]. H o w e v e r , t h e m e t h y l a lion of abnormal aspartyl residues may also be consist e n t w i t h z r e g u l a t o r y f u n c t i o n f o r this e n z y m e , s i n c e these residues may provide selective recognition sites f o r t h e P C M T [28}. A n o t h e r possibility w o u l d b e t h a t t h e G T P - y S - d c p e n d e n t carboxyl m e t h y l a t i o n s y s t e m d c s c n ~ c d hcrc r e p r e s e n t s t h e a c t i o n o f a distinct ciags of methylating enzs'mes. Elucidation of the functions of the substratc proteins which undergo enhanced carboxs'l mcth?,'la'ion f o l l m v i n g a c t i v a t i o n o f g u a n i n e n u cl~:otide-binding p r o t e i n s m a y p r o v i d e n e w i n s i g h l s i n t o t h e a s ~'ct u n d e f i n e d role o f p r o t e i n carboxyl m e t h y l a lion in t h e p h y s i o l o g i c a l f u n c t i o n s o f t h e k i d n e y . Admo~led~ments W e w o u l d like t o t h a n k L i s ¢ T r e m b l a y a n d F r a n g o i s Gauthier for di~ussions on G-protein action and Dr. V i n c e n t V a c h o n f o r his critical r e a d i n g o f t h e manuscript. The financial support of the Kidney Found a t i o n o f C a n a d a a n d t h e F o n d a t i o n U Q A M is g r a t e fully a c k n o w l e d g e d .

References I Clarke. S. (lYe;5) Ann. Rev. BhK:hcm 54. 479-5lh~. 2 McFadden, P. and Ch:rkc. S. (1982) Proc. Nail. Acad. Sci. USA 79. 2460-2464. 3 Aswad. D.W. and Johwon. B.A. (19,~7)Trends Biochem. SO. I Z 155-158. 4 LowcrL,,~n. J. and Clarke. S. (19,',k'~)Blood CulLs 14. 11}3-117.

5 Johnson. B~%. Langrnark. E . L and A~,~ad. D.W. (19871J. Biol.

(_'h¢~. Z6"~ 12_~3-1q'~.~7. ,'J Clal'k¢. S- *v'ogc~ J.P_ ~k.",,chcrl¢~ R J and Stock. J. (|¢)~$) Proc. Natl. ,~mi. Sci. USA 2~5.4f~3--16~7. 7 []~'sch~ncs. R_I_ [.LmmcL J.B.. Clarke. S.. Stock. J. and Broach. JR- i1%'9) J. Biol. C"hcm. 264. 111465-111473.

K Gut/,cnrez. L . Magt.~c. A-L .MaP~halL C J . and Hancock, J.F. (1~,9) E.MBO ,I. X. 1093- 11198. 9 Cn=bl,% D_ Ohon. J.F. and K~r,hlamL D.E.. Jr. (1987) ,I. Biol. Chem. 2.b~_ 4303-4309. lip Fang. B.K.-K. Y~mane. It.K_. Ola. I.M. aad (.3arks. S. tIM'D F'EBS Len. 260. 313-317. II Ong. O.C.. Ota. I_M_ C l a ~ . S. and Fung. B.K.-K. 0989) Proc. Nail. Ac~_d. S~..'. USA ge, 9Z~C-924Z 12 HaococL J.F_ Mag¢~. A . L Childs..I.E. and MaP~hall. C J . (198Y) C~.II 57. 1167-11T7. 13 Stcphcnson. R-C. and Oarke. S. (199t)) J. Biol. Chem. 265.

1624/g-262_'g4. 14 Backlund. P~_ Jr. and ~.,amiL R.R. (1988| J. Biol. Chem. 263. 15~6J,- 15~67. 15 lluzoor-tlddrar. Wir.cgar. D.A. and Lapetina. E.G. 0 9 9 | ) J, Biol. C'i'a.~'n. 2t~6. 43.g7-4391. 16 Kinn¢. R_ IItPdO) in B~onnct. F. and Klcinzcllcr. A. teals.), Cunenl Topics in Membram.'s and Tran_spo~, Vol. 13. pp. 303313. 17 Shlalz. L D _ .Schwartz. I.L. Kinn,:-Saffran. E. and Kinnc. R. lieD'S) J. Men'hhrane Biol. 24. 131-1-1-4. 18 Gingras. D_ M6.nard. P. and Bdli,.cau. R. (1~1) B/ochim. Bioph~,s. Ac'la 1066. 261-267. I~' Booth. A.G. and Kcnn~'. A J . 0974) Biochcm. J. 14"P 575-581. ~) Xie. H_ Yamane. H.K.. Sk'phcnsoa. R.C_ Ong. O.C.. lung. B.K.-K. and Clarke. S. (Iqgl}) M¢lhods: A companion to Methods iq Enz~mology I. 27b-2i~Z 21 Ingrosso. D.o Fo,,,dcr. A.V_ Bl¢ibaum.. J. and Clarke. S. tWiCe) J. Biol. (._'hem. 264. ~}131-20139. Gilman. t~LG. (1987) Ann. Ro'. B/o~Lm. 56. 615--649. Willum~cn. B.M_ Norris. K.. FaFagcc, rg::. ,,'LG.. HubL'-crt. N.L and Lt,~-:i. D.R. t I~Pt,.,~)EMBO J. 3. 25~1-2~,~5. 24 Lynch. CJ.. Morbach. L . Blackmor¢. P.F. aad Exton. ,I.H. 0988) FEBS Leu. 200. 333-336. L¢, Milligan~ G. and Ur'.~on. C. 0989) Biocherq ~. ~Ja0.837-841. 26 Nakamura. 1". and UL M tingS) J. B/oL ~ . 260. 3584-3593. 27 Bokoch. G.M_ Bickford. K. and BohL B.P. (Iq~8) J. Cell Biol.

loft,. 19Z/- 1936. As'~ad. D.W. (1984)J. Biol. Chem. ~59. 11)714-1072.1.