Communication Val. 266 No. 8, Issue of March 15, pp. 4673-4676,1991 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Printed in U. S A .

THE JOURNAL OF BIOLOGICAL CHEMISTRY

A Dominant Negative Gas Mutant Is Rescued by Secondary Mutation of the a Chain Amino Terminus*

(Received for publication, October 16, 1990)

Shoji OsawaS and Gary L. Johnson5 From the Division of Basic Sciences, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206 and the Department of Pharmacology, University of Colorado Medical School, Denver, Colorado 80262

The G. protein a subunit, as, stimulates the activity of adenylyl cyclase. The sequence 223Asp-Val-Gly-Gly- GlnZ2’ in the a. polypeptide is predicted to interact with the y-phosphate of GTP and mediate the conforma- tional change involved in a. activation. Mutation of the as polypeptide within this region at GlyZ2‘+Thr had two demonstrative phenotypic effects when expressed in COS-1 cells: the mutant as chain was ineffective in activating adenylyl cyclase and inhibited in a concen- tration-dependent manner the 8-adrenergic receptor stimulation of cAMP synthesis. Thus, the G l ~ ~ ’ ~ + T h r mutation alters the ability of GTP to activate the a. chain and when overexpressed the mutant polypeptide exerts a dominant negative phenotype. Mutation at the amino terminus which creates a constitutively active a. rescued the inhibited state of the GlyZz5-+Thr mutant when both mutations were encoded in the same poly- peptide. This finding defines the amino terminus as a functional regulatory domain controlling the proper- ties of the GTP/GDP binding site of G protein a subunit polypeptide chains.

The functions of G protein a subunits, elongation factors such as elongation factor-Tu and the ras p21 polypeptides are allosterically regulated by the binding of GTP (1, 2). The amino acid sequences forming the GTP/GDP binding site within these polypeptides are highly conserved (3) and crys- tallographic studies have been carried out for elongation fac- tor-Tu (3) and ras p21 (4-6). Among the amino acids that form the GTP/GDP binding domain is the invariant Asp-X2- Gly sequence corresponding to residues 57-60 in ras p21 and 223-226 in as, the G. a chain that activates adenylyl cyclase. The crystal structure of ras p21 indicates that Asp-57 is involved in the coordination of the essential Mg2+ and Gly- 60 functions as a pivot point that alters the conformation of the next 15-17 amino acids within the polypeptide chain that

* The work was supported by National Institutes of Health Grants GM 30324 and DK 37871 and by the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address: Dept. of Cell Biology and Anatomy, University of North Carolina Medical School, Chapel Hill, NC 27599.

I To whom correspondence should be addressed Div. of Basic Sciences, National Jewish Center for Immunology and Respiratory Medicine, 1400 Jackson St., Denver, CO 80206. Tel.: 303-398-1504; Fax: 303-398-1225.

is involved in GTP-induced allosteric activation of the protein (2,5,6). It is proposed that the invariant Asp-XZ-Gly sequence and the following 15-17 amino acids in ras p21 interact with GAP or other effector molecules (2, 5, 6).

The importance of the invariant Asp-X,-Gly region in GTP-binding protein function has led to extensive mutational analysis of ras p21 and a, to define mutants with altered regulatory properties. Analysis of ras p21 and a. mutants has indicated that the respective mutation of Gly-60 or Gly-226 inhibits the activity of both polypeptides because they are unable to pivot and assume the GTP-liganded conformation normally induced by GTP (2, 7, 8). In addition, mutation of the adjacent Gln to Leu (residues 61 and 227 in ras p21 and as, respectively) results in constitutive activation of the poly- peptide because the mutation induces a GTP-stabilized con- formation whose GTPase activity is markedly inhibited (9, 10, 11). In ras p21 mutation of Ala5’-+Thr (A59T) also acti- vates the polypeptide and inhibits its GTPase activity (7, 9). Both ras p21A59T and ras p21Q61L have strong transforming activity in cell transformation assays.

Based on the strong activating character of the Ala5’+Thr mutation in p2lras, it was predicted that the corresponding mutation would inhibit GTPase activity and activate the CY, polypeptide. In this report we demonstrate that the GlyZz5+ Thr (G225T) mutation is actually inhibitory to p-adrenergic receptor-mediated activation of adenylyl cyclase and the a,G225T mutant functions as a dominant negative a, poly- peptide.

MATERIALS AND METHODS

Site-directed mutagenesis was performed as previously described (12). All mutations were verified by DNA sequencing. The different rat G protein a subunit cDNAs in the pCWl expression plasmid were transiently expressed in COS-1 cells using the DEAE-dextran trans- fection procedure (13, 14). Cells were used for cAMP analysis and immunoblots 65 h after transfection. For immunoblotting, 70 pg of membrane protein from transfected COS-1 cells was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% ac- rylamide), transferred to nitrocellulose, and probed with antibodies specific for the carboxyl terminus of either a. or ai (13, 15, 16). For cAMP measurements transfected COS-1 cells in 60-mm Petri dishes were incubated for 10 min at room temperature in the presence of 500 pM methyl isobutylxanthine. Cellular cAMP was then extracted with 2 ml of ice-cold 65% ethanol and lyophilized. Cyclic AMP was then measured using a radioimmunoassay kit from Amersham Corp. according to the manufacturer’s directions (15). We have previously demonstrated (12, 13, 15, 19) in transfected COS-1 cells that the cAMP measurements in the presence of methyl isobutylxanthine, which inhibits phosphodiesterase activity, are a valid assay of the adenylyl cyclase activity in intact cells. Measurement of cAMP levels and immunoblotting therefore provides a quantitative comparison of the functional activity of the expressed a, polypeptides.

RESULTS AND DISCUSSION

Transient expression in COS cells of the wild-type as poly- peptide resulted in greater than a 5-fold increase in cAMP synthesis relative to transfection with the pCWl expression plasmid without an aB cDNA insert (Fig. 1). Expression of the alp polypeptide had no effect on basal cAMP levels indicating that the stimulation of adenylyl cyclase activity is a direct consequence of the expressed a, polypeptide (12, 13, 15, 19).

Amino acid substitutions in the a, polypeptide coding se- quence were generated at specific residues predicted to be

4673

Dominant Negative Gas Mutant 4674

A.

0 ln h

-n ln m

21), had little effect on the ability of the mutant a. polypeptide to activate cAMP synthesis. This is in contrast to the corre- sponding mutation in p21 ras which enhances its transforming activity due to an increased rate of GDP dissociation (20). The findings indicate that despite conservation of the amino acid sequences of ras p21 and as which form the GTP/GDP binding domain, differences exist in their respective regula- tion. These differences presumably are due to the unrelated primary sequences of the two polypeptides outside the GTP/ GDP binding domains.

Further analysis of the phenotypic consequence of a,G225T indicated that expression of the mutant polypeptide inhibited P-adrenergic receptor activation of cAMP synthesis (Figs. 2

A. Basal El Isoproterenol

G226A

I a \ G49V D295A

FIG. 1. A, regulation of cAMP synthesis in COS-I cells after expression of mutant a. polypeptides. COS1 cells were transfected with the designated wild-type as and (yi2 or mutant a, cDNA as described under “Materials and Methods.” Mock refers to transfection without plasmid and pCWl represents cells transfected with the pCWl plasmid without a cDNA insert. Cells were transfected with 0.8 pg/plate of the wild-type (as), Gly4’+Val (G49V), Gly’Z5+Thr (G225T), GlyZZfi+Ala (G226A), GlnzZi+Leu (Q227L), and AspZ“+ Ala (D295A) mutant a. or wild-type ai2 cDNAs. Cyclic AMP levels in intact cells was measured in the absence of hormonal stimulation but in the presence of 500 p~ methyl isobutylxanthine, a phosphodies- terase inhibitor, 65 h after transfection as described under “Materials and Methods.” Data represent the mean of triplicate determinations that varied by less than 10% and are representative of three inde- pendent experiments. Cyclic AMP values are shown above the bar for each CY subunit transfection. B, map of the single amino acid mutations in the 52-kDa a, polypeptide encoded by the cDNAs used for transfection and analysis of adenylyl cyclase regulation.

involved in formation of the GTP/GDP binding domain (17, 18). Fig. 1 shows the location within the as polypeptide of five different mutations and the ability of each mutant as to activate adenylyl cyclase. Three of the mutations (Gly4’+Val, Gly22s+Thr, and Asp2”+Ala) involve amino acids in the GTP/GDP binding domain. Gly-226 and Gln-227 are thought to be involved in mediating the conformational changes in- duced by the binding of GTP to the a chain polypeptide (2, 4-6,10,11). As previously demonstrated (10,11,19), mutation of Gly4’+Val (G49V) or GlnZz7+Leu (Q227L) resulted in the stimulation of adenylyl cyclase due to their inhibition of a, GTPase activity. Unexpectedly, mutation of GlyZ2’+Thr within the invariant 223Asp-X2-Gly226 sequence of the a, poly- peptide inhibited approximately 70% the ability of the mutant to activate adenylyl cyclase relative to the activity of the wild- type a. polypeptide chain. This is in contrast to the analogous mutation in ras p21 (Ala5’+Thr) which is an activating mutation resulting in a transformed phenotype when ex- pressed in fibroblasts (7). Mutation of GlyZZ6+Ala (G226A) also resulted in a mutant a. that was ineffective in stimulating cAMP synthesis. The a,G226A mutant was inhibited greater than 85% compared with an approximate 70% inhibition of the a,G225T mutant in their respective abilities to activate adenylyl cyclase relative to the wild-type a, polypeptide. Mu- tation of AspZgs+Ala, which is the residue predicted to ioni- cally interact with the 2-amino group of GDP and GTP (20,

N 0 0

L

0 0 0 -

i.l 0 v) 01 In1 7

G226A

ti G225T, 1 Q227L I I a

e 52 kDa

FIG. 2. Phenotypic consequence of a. mutations within the 22aAsp-Val-Gly-Gly-Gln22’ sequence on adenylyl cyclase stim- ulation. A , COS-1 cells were transfected using DEAE-dextran with 0.8 pg of the appropriate wild-type or mutant 0 1 ~ cDNA in the expression plasmid pCWl as described under “Materials and Meth- ods.” Mock refers to transfection without plasmid and pCW2 are cells transfected using the pCWl plasmid without a cDNA insert. Cells were also transfected with pCWl containing the wild-type (01.) or Glyz2’+Thr (G225T), Gly””+Ala (G226A), or Gln22i+Leu (Q227L) mutant 01. cDNAs. Cells were used for cAMP and immunoblotting analysis 65 h after transfection. For cAMP measurements cells were incubated for 10 min in the presence of methyl isobutylxanthine (500 p ~ ) . Data represent the mean of triplicate determinations that varied by less than 10% and are representative of three independent exper- iments. Cyclic AMP values (picomoles/mg protein) are shown above the bar for each construct. B, conditions were similar to A except the cells were challenged with 1 p~ isoproterenol for the measurement of P-adrenergic receptor-stimulated cAMP synthesis. C, map of the single amino acid mutations in the 52-kDa a. polypeptide encoded by the cDNAs used for transfection and analysis of adenylyl cyclase regulation. D, immunoblot analysis of wild-type and mutant a. poly- peptides in transfected COS-1 cells (see “Materials and Methods” for details).

Dominant Negative Gas Mutant 4675

and 3). At similar levels of expression (Fig. 2 0 ) the magnitude of receptor-stimulated cAMP synthesis was significantly lower for the a,G225T mutant than that observed for the a,G226A mutant (Fig. 2B), both of which are ineffective in activating basal adenylyl cyclase (Fig. 2 A ) . Inhibition of iso- proterenol-stimulated cAMP synthesis by the a,G225T mu- tant was dependent on the concentration of plasmid used for transfection (Fig. 3). The 30-35% inhibition of isoproterenol- stimulated cAMP synthesis indicated that a,G225T was an effective inhibitor of receptor-stimulated adenylyl cyclase ac- tivity in the fraction of COS cells expressing the mutant polypeptide. The a,G225T polypeptide, therefore, phenotyp- ically functions as a dominant negative mutant which is inefficient in activating adenylyl cyclase and inhibits the ability of @-adrenergic receptors to stimulate the endogenous wild-type as polypeptide. In contrast, a fraction of the a,Q227L mutant was coupled to @-adrenergic receptors (10) and as a result of the inhibited GTPase activity the isopro- terenol stimulation of cAMP synthesis in a,Q227L expressing cells was greater than that with expression of the wild-type a. chain. Thus, within a 3-amino acid sequence of the a. polypeptide, appropriate mutation results in phenotypes rang- ing from dominant negative inhibition of receptor stimulation to strong constitutive activation of adenylyl cyclase.

The Gly2*’-Thr mutation results in a mutant a. chain whose functional phenotype is similar to the Gly22G+Ala substitution in that they both have a diminished ability to activate adenylyl cyclase. The a,G225T mutant, however, appears to be stronger in its inhibition of @-adrenergic recep- tor-stimulated cAMP synthesis than the a,G226A mutant (Figs. 2 and 3). Based on the crystallographic analysis of the ras p21 GTP/GDP binding site (4-6), it is predicted that Gly- 226 in the a, polypeptide functions as a pivot point for GTP- induced conformational changes. I t is possible that a Gly at position 225 as well as residue 226 is necessary for the GTP- induced conformational change of the as polypeptide and substitution of Gly by a Thr at position 225 inhibits the action

- as G225T

I 0.1 0.4

I 0.8 I

FIG. 3. Stimulation of CAMP synthesis in COS-1 cells is dependent on the level of as or a,G225T expression. COS-1 cells were transfected with the indicated plasmid concentration ex- pressing either the wild-type CY*, n,G226A, or aSG225T polypeptides. pCW1 represents cells transfected with the expression plasmid with- out a cDNA insert. Cells were assayed for cAMP levels 65 h after transfection in the presence of 1 pM isoproterenol and 500 pM methyl isohutylxanthine.

4 - 7

3 -

2 -

1 -

rn Q -. Q

VI P

rn

A

Y .

FIG. 4. Rescue of adenylyl cyclase activation by placement of the ai(S4)k chimeric sequence in the a.G225T mutant poly- peptide. A, expression of wild-type or mutant (Y, cDNAs in COS-I cells was accomplished as described under “Materials and Methods.” Intracellular cAMP levels were determined after a 10-min incubation with 500 p~ methyl isobutylxanthine 65 h after the COS-1 cell transfections. Expression of the cqS4)/* polypeptide gave a 2.3-fold greater cAMP accumulation than that observed with the wild-type CY$

chain. Placement of the chimeric sequence in the Gly”’+Thr (G225T), GlyY“iAla (G226A), and Gln”’+Leu (Q227L) mutant polypeptides was accomplished using the appropriate EcoRI restric- tion site fragments from the different CY, mutants and ligation to form the (Y~(w/~/(:~JST, @iIS4)/.~/0226A, and %54)/s/Q227L cDNAs. Cyclic AMP ac- cumulations (picomoles/mg protein) for each condition were: pCW1,

(Yi(54)/s/C.226A, 287; Q227L, 1482; CY^(^^^/^/^^^^^,, 3048. Values are represent- ative of two independent experiments. B, map of the cx(S41/r and single amino acid mutations in the 52-kDa CY, polypeptide encoded by the cDNAs used for transfection and analysis of adenylyl cyclase regu- lation. c, immunoblot analysis of the ( ~ i ( ~ ~ ) / ~ and CYi(54)/?.//r.)25T, q ( ~ ~ ) / ~ / 0226Tr and ( ~ i ~ 5 ~ ) / ~ / ~ ? 2 i l , polypeptides expressed in transfected COS-I cells. COS cells normally express equivalent levels of the endogenous 45- and 52-kDa forms of the CY, polypeptide as determined by immu- noblotting (12, 13,15,19). The CY* cDNA used for transfection encodes only the 52-kDa CY, form which was increased approximately 3-fold in this transfection. The lower band of the immunoblot is the 45-kDa CY% polypeptide whose expression is unchanged with transfection of the cDNA encoding the 52-kDa (Y, subunit.

38; CYs, 580 Qi(54)la, 1268; G225T, 159; (Yi(54)/r/C.225T, 1079; G226A, 97;

4676 Dominant Negative Gas Mutant

of GTP. However, the properties of the aSG225T mutant were distinguished from the Gly226+Ala substitution by secondary mutations in the a. amino terminus (Fig. 4).

Mutation of the a, amino terminus was achieved using the a;(54)/n chimeric a chain which has the first 61 amino acids of a, substituted with the amino-terminal 54 residues of aiX (13). The ( ~ i ( 5 4 ) / ~ chimera results in the loss of seven unique a, amino acids, and 16 of the first 34 cyiz residues are noncon- served relative to the as sequence. The last 20 ai2 amino acids within the chimera are identical or highly conserved when compared with the a, sequence. The ai(s4)ls chimera behaves as a constitutively active a, polypeptide which has an en- hanced rate of GDP dissociation resulting from the mutations introduced at the amino terminus of the polypeptide. This ~ i ( , 5 4 ) / ~ polypeptide was shown to influence the GTP/GDP binding domain without affecting the intrinsic GTPase activ- ity (13). Introduction of the ai(54)/. sequence in the a8G225T mutant polypeptide resulted in an a chain which functionally activated adenylyl cyclase to levels similar to that for ai(54)/s alone (Fig. 4). In contrast, the cyi(54)/s sequence was unable to rescue the inactive a,G226A phenotype. Interestingly, the CY,(^^)/^ and Gln-227+Leu mutations were additive with one another because they independently influence the two deter- mining steps in as activation, GDP dissociation and GTP hydrolysis.

The failure of the sequence to rescue the activity of the GlyZz6+Ala mutant indicates Gly-226 is essential for as to assume the GTP-induced active conformation similar to that for Gly-60 in ras p21 (2, 5, 6). In contrast, the results show that Gly at residue 225 is not critical as a pivot point in the GTP-induced conformational changes of the polypeptide because a second mutation compensates for the amino acid substitution. Rather, the findings indicate that Gly-225 in the as polypeptide must be involved in the tertiary structural interactions that regulate GDP dissociation, GTP binding, and activation of adenylyl cyclase but is not an essential pivot for GTP-induced conformational changes. Based on the res- cue of adenylyl cyclase stimulation by the cyi(54)/&225T mutant, the as amino terminus appears to be a region that influences GTP/GDP interactions with the 223Asp-Val-Gly-Gly-Glnz27 sequence. Structural homologies between the GTP/GDP binding domains of ras p21 and as support the genetic evi- dence for the a chain amino terminus to function as a regu- lator of the 223Asp-Val-Gly-Gly-Gln“27 sequence. Crystallo- graphic studies of ras p21-Gpp(NH)p indicate that GIy-12, which is involved i? the formation of the phosphate binding loop, is within 5.7 A of Gln-61 (5). It is predicted that Gly-49, which is equivalent to Gly-12 in ras p21, would be similarly distanced from Gln-227 in the a, tertiary structure. Thus, mutations, introduced in CY, by the sequence which contains the conserved phosphate binding loop including Gly- 49 (17, 18), are in proximity to the 2”Asp-Val-Gly-Gly-Gln227 sequence and could easily influence the structure of the GTP/ GDP binding domain. The accelerated GDP dissociation and GTP activation observed with the ai(54)/a chimera (13) indi- cates that the regulation of the GTP/GDP binding domain is indeed altered without influencing the GTPase activity of the polypeptide.

The constitutively active phenotype of the chimera and its ability to rescue the a,G225T mutant with respect to adenylyl cyclase stimulation defines the amino terminus as a regulatory sequence involved in the allosteric control of as activity by GDP and GTP. We have assigned the amino- terminal regulatory sequence as having an attenuator function (12,13), where mutation of this region relieves the attenuation

control of GDP dissociation. Several properties of G protein regulation are consistent with the a chain amino terminus controlling GDP dissociation. First, the amino terminus is involved in controlling the interaction of the a chain with the & subunit complex (22), which is required for efficient recep- tor-catalyzed GDP/GTP exchange (18,22,23). Second, the a chain amino- and carboxyl termini appear to be in close proximity to one another (24), and the carboxyl terminus is a major contact site for receptor interaction (25). Thus, it is predicted that the proximity of the amino terminus with receptor and & contact sites is involved in transmitting conformational changes that alter the interaction of the a chain amino terminus with the GTP/GDP binding domain resulting in enhanced GDP dissociation. Consistent with this prediction is the same functional consequence of accelerated GDP dissociation that is observed upon either receptor acti- vation or mutation of the amino terminus in the chi- mera (13,26).

The dominant negative character of the a$225T mutant in inhibiting receptor activation of CAMP synthesis also pro- vides a unique and necessary strategy to define hormone and neurotransmitter receptor coupling to specific G protein a chains in cells. The 2”Asp-Val-Gly-Gly-Gln2z7 sequence is conserved in all G protein a chains sequenced to date. Thus, similar mutations to Gl~’~~-.+Thr should generate dominant negative mutants in other G protein a chains. Overexpression of appropriate mutant a chains can be used to inhibit receptor activation of specific G proteins and signalling pathways and permit genetic dissection of receptor-G protein coupling in cells and tissues.

REFERENCES 1. Bosch, L., Kraal, B., and Parmeggiani, A. (eds) (1989) The Guanine Nu-

cleotide-binding Proteins: Common Structural and Functional Properties, Plenum Publishing Corp., New York

2. Jurnak, F., Heffron, S., and Bergmann, E. (1990) Cell 60,525-528

4. de Vos, A. M., Tong, L., Milburn, M. V., Matias, P. M., Jancarik, J., 3. Jurnak, F. (1985) Science 2 3 0 , 32-36

Noguchi, S., Nishimura, S., Miura, ti., Ohtsaka, E., and Kim, S.-H.

5. Pai. E. F.. Kahsch. W.. Kreneel. U.. Holmes. K. C.. John. J.. and Wittine- (1988) Science 239,88&893

~ ~~

hofer, A. (1989)’Nature 34’i,‘209-214. ’ 6. Schlichting, I., Alrno, S. C., Rapp, G., Wllson, K., Petratos, ti., Lentfer, A.,

Wittinrrhofer. A.. Kabsch. W., Pai, E. F.. Petsho, G. A., and Goody, R. S.

I . , -

8. Miller, R. T., Masters, S. B., Sullivan, K. A,, Beiderman, B., and Bourne,

9. Gibbs, J. B., Sigal, I. S., Poe, M., and Scolnick, E. M. (1984) Proc. Natl.

869

H. R. (1988) Nature 3 3 4 , 712-715

Acad. Set. U. S. A. 81.5704-5708 10. Masters, S. B. , Miller, T. R., Chi, M.-H., Chan , F.-H., Beiderman, B.,

Lopez, N. G., and Bourne, H. R. (1989) J. Biol. &em. 264,15467-15474 11. Graziano, M. P., and Gilman, A. G. (1989) J. Biol. Chem. 2 6 4 , 15475-

12. Osawa, S., Dhanasekaran, N., Woon, C. W., and Johnson, G. L. (1990) Cell 15482

13. Osawa, S., Heasley, L. E., Dhanasekaran, N., Gupta, S. ti., Woon, C. W., 63,697-706

14. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Smith, J. A., Berlot, C., and Johnson, G. L. (1990) Mol. CeU. Bid. 10,2931-2940

Seidman, J. G., and Strubl, K. (1987) Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York

15. Woon, C. W. Soparkar, S., Heasley, L. E., and Johnson, G. L. (1988) J. Biol. Chem: 264,5687-5693

16. Goldsmith, P., Gierschik, P., Milligan, G., Unson, C. G., Vinitsky, R., Malech, H. L., and Spiegel, A. M. (1987) J. Bid. Chern. 262, 14683- 14688

17. Loch;;;, M. A., and Simon, M. I. (1988) Biochemistry 27,4957-4965 18. Gilman, A. G. (1987) Annu. Reo. Biochem. 56,615-649 19. Woon, C. W., Heasley, L., Osawa, S., and Johnson, G. L. (1989) Biochem-

istry 28,4547-4551 20. Sigal, I. S., Gibbs, J. B., DAIonzo, J. S., Temeles, G. L., Wolanski, B. S.,

83,952-956 Sacher, S. H., and Scolnick, E. M. (1986) Proc. Natf. Acad. Sci. U. S. A.

21. Kelleher, D. J., Dudycz, L. W., Wright, G. E., and Johnson, G. L. (1986)

22. Fung, B. K.-K. (1983) J. Biol. Chem. 2 5 8 , 10495-10502 Mol. Pharmacol. 30,603-608

23. Kelleher, D. J. and Johnson G. L. (1988) Mol. Pharmacol. 34,452-460 24. Hingorani, V. N., and Ho, L.’K. (1988) J. Biol. Chem. 263,19804-19808 25. Masters, S. 3.. Sullivan, K. A.. Miller, R. T., Beiderman, B., Lopez, N. G.,

26. Johnson, G. L., and Dhanasekaran, N. (1989) Endocr. Reu. 10,317-331 Ramachandran, J., and Bourne, H. R. (1988) Science 241,448-451