MOLECULAR AND CELLULAR BIOLOGY, OCt. 1991, p. 4830-4838 0270-7306/91/104830-09$02.00/0 Copyright © 1991, American Society for Microbiology
Vol. 11, No. 10
A Mutation in the Putative Mg2"-Binding Site of Gs (x Prevents Its Activation by Receptors JOHN D. HILDEBRANDT,1 REGINA DAY,2 CHARLES L. FARNSWORTH,2 AND LARRY A. FEIG2*
Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545,1 and Department of Biochemistry, Tufts University Health Science Campus, Boston, Massachusetts 021112 Received 1 May 1991/Accepted 27 June 1991
The properties of a G. a mutant with an Asn substituted for Ser at position 54, designated mutant 54Asn aos, studied after expression in S49 as-deficient (cyc) cells. Ser-54 in a. is comparable to Ser-17 in Ras, which is involved in binding Mg2+ associated with bound nucleotide. 54Asn as did not restore either hormone-induced cyclic AMP production in intact cyc- cells or hormone-induced adenylyl cyclase activation in membranes isolated from these cells. The defect was a failure of ligand-bound receptor to activate 54Asn as, since the mutant protein retained the ability to activate adenylyl cyclase in isolated membranes in the presence of GTP or GTP-yS. Guanine nucleotide regulation of mutant ot suggested that it has increased guanine nucleotide exchange rates and an increased preference for diphosphates over triphosphates. Hormone stimulation magnified the preference of 54Asn a. for diphosphates, which could account for its inability to be activated by receptor. The properties of this mutant are discussed in terms of similarities to and differences with the analogous RasH mutant, which has been shown to interfere with endogenous Ras function in cells. were
GTP-binding proteins are involved in the regulation of a wide variety of cellular functions, including signal transduction across the plasma membrane, protein synthesis, translocation of nascent polypeptides into the endoplasmic reticulum, and vesicular traffic (5, 6). Despite their diverse cellular activities, all GTP-binding proteins go through the same cycle of reactions. Inactive protein becomes active by replacing bound GDP with GTP. This event is catalyzed by a nucleotide exchange protein. Deactivation of the protein occurs by hydrolysis of bound GTP to GDP, a process that for many GTP-binding proteins is catalyzed by a GTPaseactivating protein (GAP). These common mechanisms are reflected in amino acid sequence conservation between family members in regions of the proteins that interact with guanine nucleotides (6). Two of the most extensively studied GTP-binding proteins are GS, which couples hormone receptors to adenylyl cyclase and ion channels (2, 3, 16), and Ras, which is involved in growth factor signal transduction pathways (1, 5). These studies have revealed strong similarities in the way these proteins function. For example, external signals promote the GTP-bound form of both G, (2, 16) and Ras (10, 17, 39) in cells, and these activated proteins propagate a signal to downstream targets. Moreover, both proteins become constitutively activated when their intrinsic GTPase activity is inhibited (1, 19, 30, 49). Their structural similarity is reflected in the fact that GTPase-deficient Gs can be generated by mutations comparable to those that produce GTPasedeficient Ras proteins (19, 28, 30, 49). There are also clear differences between Ras and Gs. First, whereas Ras is a single polypeptide, G, is a trimer containing a nucleotide-binding as subunit and an inhibitory 3-y dimer (22) thought to dissociate from cxs when activated by GTP (16). Second, the intrinsic GTPase activity of Gs is greater than that of Ras and does not change upon interactions with other proteins (6). In contrast, the GTPase activity of Ras is stimulated by its interaction with Ras GAP, resulting in a *
GTPase rate greater than that of Gs (47). Third, whereas Ras is only about 21 kDa, osx is about 45 kDa. This difference is due in large part to additional sequences in as between the first two GTP-binding domains (6). This region may control the GTPase activity of as, since either ADP ribosylation or mutation in this region leads to GTPase-deficient protein (3, 16). Finally, Gs is activated directly by ligand-bound receptors that promote GDP-to-GTP exchange. Available data suggest that Ras is activated upon tyrosine kinase receptor activation via inhibition of GAP (10), which may be mediated by mitogenic lipids (50). X-ray crystallography of Ras shows that the hydroxyl sidegroup of Ser-17 participates in the complexing of Mg2+ associated with bound GTP and GDP (32, 41). This amino acid resides in the first GTP binding loop (amino acids 10 to 17) that is involved in interactions with phosphates and is part of a GX4GK(S/T) consensus sequence found in all GTP-binding proteins (6). Mutating Ser-17 to Asn generates an extremely interesting phenotype in Ras. First, the mutant switches its nucleotide specificity from equal affinity for GTP and GDP to preferential affinity for GDP (13). Second, expression of the mutant blocks the activity of endogenous Ras in cells (8, 13, 45). Here it is shown that the properties of the corresponding as mutant, designated 54Asn as, display many similarities to the mutant 17Asn Ras and a fundamental difference. Like 17Asn Ras, the as mutant did not respond normally to external stimulation. Moreover, altered interactions with guanine nucleotides were similar in both proteins. Unlike its Ras counterpart, however, 54Asn as retained the ability to stimulate downstream targets when bound to GTP. MATERIALS AND METHODS Materials. [a-32P]ATP was from Amersham Corp., and [3H]cyclic AMP ([3HJcAMP) and 125I-protein A were from Du Pont-New England Nuclear. Preparation of expression vectors, cell infection, and isolation of as-expressing cell lines. A 650-bp amino-terminal EcoRI fragment was excised from a full-length cDNA (25)
Corresponding author. 4830
54Asn Gs a MUTANT
VOL . 1 l, 1991
encoding the high-molecular-weight form of as. It was inserted into M13 mpl8, and site-directed mutagenesis was used to change codon 54 of as from Ser to Asn. This mutant EcoRI fragment was excised from M13 mpl8 and used to replace the comparable fragment of a full-length wild-type as cDNA that was excised from pUC12 with HindlIl and subcloned into the HindlIl site of pKSV10 (Pharmacia). The reconstituted as gene was then subcloned into the retrovirus vector pMV7 (26) at a HindlIl site. Normal and mutant genes were then transfected into psi-2 packaging cells, and stable transformants, selected in G418, were used to generate virus stocks. S49 as-deficient (cyc-) cells were infected by incubation of 106 cells with 300 ,ul of virus stock in the presence of Polybrene and selected in soft agar cultures as previously described (30). In some cases, cells were selected in liquid culture containing G418 and then cloned in soft agar. S49 cell growth and membrane isolation. Wild-type S49 mouse lymphoma cells, its related cyc- mutant cell line, and recombinant cell lines prepared from the cyc- cell line were grown as described previously (9, 48). Membranes were prepared from logarithmically growing cultures at cell densities of less than 2 x 106 cells per ml by nitrogen cavitation (37) as previously described (24). The experiments reported here were conducted with three independent membrane preparations from a single cyc- clonal cell line infected with normal as and with three independent membrane preparations from two different cyc- clonal cell lines infected with the mutant 54Asn as. There were no substantial differences in the properties of the various membrane preparations from a single cell line or in the properties of the two different clonal cell lines expressing the mutant protein. Northern (RNA) blot analysis. Cytoplasmic RNA was isolated as described previously (13), and 15 p.g from each cell line was electrophoresed in 1% agarose-formaldehyde gels and then transferred to nitrocellulose filters. The filters were probed with a nick-translated, 650-bp EcoRI fragment of as described above. Hybridization was carried out as described previously (13), and filters were autoradiographed for 3 days. Transcript sizes were estimated by comparison with the migration of rRNAs. Immunological detection of a. proteins. Expression of as or 54Asn as in S49 cell membranes was determined with the as-specific antiserum RM-1 (43) after transfer of proteins separated on polyacrylamide gels to nitrocellulose (46). Samples contained 50 p,g of membrane protein and were separated on 11% polyacrylamide gels (27). Immunologic detection of a, proteins with use of 125I-protein A was done essentially as described by Gierschik et al. (18), using a 1/200 dilution of affinity-purified RM-1 antibody. Adenylyl cyclase assays. Adenylyl cyclase activity was determined by measuring the conversion of [a-32P]ATP to [32P]cAMP as described previously (23), using the separation system described by Salomon (38). Unless indicated otherwise, enzyme activity was measured for 10 min at 32°C in the presence of 0.1 mM ATP, 2.0 mM MgCl2, 1 mM EDTA, 100 mM NaCl, 20 mM sodium N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid (HEPES; pH 8.0), and 3 to 10 ,ug of membrane protein in a total volume of 50 ,ul. Assay of cAMP levels. S49 cells, cyc- cells, and recombinant cyc- cells grown to 2 x 106/ml were washed and resuspended in Hanks balanced salt solution, pH 7.4, and then stimulated for 20 min at 37°C with 0.5 mM IBMX (3-isobutyl-1-methylxanthine) with or without 10 ,uM isoproterenol. Cells were harvested and extracted in 6% trichloroacetic acid. Extracts were washed four times in 5 volumes of
A
a
b c
4831
d e
-f S -5.6Kb
1.0Kb B a
b
c
d
e
f
U' FIG. 1. Expression of as mRNA and protein in S49 cells. (A) Northern blot; (B) Western blot. Lanes: a and f, wild-type S49 cells; b, cyc- S49 cells; c, cyc-/oa5 cells; d and e, two different cloned cyc-/54Na. cell lines.
water-saturated ethyl ether and dried. Intracellular cAMP was measured by using the Rianin (1251) radioimmunoassay kit (Dupont). Miscellaneous assays. Protein was determined by the method of Bradford (7), using gamma globulin as a standard, with the Bio-Rad protein assay. Statistical analysis of results. All experiments were conducted at least twice, and generally three or more times, with at least two different membrane preparations. Results are expressed, where appropriate, as the means ± standard errors of replicate samples. The half-maximal effective concentration (EC50) of guanine nucleotides or hormones for the stimulation of adenylyl cyclase activity was estimated by fitting the observed enzyme activity (v) to equation 1, v = vo + (Vmax * c)/(c + EC50), where vo is the activity in the absence of added guanine nucleotide (or hormone), Vmax is the maximum observed enzyme activity, and c is the concentration of stimulating ligand. The half-maximal concentration of GDP,BS required to block stimulation of adenylyl cyclase by GTPyS (IC50) was estimated by fitting the observed enzyme activity (v) to equation 2, v = v1 + (vO IC50)C(c + IC50), where v0 is the activity in the absence of GDPIS that can be inhibited by the nucleotide, v, is the activity at maximally effective concentrations of GDPPS, and c is the concentration of GDPPS. Data were fit to equations 1 and 2 by using the nonlinear curve-fitting program NFIT (Island Products, Galveston, Tex.). RESULTS
Expression of at and M"Asn a, in cyc- S49 cells. The phenotypes of normal and 54Asn a, were studied by their expression in as-deficient cyc- S49 cells (20). Cyc- cells were infected with replication-defective retroviruses encoding as and Neor genes, and G418-resistant cell lines were isolated in soft agar. One cell line expressing normal as (cyc-/ao) and two cell lines expressing 54Asn as (cyc-/ 54Nao) were used in the following studies. The expression of exogenous as genes in these cell lines was confirmed by Northern blotting and Western immunoblotting. Figure IA shows the expected -1-kb endogenous a, RNA transcript in wild-type S49 cells (lane a) and its absence in cyc- cells (lane
4832
HILDEBRANDT ET AL.
I
4=,
15.0 U U
MOL. CELL. BIOL.
Basal 0 Isoproterenol
10.0
~2.0
enol qualitatively similar to that of wild-type S49 cells. The lower magnitude of the response was consistent with the lower level of a. protein present in these cells. Cyc-/54Nac cells had baseline levels of cAMP comparable to those of cyc-/as cells, but they did not respond to isoproterenol. These results suggest that 54Asn as can support baseline levels of cAMP but cannot transmit a signal from cell surface receptors. Adenylyl cyclase activities in membranes from reconstituted cyc- cells. The failure of 54Asn as to reconstitute a hormone-
0.0I
cyc S49
cyc-/as cyc/54Nas S49 Cell Line wt S49
FIG. 2. Basal and isoproterenol-stimulated cAMP levels in intact S49 cells. cAMP levels were determined in the indicated cell lines as described in Materials and Methods in the absence or presence of 10 ,uM isoproterenol. The results are means ± standard errors of three experiments.
b). In contrast, only -6-kb bands characteristic of viral transcripts encoding as were observed in infected cells (lanes c to e). Western blot analysis (Fig. 1B) using the as-specific antiserum RM-1 (43) showed the expected aos doublet in wild-type S49 cells (lanes a and f), representing two alternatively spliced forms of the protein, and their absence in cyccells (lane b). Cyc- cells expressing exogenous normal as (lane c) and 54Asn as (lanes d and e) contained comparable levels of only the upper band, consistent with the specific cDNA used. Regulation of cAMP levels in reconstituted cyc- cells. To assess the ability of 54Asn as to restore a functional hormone-stimulated adenylyl cyclase, cAMP levels in the various cell lines were measured in the absence and presence of the ,B-adrenergic agonist isoproterenol (Fig. 2). The results for the two cyc-/54Nao cell lines were similar and therefore were averaged. As expected, cyc- cells had a low baseline level of cAMP that was not affected by isoproterenol. Moreover, wild-type S49 cells displayed higher baseline cAMP levels that were increased nearly 30-fold after hormone treatment. Cyc-/as cells had baseline cAMP levels greater than those of cyc- cells and a response to isoproter-
IA
I B
stimulated adenylyl cyclase in cyc- cells could have been due either to abnormal coupling of the mutant protein to the ligand-bound receptor or to an abnormal interaction between the mutant protein and the adenylyl cyclase enzyme. To distinguish between these possibilities, adenylyl cyclase activities were assayed in membranes from the various cell lines (Fig. 3). Results of control experiments were qualitatively similar to those with intact cells. For example, in cycmembranes (Fig. 3A), adenylyl cyclase was not activated either by direct stimulators of GTP-binding proteins (GTP, GTPyS, and NaF/AlCl3) or by isoproterenol, even though it was activated by direct stimulation of the enzyme with forskolin (42). Moreover, membranes from cyc-/as cells differed from wild-type S49 cell membranes only in the magnitude of responses (Fig. 3B and D). Adenylyl cyclase activity in membranes from cyc-/54Nax cells (Fig. 3C) was above the baseline observed for cyc(Fig. 3A) and comparable to that observed for cyc-/as (Fig. 3B). Importantly, the activity in cyc-/54Nas cell membranes was stimulated either by GTP or by its nonhydrolyzable analog, GTP-yS. These results show that 54Asn ot can stimulate adenylyl cyclase when activated by a guanine nucleotide triphosphate. Consistent with the in vivo experiments (Fig. 2), isoproterenol did not stimulate adenylyl cyclase in membranes containing 54Asn as. This was true for isoproterenol concentrations varying over 5 orders of magnitude and despite the fact that the enzyme in cyc-/as cells responded similarly to that in wild-type S49 cells (Fig. 4). Altered guanine nucleotide exchange properties of 54Asn x,s expressed in cyc- cells. The data in Fig. 3 and 4 suggested that 54Asn a lacks function in vivo because it does not respond to ligand-bound receptor, not because of its inherent
I
C
.I
V 1-1
'0
U
FIG. 3. Adenylyl cyclase activities of S49 cell membranes. The assay conditions were as described in Materials and Methods except that the MgCl2 concentration was 10 mM. Triplicate samples were incubated with the stimulators indicated: GTP, 10 ,uM; isoproterenol (Iso), 10 F.M; GTPyS, 10 ,uM; NaF, 10 mM; AIC12, 50 ,uM; forskolin, 100 ,uM.
54Asn Gs
VOL. 11, 1991
'o.1Pt
*5,
I
10-
0
.001
.01
.1
1
10
10
Isoproterenol Concentration ( IIM) FIG. 4. Effects of isoproterenol concentration on stimulation of adenylyl cyclase in S49 cell membranes. The assay conditions were as described in Materials and Methods except that all of the samples also contained 10 ,uM GTP. The inset shows data generated in the same experiment for wild-type S49 cell membranes. The ECQ0 for isoproterenol stimulation, determiined by fitting equation 1, was 77 nM for wild-type S49 and 43 nM for cyc-/a5. inability to stimulate adenylyl cyclase. However, the regulation of 54Asn as was clearly not normal even in the absence of hormone (Fig. 3). For example, GTP alone stimulated adenylyl cyclase in membranes containing 54Asn as but not normal as (compare Fig. 3B and C). In wild-type S49 cell membranes, adenylyl cyclase is not stimulated by GTP unless a hormone such as isoproterenol is also present (37). to GTP is also evident in The responsiveness of cycJ54Nac the data in Fig. 4, which show that the activity in the absence of isoproterenol is severalfold greater than that ofcyc/aM, even though they express comparable levels of a-subunit protein (Fig. 1). Interestingly, in addition to the abnormal response to GTP, NaF/AICl3 failed to stimulate 54Asn a5 (Fig. 3). An AlFi4 complex is thought to mimico the y-phosphate of GTP and thereby stimulate as with bound GDP. These results seemed to point to a fundamental change in the way guanine nucleotides interact with 54Asnas as the underlying cause of its failure to respond to hormone.
A
wt S49 ISO
4
0-
e5
20 time
4833
B sI CYC- /54N a
2- OA.
30 40 (mini)
50
-
0.2
0.8.
Vol. 11, No. 10
A Mutation in the Putative Mg2"-Binding Site of Gs (x Prevents Its Activation by Receptors JOHN D. HILDEBRANDT,1 REGINA DAY,2 CHARLES L. FARNSWORTH,2 AND LARRY A. FEIG2*
Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545,1 and Department of Biochemistry, Tufts University Health Science Campus, Boston, Massachusetts 021112 Received 1 May 1991/Accepted 27 June 1991
The properties of a G. a mutant with an Asn substituted for Ser at position 54, designated mutant 54Asn aos, studied after expression in S49 as-deficient (cyc) cells. Ser-54 in a. is comparable to Ser-17 in Ras, which is involved in binding Mg2+ associated with bound nucleotide. 54Asn as did not restore either hormone-induced cyclic AMP production in intact cyc- cells or hormone-induced adenylyl cyclase activation in membranes isolated from these cells. The defect was a failure of ligand-bound receptor to activate 54Asn as, since the mutant protein retained the ability to activate adenylyl cyclase in isolated membranes in the presence of GTP or GTP-yS. Guanine nucleotide regulation of mutant ot suggested that it has increased guanine nucleotide exchange rates and an increased preference for diphosphates over triphosphates. Hormone stimulation magnified the preference of 54Asn a. for diphosphates, which could account for its inability to be activated by receptor. The properties of this mutant are discussed in terms of similarities to and differences with the analogous RasH mutant, which has been shown to interfere with endogenous Ras function in cells. were
GTP-binding proteins are involved in the regulation of a wide variety of cellular functions, including signal transduction across the plasma membrane, protein synthesis, translocation of nascent polypeptides into the endoplasmic reticulum, and vesicular traffic (5, 6). Despite their diverse cellular activities, all GTP-binding proteins go through the same cycle of reactions. Inactive protein becomes active by replacing bound GDP with GTP. This event is catalyzed by a nucleotide exchange protein. Deactivation of the protein occurs by hydrolysis of bound GTP to GDP, a process that for many GTP-binding proteins is catalyzed by a GTPaseactivating protein (GAP). These common mechanisms are reflected in amino acid sequence conservation between family members in regions of the proteins that interact with guanine nucleotides (6). Two of the most extensively studied GTP-binding proteins are GS, which couples hormone receptors to adenylyl cyclase and ion channels (2, 3, 16), and Ras, which is involved in growth factor signal transduction pathways (1, 5). These studies have revealed strong similarities in the way these proteins function. For example, external signals promote the GTP-bound form of both G, (2, 16) and Ras (10, 17, 39) in cells, and these activated proteins propagate a signal to downstream targets. Moreover, both proteins become constitutively activated when their intrinsic GTPase activity is inhibited (1, 19, 30, 49). Their structural similarity is reflected in the fact that GTPase-deficient Gs can be generated by mutations comparable to those that produce GTPasedeficient Ras proteins (19, 28, 30, 49). There are also clear differences between Ras and Gs. First, whereas Ras is a single polypeptide, G, is a trimer containing a nucleotide-binding as subunit and an inhibitory 3-y dimer (22) thought to dissociate from cxs when activated by GTP (16). Second, the intrinsic GTPase activity of Gs is greater than that of Ras and does not change upon interactions with other proteins (6). In contrast, the GTPase activity of Ras is stimulated by its interaction with Ras GAP, resulting in a *
GTPase rate greater than that of Gs (47). Third, whereas Ras is only about 21 kDa, osx is about 45 kDa. This difference is due in large part to additional sequences in as between the first two GTP-binding domains (6). This region may control the GTPase activity of as, since either ADP ribosylation or mutation in this region leads to GTPase-deficient protein (3, 16). Finally, Gs is activated directly by ligand-bound receptors that promote GDP-to-GTP exchange. Available data suggest that Ras is activated upon tyrosine kinase receptor activation via inhibition of GAP (10), which may be mediated by mitogenic lipids (50). X-ray crystallography of Ras shows that the hydroxyl sidegroup of Ser-17 participates in the complexing of Mg2+ associated with bound GTP and GDP (32, 41). This amino acid resides in the first GTP binding loop (amino acids 10 to 17) that is involved in interactions with phosphates and is part of a GX4GK(S/T) consensus sequence found in all GTP-binding proteins (6). Mutating Ser-17 to Asn generates an extremely interesting phenotype in Ras. First, the mutant switches its nucleotide specificity from equal affinity for GTP and GDP to preferential affinity for GDP (13). Second, expression of the mutant blocks the activity of endogenous Ras in cells (8, 13, 45). Here it is shown that the properties of the corresponding as mutant, designated 54Asn as, display many similarities to the mutant 17Asn Ras and a fundamental difference. Like 17Asn Ras, the as mutant did not respond normally to external stimulation. Moreover, altered interactions with guanine nucleotides were similar in both proteins. Unlike its Ras counterpart, however, 54Asn as retained the ability to stimulate downstream targets when bound to GTP. MATERIALS AND METHODS Materials. [a-32P]ATP was from Amersham Corp., and [3H]cyclic AMP ([3HJcAMP) and 125I-protein A were from Du Pont-New England Nuclear. Preparation of expression vectors, cell infection, and isolation of as-expressing cell lines. A 650-bp amino-terminal EcoRI fragment was excised from a full-length cDNA (25)
Corresponding author. 4830
54Asn Gs a MUTANT
VOL . 1 l, 1991
encoding the high-molecular-weight form of as. It was inserted into M13 mpl8, and site-directed mutagenesis was used to change codon 54 of as from Ser to Asn. This mutant EcoRI fragment was excised from M13 mpl8 and used to replace the comparable fragment of a full-length wild-type as cDNA that was excised from pUC12 with HindlIl and subcloned into the HindlIl site of pKSV10 (Pharmacia). The reconstituted as gene was then subcloned into the retrovirus vector pMV7 (26) at a HindlIl site. Normal and mutant genes were then transfected into psi-2 packaging cells, and stable transformants, selected in G418, were used to generate virus stocks. S49 as-deficient (cyc-) cells were infected by incubation of 106 cells with 300 ,ul of virus stock in the presence of Polybrene and selected in soft agar cultures as previously described (30). In some cases, cells were selected in liquid culture containing G418 and then cloned in soft agar. S49 cell growth and membrane isolation. Wild-type S49 mouse lymphoma cells, its related cyc- mutant cell line, and recombinant cell lines prepared from the cyc- cell line were grown as described previously (9, 48). Membranes were prepared from logarithmically growing cultures at cell densities of less than 2 x 106 cells per ml by nitrogen cavitation (37) as previously described (24). The experiments reported here were conducted with three independent membrane preparations from a single cyc- clonal cell line infected with normal as and with three independent membrane preparations from two different cyc- clonal cell lines infected with the mutant 54Asn as. There were no substantial differences in the properties of the various membrane preparations from a single cell line or in the properties of the two different clonal cell lines expressing the mutant protein. Northern (RNA) blot analysis. Cytoplasmic RNA was isolated as described previously (13), and 15 p.g from each cell line was electrophoresed in 1% agarose-formaldehyde gels and then transferred to nitrocellulose filters. The filters were probed with a nick-translated, 650-bp EcoRI fragment of as described above. Hybridization was carried out as described previously (13), and filters were autoradiographed for 3 days. Transcript sizes were estimated by comparison with the migration of rRNAs. Immunological detection of a. proteins. Expression of as or 54Asn as in S49 cell membranes was determined with the as-specific antiserum RM-1 (43) after transfer of proteins separated on polyacrylamide gels to nitrocellulose (46). Samples contained 50 p,g of membrane protein and were separated on 11% polyacrylamide gels (27). Immunologic detection of a, proteins with use of 125I-protein A was done essentially as described by Gierschik et al. (18), using a 1/200 dilution of affinity-purified RM-1 antibody. Adenylyl cyclase assays. Adenylyl cyclase activity was determined by measuring the conversion of [a-32P]ATP to [32P]cAMP as described previously (23), using the separation system described by Salomon (38). Unless indicated otherwise, enzyme activity was measured for 10 min at 32°C in the presence of 0.1 mM ATP, 2.0 mM MgCl2, 1 mM EDTA, 100 mM NaCl, 20 mM sodium N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid (HEPES; pH 8.0), and 3 to 10 ,ug of membrane protein in a total volume of 50 ,ul. Assay of cAMP levels. S49 cells, cyc- cells, and recombinant cyc- cells grown to 2 x 106/ml were washed and resuspended in Hanks balanced salt solution, pH 7.4, and then stimulated for 20 min at 37°C with 0.5 mM IBMX (3-isobutyl-1-methylxanthine) with or without 10 ,uM isoproterenol. Cells were harvested and extracted in 6% trichloroacetic acid. Extracts were washed four times in 5 volumes of
A
a
b c
4831
d e
-f S -5.6Kb
1.0Kb B a
b
c
d
e
f
U' FIG. 1. Expression of as mRNA and protein in S49 cells. (A) Northern blot; (B) Western blot. Lanes: a and f, wild-type S49 cells; b, cyc- S49 cells; c, cyc-/oa5 cells; d and e, two different cloned cyc-/54Na. cell lines.
water-saturated ethyl ether and dried. Intracellular cAMP was measured by using the Rianin (1251) radioimmunoassay kit (Dupont). Miscellaneous assays. Protein was determined by the method of Bradford (7), using gamma globulin as a standard, with the Bio-Rad protein assay. Statistical analysis of results. All experiments were conducted at least twice, and generally three or more times, with at least two different membrane preparations. Results are expressed, where appropriate, as the means ± standard errors of replicate samples. The half-maximal effective concentration (EC50) of guanine nucleotides or hormones for the stimulation of adenylyl cyclase activity was estimated by fitting the observed enzyme activity (v) to equation 1, v = vo + (Vmax * c)/(c + EC50), where vo is the activity in the absence of added guanine nucleotide (or hormone), Vmax is the maximum observed enzyme activity, and c is the concentration of stimulating ligand. The half-maximal concentration of GDP,BS required to block stimulation of adenylyl cyclase by GTPyS (IC50) was estimated by fitting the observed enzyme activity (v) to equation 2, v = v1 + (vO IC50)C(c + IC50), where v0 is the activity in the absence of GDPIS that can be inhibited by the nucleotide, v, is the activity at maximally effective concentrations of GDPPS, and c is the concentration of GDPPS. Data were fit to equations 1 and 2 by using the nonlinear curve-fitting program NFIT (Island Products, Galveston, Tex.). RESULTS
Expression of at and M"Asn a, in cyc- S49 cells. The phenotypes of normal and 54Asn a, were studied by their expression in as-deficient cyc- S49 cells (20). Cyc- cells were infected with replication-defective retroviruses encoding as and Neor genes, and G418-resistant cell lines were isolated in soft agar. One cell line expressing normal as (cyc-/ao) and two cell lines expressing 54Asn as (cyc-/ 54Nao) were used in the following studies. The expression of exogenous as genes in these cell lines was confirmed by Northern blotting and Western immunoblotting. Figure IA shows the expected -1-kb endogenous a, RNA transcript in wild-type S49 cells (lane a) and its absence in cyc- cells (lane
4832
HILDEBRANDT ET AL.
I
4=,
15.0 U U
MOL. CELL. BIOL.
Basal 0 Isoproterenol
10.0
~2.0
enol qualitatively similar to that of wild-type S49 cells. The lower magnitude of the response was consistent with the lower level of a. protein present in these cells. Cyc-/54Nac cells had baseline levels of cAMP comparable to those of cyc-/as cells, but they did not respond to isoproterenol. These results suggest that 54Asn as can support baseline levels of cAMP but cannot transmit a signal from cell surface receptors. Adenylyl cyclase activities in membranes from reconstituted cyc- cells. The failure of 54Asn as to reconstitute a hormone-
0.0I
cyc S49
cyc-/as cyc/54Nas S49 Cell Line wt S49
FIG. 2. Basal and isoproterenol-stimulated cAMP levels in intact S49 cells. cAMP levels were determined in the indicated cell lines as described in Materials and Methods in the absence or presence of 10 ,uM isoproterenol. The results are means ± standard errors of three experiments.
b). In contrast, only -6-kb bands characteristic of viral transcripts encoding as were observed in infected cells (lanes c to e). Western blot analysis (Fig. 1B) using the as-specific antiserum RM-1 (43) showed the expected aos doublet in wild-type S49 cells (lanes a and f), representing two alternatively spliced forms of the protein, and their absence in cyccells (lane b). Cyc- cells expressing exogenous normal as (lane c) and 54Asn as (lanes d and e) contained comparable levels of only the upper band, consistent with the specific cDNA used. Regulation of cAMP levels in reconstituted cyc- cells. To assess the ability of 54Asn as to restore a functional hormone-stimulated adenylyl cyclase, cAMP levels in the various cell lines were measured in the absence and presence of the ,B-adrenergic agonist isoproterenol (Fig. 2). The results for the two cyc-/54Nao cell lines were similar and therefore were averaged. As expected, cyc- cells had a low baseline level of cAMP that was not affected by isoproterenol. Moreover, wild-type S49 cells displayed higher baseline cAMP levels that were increased nearly 30-fold after hormone treatment. Cyc-/as cells had baseline cAMP levels greater than those of cyc- cells and a response to isoproter-
IA
I B
stimulated adenylyl cyclase in cyc- cells could have been due either to abnormal coupling of the mutant protein to the ligand-bound receptor or to an abnormal interaction between the mutant protein and the adenylyl cyclase enzyme. To distinguish between these possibilities, adenylyl cyclase activities were assayed in membranes from the various cell lines (Fig. 3). Results of control experiments were qualitatively similar to those with intact cells. For example, in cycmembranes (Fig. 3A), adenylyl cyclase was not activated either by direct stimulators of GTP-binding proteins (GTP, GTPyS, and NaF/AlCl3) or by isoproterenol, even though it was activated by direct stimulation of the enzyme with forskolin (42). Moreover, membranes from cyc-/as cells differed from wild-type S49 cell membranes only in the magnitude of responses (Fig. 3B and D). Adenylyl cyclase activity in membranes from cyc-/54Nax cells (Fig. 3C) was above the baseline observed for cyc(Fig. 3A) and comparable to that observed for cyc-/as (Fig. 3B). Importantly, the activity in cyc-/54Nas cell membranes was stimulated either by GTP or by its nonhydrolyzable analog, GTP-yS. These results show that 54Asn ot can stimulate adenylyl cyclase when activated by a guanine nucleotide triphosphate. Consistent with the in vivo experiments (Fig. 2), isoproterenol did not stimulate adenylyl cyclase in membranes containing 54Asn as. This was true for isoproterenol concentrations varying over 5 orders of magnitude and despite the fact that the enzyme in cyc-/as cells responded similarly to that in wild-type S49 cells (Fig. 4). Altered guanine nucleotide exchange properties of 54Asn x,s expressed in cyc- cells. The data in Fig. 3 and 4 suggested that 54Asn a lacks function in vivo because it does not respond to ligand-bound receptor, not because of its inherent
I
C
.I
V 1-1
'0
U
FIG. 3. Adenylyl cyclase activities of S49 cell membranes. The assay conditions were as described in Materials and Methods except that the MgCl2 concentration was 10 mM. Triplicate samples were incubated with the stimulators indicated: GTP, 10 ,uM; isoproterenol (Iso), 10 F.M; GTPyS, 10 ,uM; NaF, 10 mM; AIC12, 50 ,uM; forskolin, 100 ,uM.
54Asn Gs
VOL. 11, 1991
'o.1Pt
*5,
I
10-
0
.001
.01
.1
1
10
10
Isoproterenol Concentration ( IIM) FIG. 4. Effects of isoproterenol concentration on stimulation of adenylyl cyclase in S49 cell membranes. The assay conditions were as described in Materials and Methods except that all of the samples also contained 10 ,uM GTP. The inset shows data generated in the same experiment for wild-type S49 cell membranes. The ECQ0 for isoproterenol stimulation, determiined by fitting equation 1, was 77 nM for wild-type S49 and 43 nM for cyc-/a5. inability to stimulate adenylyl cyclase. However, the regulation of 54Asn as was clearly not normal even in the absence of hormone (Fig. 3). For example, GTP alone stimulated adenylyl cyclase in membranes containing 54Asn as but not normal as (compare Fig. 3B and C). In wild-type S49 cell membranes, adenylyl cyclase is not stimulated by GTP unless a hormone such as isoproterenol is also present (37). to GTP is also evident in The responsiveness of cycJ54Nac the data in Fig. 4, which show that the activity in the absence of isoproterenol is severalfold greater than that ofcyc/aM, even though they express comparable levels of a-subunit protein (Fig. 1). Interestingly, in addition to the abnormal response to GTP, NaF/AICl3 failed to stimulate 54Asn a5 (Fig. 3). An AlFi4 complex is thought to mimico the y-phosphate of GTP and thereby stimulate as with bound GDP. These results seemed to point to a fundamental change in the way guanine nucleotides interact with 54Asnas as the underlying cause of its failure to respond to hormone.
A
wt S49 ISO
4
0-
e5
20 time
4833
B sI CYC- /54N a
2- OA.
30 40 (mini)
50
-
0.2
0.8.
