Identification of Common and Distinct Residues Involved in the Interaction of alpha i2 and alpha s with Adenylyl Cyclase*

(Received for publication, January 22, 1997, and in revised form, April 7, 1997)

Galina Grishina and Catherine H. Berlot Dagger

From the Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520-8026

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The G protein alpha  subunits, alpha s and alpha i2, have stimulatory and inhibitory effects, respectively, on a common effector protein, adenylyl cyclase. These effects require a GTP-dependent conformational change that involves three alpha  subunit regions (Switches I-III). alpha s residues in three adjacent loops, including Switch II, specify activation of adenylyl cyclase. The adenylyl cyclase-specifying region of alpha i2 is located within a 78-residue segment that includes two of these loops but none of the conformational switch regions. We have used an alanine-scanning mutagenesis approach within Switches I-III and the 78-residue segment of alpha i2 to identify residues required for inhibition of adenylyl cyclase. We found a cluster of conserved residues in Switch II in which substitutions cause major losses in the abilities of both alpha i2 and alpha s to modulate adenylyl cyclase activity but do not affect alpha  subunit expression or the GTP-induced conformational change. We also found two regions within the 78-residue segment of alpha i2 in which substitutions reduce the ability of alpha i2 to inhibit adenylyl cyclase, one of which corresponds to an effector-activating region of alpha s. Thus, both alpha i2 and alpha s interact with adenylyl cyclase using: 1) conserved Switch II residues that communicate the conformational state of the alpha  subunit and 2) divergent residues that specify particular effectors and the nature of their modulation.


INTRODUCTION

Upon activation by cell surface receptors, heterotrimeric G proteins transmit signals to effector proteins that regulate a wide variety of cellular processes (1-4). Receptors activate G proteins by catalyzing the replacement of GDP bound to the alpha subunit with GTP, resulting in dissociation of alpha ·GTP from the beta gamma subunits. The GTPase activity of the alpha  subunit regulates the timing of deactivation and reassociation of the G protein subunits. The fidelity of cellular signaling requires that alpha  subunits modulate effector proteins only when bound to GTP and that only the appropriate alpha  subunit-effector pairs interact. GTP-dependent effector interaction most likely involves one or more of the three alpha  subunit regions that change conformation during the GTPase cycle (Switches I-III), identified by comparison of the x-ray crystal structures of the GTPgamma S-bound1 (active) and GDP-bound (inactive) forms of alpha t (5, 6) and alpha i1 (7, 8). Differences in the amino acid sequences of the structurally conserved alpha  subunits (40% identity at the amino acid level, with 60-90% identity within subfamilies) determine the specificity and nature of their interactions with effector proteins (9). However, the relationship between the molecular determinants of effector specificity and of GTP-dependent effector regulation is poorly understood.

Regulation of adenylyl cyclase by the G protein alpha  subunits, alpha s and alpha i, raises issues specific for this alpha  subunit-effector interaction. alpha s and alpha i, which are relatively poorly conserved among the family of alpha  subunits (~40% identical amino acids), both bind to adenylyl cyclase but have opposite effects on activity. Inhibition of adenylyl cyclase by alpha i requires prior activation by alpha s, forskolin, or calmodulin (10, 11). Since adenylyl cyclase can be inhibited by alpha i in the absence of alpha s, inhibition does not appear to be due to competition between alpha i and alpha s for binding to adenylyl cyclase. Indeed, there is evidence that suggests that adenylyl cyclase has distinct binding sites for alpha s and alpha i (11). Key questions that arise are: why does alpha s activate and alpha i inhibit, and why do only alpha s and alpha i, but not other alpha  subunits, modulate adenylyl cyclase activity?

The alpha s residues that specify activation of adenylyl cyclase are located in three adjacent loops, one of which includes Switch II (12). The location of a conformational switch region within the effector-specifying surface of alpha s provides a simple mechanism for the GTP-dependence of the alpha s-adenylyl cyclase interaction. However, studies with chimeric alpha  subunits containing portions of alpha i2 and alpha q, which does not interact with adenylyl cyclase (13), showed that an alpha q/alpha i2/alpha q chimera containing only 78 residues of alpha i2 (residues 245-322) inhibits adenylyl cyclase as well as alpha i2 does (14). This 78-residue effector-specifying segment includes residues homologous to two of the three clusters of alpha s residues that specify activation of adenylyl cyclase (12, 15) but does not include any of the conformational switch regions. This was a surprise since the GTP-bound form of alpha i is much more effective at inhibiting adenylyl cyclase than the GDP-bound form is (11). However, the importance of the conformational switch regions might have been missed using a chimeric alpha  subunit approach due to the high degree of sequence similarity in these regions between alpha q and alpha i2.

To determine whether any of the conformational switch regions are involved in inhibition of adenylyl cyclase by alpha i2, we substituted alanines for solvent-exposed residues in these regions. We tested the effect of these mutations on both the inhibition of adenylyl cyclase and the ability of the mutant proteins to achieve the activated conformation as measured by the acquisition of trypsin resistance upon binding of GTP. We identified a part of Switch II that is conserved among alpha  subunits in which alanine substitutions blocked the inhibition of adenylyl cyclase by alpha i2. We also found that substitutions of alanines for the corresponding alpha s residues specifically prevent activation of adenylyl cyclase. Thus it appears that both alpha i2 and alpha s interact with adenylyl cyclase using two types of residues: 1) conserved residues within Switch II that signal that the alpha  subunit is in the GTP-bound active conformation and 2) divergent residues that specify activation or inhibition of this effector enzyme.

To identify the alpha i2 residues involved in specifying inhibition of adenylyl cyclase, we substituted alanines for solvent-exposed residues within the 78-residue segment. We found two regions of sequence in which mutations impaired the ability of alpha i2 to inhibit adenylyl cyclase, the amino terminus of alpha 3 and the alpha 4/beta 6 loop. The alpha 4/beta 6 loop is also important for the effector interactions of alpha s (12) and alpha t (16, 17). These substitutions did not cause as much of a decrease in adenylyl cyclase inhibition as the Switch II mutations did, suggesting that Switch II residues are the primary contributors to the interaction between alpha i2 and adenylyl cyclase.


EXPERIMENTAL PROCEDURES

Generation of Plasmids

alpha i2 mutants were constructed from the mouse alpha i2 cDNA (18), and alpha s mutants were constructed from the rat alpha s cDNA (19). Two modifications were made to each of the alpha  subunits to facilitate detection of their activities and expression levels. The arginine at position 179 in alpha i2 and 201 in alpha s was mutated to cysteine to inhibit GTPase activity and produce constitutive activation (20, 21). An epitope, referred to as the EE epitope (22) was generated by mutating alpha i2 residues SDYIPTQ (166-172) to EEYMPTE and alpha s residues DYVPSD (189-194) to EYMPTE (single letter amino acid code, mutated residues are underlined). The resultant constructs were designated alpha i2RCEE and alpha sRCEE respectively. alpha oRCEE was generated from the rat alpha o cDNA (19) by mutating arginine 179 to cysteine and residues DYQPTE (167-172) to EYMPTE.

The alpha i2RCEE cDNA (gift of Ann Pace and Henry Bourne, University of California, San Francisco) was subcloned into pcDNA I/Amp (Invitrogen) as an EcoRI fragment. The alpha sEE cDNA (gift of Paul Wilson and Henry Bourne, University of California, San Francisco) was subcloned into pcDNA I/Amp as a HindIII fragment. To produce the alpha sRC cDNA, the alpha sRCHA cDNA (12), which contains the HA epitope from influenza virus (23), was digested with XbaI and EcoRI to yield a fragment containing the R201C mutation but not the HA epitope. XbaI-EcoRI restriction of alpha sEEpcDNA I/Amp removed a fragment containing the EE epitope, which was replaced by the XbaI-EcoRI fragment from the alpha sRCHA cDNA to produce alpha sRCpcDNA I/Amp. To generate alpha sRCEEpcDNA I/Amp, alpha sRCpcDNA I/Amp was digested with Alwn I to yield a fragment containing the R201C mutation, which was ligated into alpha sEEpcDNA I/Amp in place of the analogous fragment to produce an alpha s cDNA containing both the R201C mutation and the EE epitope.

All mutations were generated by oligonucleotide-directed in vitro mutagenesis (24) using the Bio-Rad Muta-Gene kit except for those in the alpha i2RCEE derivatives, Constructs 2 and 3, which were produced by polymerase chain reactions that generated DNA fragments with overlapping ends that were subsequently combined in a fusion polymerase chain reaction (25). All mutagenesis procedures were verified by restriction enzyme analysis and DNA sequencing.

cAMP Accumulation Assay

Recombinant alpha  subunits were transiently expressed in the human embryonic kidney fibroblast line, HEK-293 (American Type Culture Collection CRL-1573), using DEAE-dextran (26) under the control of the cytomegalovirus promoter in the expression vector, pcDNA pcDNA I/Amp. To measure inhibition of adenylyl cyclase, 106 cells/60-mm dish were co-transfected with 0.1 µg of vector containing alpha sRC and 0.3 µg of vector containing alpha i2RCEE, alpha oRCEE, or mutant derivatives of alpha i2RCEE. To measure activation of adenylyl cyclase, 106 cells/60-mm dish were transfected with 1.5 µg of vector containing alpha sRCEE or mutant derivatives of this construct or with vector alone. Intracellular cAMP levels in cells labeled with [3H]adenine were determined as described (14).

Membrane Preparations and Trypsin Assay

HEK-293 cells were transiently transfected with recombinant alpha  subunit constructs using DEAE dextran (26). Membranes were prepared 48 h after transfection as described (14). For the trypsin resistance assay (12), membrane proteins (70 µg) were diluted to a concentration of 6 mg/ml in a buffer containing 20 mM HEPES (pH 8.0), 10 mM MgCl2, 1 mM EDTA, 2 mM beta -mercaptoethanol, and 0.64% (w/v) of the detergent lubrol PX. Solubilized proteins were collected after centrifugation for 10 min at 4 °C in a microcentrifuge and incubated for 30 min at 30 °C in the presence or absence of 125 µM GTPgamma S. Tosylphenylalanyl chloromethyl ketone-treated trypsin (Sigma T-8642) was added to a final concentration of 5 µg/ml, and the mixture was incubated for 5 min at 30 °C. The digestion was terminated by adding soybean trypsin inhibitor to a final concentration of 1 mg/ml. The samples were then resolved by SDS-polyacrylamide electrophoresis (10%), transferred to nitrocellulose, and probed with the anti-EE monoclonal antibody (22), which was purified from hybridoma supernatants using E-Z-SEP reagents (Middlesex Sciences, Inc). The antigen-antibody complexes were detected using an anti-mouse horseradish peroxidase-linked antibody according to the ECL Western blotting protocol (Amersham Life Science, Inc.).


RESULTS

Characterization of Mutant alpha i2 Constructs Using cAMP Assay

To characterize mutant alpha i2 subunits after transient expression in HEK-293 cells, two features were included, as in a previous study (14), to enable measurement of their functions without interference from the activities of the alpha i proteins endogenous to these cells. First, a conserved arginine (R179C) was replaced by cysteine. This mutation constitutively activates alpha i2 by inhibiting its GTPase activity (20) and made it possible to measure inhibition of adenylyl cyclase without requiring receptor-mediated activation of the mutant alpha i2 subunits. Second, the alpha i2 constructs include an epitope from an internal region of polyoma virus medium T antigen, referred to as the EE epitope (22), which does not interfere with the alpha i2-adenylyl cyclase interaction (27).

We measured the ability of recombinant alpha  subunits to inhibit adenylyl cyclase in HEK-293 cells by co-expressing them with the constitutively activated alpha s mutant, alpha sRC, in which arginine 201 is mutated to cysteine (21). As in a previous study (14), transfection with 0.1 µg of vector containing alpha sRC resulted in an approximately 18-fold increase in cAMP production compared with cells transfected with vector alone. Co-transfection with 0.3 µg of vector containing alpha i2RCEE resulted in ~60% inhibition of the cAMP response to alpha sRC, while co-transfection with the same amount of vector containing alpha oRCEE inhibited the response to alpha sRC by only ~15% (Fig. 1). We used alpha oRCEE as a negative control because alpha o has been shown to have little or no ability to inhibit adenylyl cyclase (10, 11).


Fig. 1. Alanine substitutions of solvent-exposed residues in Switches I-III. The residues that were substituted by alanines in each construct and the residue ranges and sequences of Switches I-III in alpha i2 are indicated. All constructs include the GTPase-inhibiting arginine to cysteine mutation (R179C in alpha i2 and alpha o) and the EE epitope. cAMP accumulation in 106 HEK-293 cells transfected with 0.1 µg of vector containing alpha sRC and 0.3 µg of vector containing the indicated alpha  subunit constructs is shown. The amount of cAMP accumulation in cells transfected with alpha sRC alone is set at 1.0, and the values from cells co-transfected with the indicated constructs are expressed relative to this value. Asterisks indicate cAMP values of constructs with significantly decreased abilities to inhibit cAMP accumulation (p < 0.05) compared with alpha i2RCEE. cAMP levels in [3H]adenine-labeled cells were determined as described under "Experimental Procedures." Each value represents the mean ± S.E. of at least three independent experiments.
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Alanine Substitutions within Conformational Switch Regions

Since the GTP-bound form of alpha i2 inhibits adenylyl cyclase much more effectively than the GDP-bound form does (11), it was surprising that the effector-specifying region of alpha i2, as defined by the 78-residue segment, residues 245-322 (14), did not include any of the three regions, Switches I-III (6, 8), that undergo GTP-dependent conformational changes. However, the sequences of these regions are highly conserved in alpha i2 and alpha q. 7 of the 11 Switch I residues, 18 of the 21 Switch II residues, and 6 of the 12 Switch III residues are identical in the sequences of alpha i2 and alpha q. Therefore, the importance of these regions as effector binding sites could have been missed using homologous sequence substitutions.

To directly test the importance of Switches I-III as effector contact sites, we mutated solvent-exposed residues within each of these regions to alanine residues. Substitutions using alanine residues eliminate the side chain beyond the beta  carbon but generally do not alter the main chain conformation and do not impose significant electrostatic or steric effects (28). We identified clusters of solvent-exposed residues by inspection of the x-ray crystal structures of the GTPgamma S-bound forms of alpha i1 (7) and alpha t (5) and calculations of fractional accessibility values (29) from the coordinates. As shown in Fig. 1, we mutated three clusters of residues in Switch I (6 residues), five clusters of residues in Switch II (8 residues), and four clusters of residues in Switch III (7 residues).

We found that alanine substitutions of three residues in Switch II, Arg-209, Lys-210, and Ile-213, blocked alpha i2RCEE from inhibiting adenylyl cyclase (Fig. 1). These residues are located in the middle of the alpha 2 helix and are highly conserved among alpha  subunits (see Fig. 7). We previously found that substituting alpha i2 homologs for three alpha s Switch II residues located at the carboxyl terminus of alpha 2 and in the alpha 2/beta 4 loop, Gln-236, Asn-239, and Asp-240 (see Fig. 7), specifically prevents alpha s from activating adenylyl cyclase (12). Although not conserved between alpha s and alpha i2, these residues are identical in the sequences of alpha i2 and alpha q and therefore were not tested in our previous alpha i2/alpha q chimera studies (14). The alpha t and alpha i1 homologs of the first two of these residues are solvent-exposed in the structures. Alanine substitutions of the corresponding alpha i2 residues, His-214 and Glu-217, did not block the ability of alpha i2 to inhibit adenylyl cyclase (Fig. 1).


Fig. 7. Comparison of effector-interacting residues of alpha i2, alpha s, and alpha t in Switch II and in the alpha 4/beta 6 loop. Residue numbers of alpha i2, alpha s, and alpha t in the Switch II and alpha 4/beta 6 regions are indicated in parentheses. Mutations of boxed residues impaired effector interaction. Mutations of underlined residues did not impair effector interaction. Mutation of the circled glutamate residue in Switch II of alpha t caused constitutive activation of PDE. Data for alpha i2 are from Figs. 1 and 4. Data for alpha s are from Fig. 3 and Berlot and Bourne (12). Data for alpha t are from Spickofsky et al. (17), Faurobert et al. (36), and Mittal et al. (37).
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We did not obtain evidence that either the Switch I or Switch III regions of alpha i2 are specifically involved in inhibition of adenylyl cyclase (Fig. 1). The only substitutions that caused a partial loss of function were in residues Ile-185 and Glu-187 in Switch I (Fig. 1). However, these substitutions also greatly reduced the expression level of alpha i2RCEE (see below).

Criteria for Specificity of Mutations

Mutations that prevent alpha i2RCEE from inhibiting adenylyl cyclase could do so for reasons other than disruption of residues that interact with this effector. Therefore, we subjected constructs with these mutations to the following criteria for specificity. The first criterion was that the mutants should be expressed at wild-type levels in HEK-293 cell membranes. This criterion was tested by performing immunoblots on membranes prepared from cells expressing the mutants. The second criterion was that the mutants should be able to bind GTP and undergo the GTP-dependent conformational change that is detected as the acquisition of resistance to trypsin cleavage (30-32). This second criterion is quite stringent because it requires not only proper GTP binding but also the ability to respond to this binding with an activating conformational change. Under the conditions of this assay, in the presence of GTPgamma S, trypsin removes a short segment from the amino terminus but leaves most of the protein intact (Fig. 2). However, in the absence of GTPgamma S, trypsin degrades alpha i2RCEE to small fragments not seen on SDS-polyacrylamide gels.


Fig. 2. Expression and trypsin sensitivity of alpha i2 constructs containing mutations in Switches I and II. 12.5 × 106 HEK-293 cells were transfected with 2 µg/106 cells of vector alone or vector containing the indicated alpha i2 constructs, and membranes were prepared, treated with trypsin, and immunoblotted as described under "Experimental Procedures." The first lane in each set is the control (no trypsin). The second and third lanes show the result of trypsin digestion in the presence or absence, respectively, of GTPgamma S.
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We found that the Switch II mutant constructs, (R209A)alpha i2RCEE and (K210A,I213A)alpha i2RCEE, were expressed as well as alpha i2RCEE and achieved the GTP-dependent activated conformation, as measured by the trypsin assay (Fig. 2). Therefore, by our criteria, residues Arg-209, Lys-210, and Ile-213 are specifically required for interaction with adenylyl cyclase. In contrast, although the Switch I mutant construct, (I185A,E187A)alpha i2RCEE, exhibited resistance to trypsin in the presence of GTPgamma S, it was expressed very poorly (Fig. 2). The role of residues Ile-185 and Glu-187 in effector interaction is, therefore, uncertain.

In the course of these studies, we mutated the alpha i2 residue, Arg-209, that corresponds to the GTPgamma S-protected trypsin site determined by amino-terminal sequencing of tryptic peptides from alpha t and alpha o (31). Elimination of this cleavage site would be expected to result in an alpha  subunit that was resistant to trypsin cleavage in both the presence and absence of GTPgamma S. However, (R209A)alpha i2RCEE was resistant to trypsin cleavage in the presence but not the absence of GTPgamma S (Fig. 2). Similar results were obtained upon mutation of each of the other potential trypsin sites in Switch II, Arg-206,2 Lys-210 (Fig. 2), and Lys-211,2 as well as mutation of all four residues simultaneously.2 These results suggest that, although Switch II may contain cleavage sites that change conformation upon GTP binding, there are also other sites outside of this region that are preferentially cleaved by trypsin in the absence compared with the presence of GTPgamma S. Nevertheless, the ability of the trypsin assay to detect GTP-dependent conformational changes in Switch II is demonstrated by the fact that the Switch II alpha s mutant, G226Aalpha s, which is unable to undergo the activating conformational change required for dissociation from beta gamma , does not acquire trypsin resistance in the presence of GTPgamma S (32, 33).

A Conserved Region of Switch II Is Specifically Required for the Effector Interactions of Both alpha s and alpha i2

To determine whether the highly conserved middle region of Switch II (see Fig. 7) is required for the activation of adenylyl cyclase by alpha s, we tested the effects of substituting alanines for the alpha s residues (Arg-232 and Ile-235) that correspond to Lys-210 and Ile-213 in alpha i2. We introduced these substitutions into alpha sRCEE, which contains the EE epitope, previously shown to have no effect on the interaction between alpha s and adenylyl cyclase (34). The substitutions almost entirely prevented alpha sRCEE from activating adenylyl cyclase without affecting the GTP-dependent conformational change measured by the trypsin assay (Fig. 3). Thus, the same region of Switch II is required for the interaction of both alpha s and alpha i2 with adenylyl cyclase.


Fig. 3. A conserved region of Switch II is specifically required for the activation of adenylyl cyclase by alpha s. Top part of figure shows cAMP accumulation in 106 HEK-293 cells transfected with 1.5 µg of vector containing alpha sRCEE or (R232A,I235A)alpha sRCEE or with vector alone. cAMP levels in [3H]adenine-labeled cells were determined as described under "Experimental Procedures." Conversion of ATP to cAMP is expressed as [3H]cAMP/([3H]ATP + [3H]cAMP) × 1000 (44). Each value represents the mean ± S.E. of three independent experiments. Bottom part of figure shows expression and trypsin sensitivity of these constructs. 12.5 × 106 HEK-293 cells were transfected with 6 µg/106 cells of vector alone or vector containing alpha sRCEE or (R232A,I235A)alpha sRCEE, and membranes were prepared, treated with trypsin, and immunoblotted as described under "Experimental Procedures." The first lane in each set is the control (no trypsin). The second and third lanes show the result of trypsin digestion in the presence or absence, respectively, of GTPgamma S.
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Alanine Substitutions within the 78-Residue Segment

Since alpha i2, but not alpha q, inhibits adenylyl cyclase (10, 13) and an alpha q/alpha i2/alpha q chimera containing only 78 alpha i2 residues (245-322) inhibits adenylyl cyclase as well as alpha i2 does (14), the alpha i2 residues that specify inhibition of adenylyl cyclase must be located within this 78-residue segment. To identify these effector-specifying residues, we tested the effects of mutating nine clusters of solvent-exposed residues (22 residues total) to alanine residues (Fig. 4). Within the 78-residue segment of alpha i2, 65 residues are identical among the three alpha i isoforms, which have equal abilities to inhibit adenylyl cyclase (11). Of these 65 residues, 28 are different in alpha q and therefore might account for the ability of alpha i, but not alpha q, to inhibit adenylyl cyclase. 20 of the substitutions were in residues that are identical among the three alpha i subunits, and 18 were in residues that differ between alpha i2 and alpha q. The thoroughness of our mutational analysis is illustrated in Fig. 6A.


Fig. 4. Alanine substitutions of solvent-exposed residues within the 78-residue alpha i2 segment. The top sequence is that of alpha i2 residues 245-322. Below that are the sequences of alpha i1, alpha i3, and alpha q, with residues identical to alpha i2 residues represented by dashes. The numbered sequences represent individual mutant constructs with alanine substitutions at the indicated positions. All constructs include the GTPase-inhibiting arginine to cysteine mutation (R179C in alpha i2 and alpha o) and the EE epitope. Shown next to each construct is the cAMP accumulation in 106 HEK-293 cells transfected with 0.1 µg of vector containing alpha sRC and 0.3 µg of vector containing the indicated alpha  subunit construct. The amount of cAMP accumulation in cells transfected with alpha sRC alone is set at 1.0, and the values from cells co-transfected with the indicated constructs are expressed relative to this value. Asterisks indicate cAMP values of constructs with significantly decreased abilities to inhibit cAMP accumulation (p < 0.05) compared with alpha i2RCEE. cAMP levels in [3H]adenine-labeled cells were determined as described under "Experimental Procedures." Each value represents the mean ± S.E. of at least three independent experiments.
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Fig. 6.

Mapping of effector-interacting residues of alpha i2 and alpha s onto the x-ray crystal structure of the GTPgamma S-bound form of alpha i1. A, space-filling model showing alpha i2 residues required for inhibition of adenylyl cyclase. Residues that were mutated are shown in red, magenta, orange, and dark blue, as follows. Residues in Switch II specifically required for inhibition of adenylyl cyclase are red. Residues in Switch I in which mutations reduce both inhibition of adenylyl cyclase and expression level are magenta. Residues within the 78-residue segment in which mutations cause a partial loss of adenylyl cyclase inhibition are orange. Residues in which mutations do not affect inhibition of adenylyl cyclase are dark blue. Residues that were not mutated in this study are shown in green, light blue, and white, as follows. Residues outside of the 78-residue segment and Switches I-III are green. Also green are the residues within the 78-residue alpha i2 segment that are conserved between alpha i2 and alpha q. The alpha i2 residues within this segment that differ from alpha q residues but are not identical among the three alpha i isoforms are light blue. Residues in Switches I-III that were not mutated and residues within the 78-residue segment that differ from alpha q residues and are conserved among the three alpha i isoforms, but were not mutated, are white. Main chain backbone atoms are gray. The GTP is yellow. The numbers on the model refer to alpha i2 residues. The model on the right is rotated approximately 90° about the vertical axis relative to the model on the left. B, ribbon diagram showing comparison of effector-interacting surfaces of alpha i2 and alpha s. Residues important for the effector interactions of alpha i2 are red, of alpha s are blue, and of both alpha  subunits are magenta. Switches I-III are orange. The model is in the same orientation as the model on the left in panel A. Coordinates of the GTPgamma S-bound form of alpha i1 are from Coleman et al. (7). The figures were drawn using MidasPlus, developed by the Computer Graphics Laboratory at University of California, San Francisco.


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As shown in Fig. 4, substitutions of three sets of residues: His-245 (Construct 1), Lys-313, Asp-316, and Thr-317 (Construct 8), and Arg-314, Lys-315, and Glu-319 (Construct 9), significantly reduced inhibition of adenylyl cyclase. However, in contrast to the Switch II mutations, which entirely blocked the ability of alpha i2RCEE to inhibit adenylyl cyclase, the mutations in Constructs 1, 8, and 9 had only partial effects. The other six clusters of mutations (15 residues) did not significantly impair the ability of alpha i2RCEE to inhibit adenylyl cyclase.

All of the constructs that inhibited adenylyl cyclase to a similar or decreased extent compared with alpha i2RCEE were expressed in HEK-293 cell membranes and were able to undergo the GTP-dependent conformational change that results in increased resistance to trypsin digestion (Fig. 5). However, since scanning densitometry of immunoblots showed that Constructs 1, 8, and 9 were expressed at lower levels than alpha i2RCEE was, their decreased abilities to inhibit adenylyl cyclase may be due to effects of the mutations on protein folding and/or stability. Nevertheless, since we have substituted alanines for the majority of solvent-exposed residues within the effector-specifying 78-residue segment (see Fig. 6A) and the other substitutions did not significantly reduce adenylyl cyclase inhibition, the residues in Constructs 1, 8, and 9 are, by default, the most likely candidates for specifying inhibition of adenylyl cyclase.


Fig. 5. Expression and trypsin sensitivity of alpha i2 constructs containing mutations within the 78-residue alpha i2 segment. 12.5 × 106 HEK-293 cells were transfected with 2 µg/106 cells of vector alone or vector containing the indicated alpha i2 constructs, and membranes were prepared, treated with trypsin, and immunoblotted as described under "Experimental Procedures." The first lane in each set is the control (no trypsin). The second and third lanes show the result of trypsin digestion in the presence or absence, respectively, of GTPgamma S.
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Comparison of the Effector-Interacting Surfaces of alpha i2 and alpha s

We used the x-ray crystal structure of the GTPgamma S-bound form of alpha i1 (7) to map the results of our mutagenesis studies. 88% of the residues in alpha i2 can be aligned with identical residues in alpha i1, while 67% of the alpha i1 residues can be aligned with identical residues in alpha t. Since the structures of the active (GTPgamma S-bound) forms of alpha i1 (7) and alpha t (5) are virtually identical, the structure of alpha i1 is an excellent model for that of alpha i2. Our mutagenesis analysis of Switches I-III in alpha i2 and the 78-residue effector-specifying alpha i2 segment, residues 245-322, focused on solvent-exposed residues. In addition, most of the alanine substitutions in the 78-residue segment were of residues that are: 1) different from the homologous alpha q residues and 2) conserved among the alpha i isoforms. The thoroughness of this study is demonstrated by the fact that the residues in Switches I-III that were not mutated and the residues in the 78-residue segment that meet criteria 1 and 2 but were not mutated represent a very small fraction of the available surface area (shown in white in Fig. 6A).

The alanine substitutions that caused the largest decrease in the ability of alpha i2RCEE to inhibit adenylyl cyclase were in the middle of the alpha 2 helix in Switch II (red in Fig. 6A). The effector-interacting surfaces of alpha s and alpha i2 overlap exactly in this region (magenta in Fig. 6B) where the sequences of the two alpha  subunits are highly conserved (Fig. 7). However, the alpha 2/beta 4 loop at the carboxyl-terminal end of Switch II is important for the interaction of alpha s (12) but not alpha i2 (Fig. 1) with adenylyl cyclase (blue in Fig. 6B).

The alanine substitutions within the 78-residue effector-specifying segment that caused a moderate reduction in the ability of alpha i2RCEE to inhibit adenylyl cyclase (orange in Fig. 6A) were in the amino terminus of alpha 3 (Construct 1) and in the alpha 4/beta 6 loop (Constructs 8 and 9) (Fig. 6B). The amino terminus of alpha 3 (red in Fig. 6B) is important for the effector interactions of alpha i2 (Fig. 4), but not alpha s (12), while mutations in the alpha 3/beta 5 loop (blue in Fig. 6B) disrupt interaction between alpha s and adenylyl cyclase (12) but do not have a significant effect on the alpha i2-adenylyl cyclase interaction (Fig. 4). Residues in the alpha 4/beta 6 loop found to be important for specifying the effector interactions of both alpha i2 and alpha s are magenta in Fig. 6B.


DISCUSSION

The studies reported here investigated two key aspects of alpha  subunit-effector interactions, GTP-dependence and specificity. We found that in the case of alpha i2, these two components of effector interaction are mediated by distinct regions of surface residues. GTP-dependent effector interaction is mediated by Switch II residues that are conserved among alpha  subunits (Fig. 1) while specificity (inhibition of adenylyl cyclase) is mediated by nonconserved residues (the amino terminus of alpha 3 and the alpha 4/beta 6 loop) outside of the conformational switch regions (Fig. 4). In contrast, in the case of alpha s, Switch II plays a role in regulating both the GTP dependence of effector interaction as well as effector specificity. The conserved Switch II region is required for GTP-dependent activation of adenylyl cyclase (Fig. 3) while nonconserved Switch II residues, as well as residues outside of the conformational switch regions (the alpha 3/beta 5 and alpha 4/beta 6 loops), are involved in regulating effector specificity (12). In the case of alpha t, the conformational switch regions and regions that don't switch conformation (alpha 3 and the alpha 3/beta 5 loop) interact with distinct regions of the effector molecule, PDE (35).

Taken together, our results and those of others indicate that two alpha  subunit regions, Switch II and the alpha 4/beta 6 loop, may be important for effector interactions in general (Fig. 7). The conserved middle region of Switch II has been shown to be important for the interaction between alpha t and PDE. Mutation of a conserved tryptophan in alpha t reduces binding to PDE (36) while mutation of a conserved glutamate causes constitutive activation of PDE by the GDP-bound form of alpha t (37). The alpha 4/beta 6 loop is involved in specifying the effector interactions of at least three alpha  subunits (Fig. 7). We previously found that replacement of alpha s residues in this region by their alpha i2 homologs prevents alpha s from activating adenylyl cyclase without preventing the mutant protein from attaining the GTP-dependent active conformation (12). Rarick et al. (16) found that a 22-amino acid peptide (alpha t residues 293-314) activates PDE. Within this region, Spickofsky et al. (17) identified five residues in which substitutions of homologs from other alpha  subunits block PDE activation by peptides. Three of these residues are in the alpha 4 helix and two are in the alpha 4/beta 6 loop. Mutations in the alpha 4/beta 6 loop of alpha s and alpha i2, but not in alpha 4 cause decreases in effector modulation. In the case of alpha q, alpha 4 and the alpha 4/beta 6 loop have been implicated in PLC activation in studies using peptides (38). However, chimera studies showed this region could be replaced with alpha s sequence without affecting PLC activation (39).

Since alpha s and alpha i2 have opposite effects on adenylyl cyclase activity, the conserved region of Switch II required for the effector interactions of both alpha  subunits is most likely involved in regulating GTP-dependent effector binding. Of the three residues found to be important for inhibition of adenylyl cyclase by alpha i2, Arg-209 and Ile-213 are identical in the sequences of alpha s and alpha i2 (see Fig. 7). The third residue is conserved but not identical between the two alpha  subunits (Lys-210 in alpha i2, Arg-232 in alpha s). However, alpha i2/alpha s chimera studies showed that substitution of lysine for arginine at position 232 in alpha s has no effect on activation of adenylyl cyclase (12). Furthermore, the alpha q residue corresponding to Lys-210 is an arginine residue and alpha q/alpha i2 chimera studies showed that substitution of arginine at this position does not affect inhibition of adenylyl cyclase (14). Therefore, these Switch II residues do not determine the nature of adenylyl cyclase modulation by alpha s and alpha i2.

Although all alpha  subunits are conserved in this Switch II region, other alpha  subunits do not modulate adenylyl cyclase, with the exception of a weak inhibition of type I adenylyl cyclase by alpha o (11). A possible explanation for this selectivity is that other alpha  subunits contain residues that preclude a productive adenylyl cyclase interaction. If so, then replacing alpha i2 residues in the amino terminus of alpha 3 and in the alpha 4/beta 6 loop with the homologous residues from alpha q or other alpha  subunits might cause a larger reduction in ability to inhibit adenylyl cyclase than was observed for alanine substitutions.

Our studies show that the effector-specifying regions of alpha s and alpha i2 overlap but are not identical (see Fig. 6B). Studies using alpha  subunit chimeras localized the region of alpha i2 that specifies inhibition of adenylyl cyclase to a 78-residue segment (amino acids 245-322) that extends from alpha 3 to beta 6 (14). Residues corresponding to two of the three alpha s regions that specify activation of adenylyl cyclase (12, 15), the alpha 3/beta 5 and alpha 4/beta 6 loops, are included in this segment. The only region of overlap that we have found among the effector-specifying regions of alpha s and alpha i2 is in the alpha 4/beta 6 loop. Effector-specifying regions unique for alpha s are located in the alpha 3/beta 5 loop and in the carboxyl-terminal part of Switch II (12). Similarly, mutation of a single residue in the amino terminus of alpha 3 reduces the ability of alpha i2 to inhibit adenylyl cyclase but is not required for the activation of adenylyl cyclase by alpha s (12).

Since both alpha s and alpha i2 interact with adenylyl cyclase, the effector-specifying residues of each alpha  subunit presumably determine whether activation or inhibition will result from alpha  subunit binding. However, the effector-specifying residues of alpha s appear to contribute more to the interaction with adenylyl cyclase than do those of alpha i2. Substitutions in the effector-specifying segment of alpha i2 do not cause as large of a decrease in the ability to inhibit adenylyl cyclase as do substitutions in the conserved middle part of Switch II. However, mutations in two of the effector-specifying regions of alpha s, the nonconserved carboxyl-terminal part of Switch II and the alpha 3/beta 5 loop, decrease effector activation to the same extent as do mutations in the conserved Switch II region.2 Consistent with our results, Taussig et al. (11) found that replacing alpha i1 residues with alpha s homologs in the alpha 3/beta 5 loop results in an alpha  subunit that weakly activates certain adenylyl cyclase isoforms. Thus, the effector-specifying regions of alpha s appear to be dominant over those of alpha i.

Mutagenesis studies of hGH and its receptor, for which a structure of the hormone-receptor complex is available (40), have characterized the functional importance of residues in the binding interface. Individual replacements of residues in hGH (41) and its receptor (42) demonstrated that only a small subset of the residues at the center of the contact region contribute substantially to binding affinity. However, hGH residues in the periphery of the interface, which do not contribute much to the affinity of binding (41), are important for the specificity of binding (43).

In a similar manner, our studies of the interaction between alpha i2 and adenylyl cyclase implicate Switch II residues as being the major contributors to this binding interaction. Substitutions in the effector-specifying segment of alpha i2 have a more modest effect on the ability of alpha i2RCEE to inhibit adenylyl cyclase. In the absence of any structures of alpha  subunit-effector complexes, we predict that interactions between these proteins will include the conserved Switch II region as well as nonconserved specificity regions but that, as seen in the case of hGH and its receptor (41, 42), the contact surfaces may be larger than the "functional epitopes" defined by our mutagenesis studies.


FOOTNOTES

*   This work was supported by grants from the National Institutes of Health and the Patrick and Catherine Weldon Donaghue Medical Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom all correspondence should be addressed. Tel: (203)-785-3202; FAX: (203)-785-4951; E-mail: cathy_berlot{at}quickmail.cis.yale.edu.
1   The abbreviations used are: GTPgamma S, guanosine 5'-O-(thiotriphosphate); hGH, human growth hormone; PDE, cGMP phosphodiesterase; PLC, phosphoinositide phospholipase C.
2   C. H. Berlot, unpublished observations.

ACKNOWLEDGEMENTS

We thank Gernot Walter (UCSD) for the EE hybridoma; Ann Pace, Paul Wilson, and Henry Bourne (UCSF) for the alpha i2RCEE and alpha sEE constructs; and Thomas Hynes (Pfizer, Inc.) and Rolando Medina for helpful discussions and critical reading of the text.


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