Structural Basis for the Selectivity of the RGS Protein, GAIP, for Galpha i Family Members
IDENTIFICATION OF A SINGLE AMINO ACID DETERMINANT FOR SELECTIVE INTERACTION OF Galpha i SUBUNITS WITH GAIP*

Donna S. WoulfeDagger and Jeffrey M. Stadeldagger

From the Department of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania 19104 and the Department of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

GAIP is a regulator of G protein signaling (RGS) that accelerates the rate of GTP hydrolysis by some G protein alpha  subunits. In the present studies, we have examined the structural basis for the ability of GAIP to discriminate among members of the Galpha i family. Galpha i1, Galpha i3, and Galpha o interacted strongly with GAIP, whereas Galpha i2 interacted weakly and Galpha s did not interact at all. A chimeric G protein composed of a Galpha i2 N terminus and a Galpha i1 C terminus interacted as strongly with GAIP as native Galpha i1, whereas a chimeric N-terminal Galpha i1 with a Galpha i2 C terminus did not interact. These results suggest that the determinants responsible for GAIP selectivity between these two Galpha is reside within the C-terminal GTPase domain of the G protein. To further localize residues contributing to G protein-GAIP selectivity, a panel of 15 site-directed Galpha i1 and Galpha i2 mutants were assayed. Of the Galpha i1 mutants tested, only that containing a mutation at aspartate 229 located at the N terminus of Switch 3 did not interact with GAIP. Furthermore, the only Galpha i2 variant that interacted strongly with GAIP contained a replacement of the corresponding Galpha i2 Switch 3 residue (Ala230) with aspartate. To determine whether GAIP showed functional preferences for Galpha subunits that correlate with the binding data, the ability of GAIP to enhance the GTPase activity of purified alpha  subunits was tested. GAIP catalyzed a 3-5-fold increase in the rate of GTP hydrolysis by Galpha i1 and Galpha i2(A230D) but no increase in the rate of Galpha i2 and less than a 2-fold increase in the rate of Galpha i1(D229A) under the same conditions. Thus, GAIP was able to discriminate between Galpha i1 and Galpha i2 in both binding and functional assays, and in both cases residue 229/230 played a critical role in selective recognition.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Heterotrimeric G proteins associate with the cytoplasmic surfaces of 7-transmembrane spanning receptors and function to transduce signals from receptors activated by extracellular ligands to intracellular effectors (1). One of the most recent developments in the study of G protein regulation is the identification of a novel family of proteins known as regulators of G protein signaling or RGS proteins (2). RGS proteins are characterized by the presence of an RGS domain that is structurally conserved across evolution (3, 4). These molecules function to desensitize G protein-coupled responses in organisms from yeast to man by directly interacting with the alpha  subunit of heterotrimeric G proteins and increasing their rate of GTP hydrolysis (5). Direct interaction between G protein alpha  subunits and RGS molecules was first demonstrated by DeVries et al. (6), who isolated the cDNA for the RGS GAIP (G alpha interacting protein) using a yeast two-hybrid screen for Galpha i3-interacting proteins. A number of studies quickly followed revealing GAP (GTPase-activating protein)1 activity to be the mechanism by which RGSs turned off G protein activation (7-10). Both the structural interaction between RGS and Galpha subunits and the mechanism of RGS GAP activity were further elucidated by the co-crystallization of RGS4 with Galpha i1 (11). However, much remains to be revealed about the function of individual members of the RGS family, their specificities for interacting proteins, and the structural determinants that define these interactions.

Most of the initially described RGS proteins showed both binding and functional selectivity for the Galpha i family of G proteins (7-9, 12). More recently, a number of RGS molecules have demonstrated binding or functional interactions with Galpha q and/or Galpha s signaling pathways (13-17), and p115RhoGEF was shown to be a functional RGS for the Galpha 12/Galpha 13 family of G proteins (18-20). However, there has been little information about the ability of any RGS to discriminate among the closely related members of the Galpha i family. Evidence for some specificity of RGS binding to distinct Galpha i family members was demonstrated by DeVries et al. (6), who showed strong interaction of GAIP with Galpha i1, Galpha i3, and Galpha o but weak interaction with Galpha i2 and no interaction with Galpha s. The differential binding characteristics of Galpha i1 and Galpha i2 are particularly intriguing because these two G proteins are highly homologous, having an amino acid sequence identity of 88%. Differences in RGS binding may reveal structural differences in these two G proteins that have implications for their ability to differentially activate divergent downstream signaling pathways.

To evaluate the structural basis for the selectivity of the RGS GAIP for individual members of the Galpha i family, we have expressed native, chimeric, and mutant Galpha proteins and compared their abilities to bind GAIP and act as substrates for GAIP GAP activity. The results show a preference of GAIP for Galpha i1 over Galpha i2 in both binding assays and GAP assays. This preference was reversed by mutating residue Asp229 in Galpha i2 to alanine and making the reciprocal mutation (A230D) in Galpha i1. Interestingly, the selectivity of GAIP for Galpha i1 over Galpha i2 was lost when GTPase-deficient mutants of these two Galpha is were tested for GAIP binding. Thus, the structural preference of GAIP for Galpha i1 versus Galpha i2 in their ground (presumably GDP-bound) states has functional consequences in their respective GAP activities.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Generation of Yeast Two-hybrid Fusion Constructs-- Rat G protein alpha  subunits were PCR amplified with oligonucleotides containing 5' EcoRI restriction sites and 3' SalI restriction sites. PCR products were then subcloned into the pCRII vector (Invitrogen, Carlsbad, CA) and sequenced to ensure fidelity to the template. Inserts were excised with EcoRI and SalI and subcloned into the pGBT9 Gal 4 DNA-binding domain fusion vector (CLONTECH).

Human GAIP was PCR amplified from a human heart cDNA library using oligonucleotides containing a 5' NarI restriction site and a 3' SalI restriction site. PCR products were subcloned and sequenced as above, then removed from pCRII with Nar I and SalI, and subcloned into the pGAD Gal 4 activation domain fusion vector (CLONTECH).

Generation of G Protein alpha  Subunit Chimeras-- The Galpha s/i3 chimera was generated by removing the N-terminal BamHI site in the Galpha s cDNA via site-directed mutagenesis (see below) and then ligating the BamHI-digested N-terminal 700-bp fragment of Galpha s to the 430-bp C-terminal fragment of BamHI-digested Galpha i3 cDNA. The Galpha i3/s chimera was generated by ligating the N-terminal 630-bp Galpha i3 fragment to the C-terminal 516-bp Galpha s fragment of the same digestions. Both chimeras were subcloned into the pGBT9 vector and characterized with BamHI and EcoRI as well as with BamHI and SalI digestions to ensure correct constructions.

Galpha i1/i2 and Galpha i2/i1 chimeras were made by engineering a BamHI site into the Galpha i1 cDNA at the same site as a naturally occurring BamHI in Galpha i2. Galpha i2 and mutant Galpha i1 cDNAs were digested with BamHI, and the N-terminal 635-bp fragment of Galpha i1 was ligated to the C-terminal 433-bp fragment of Galpha i2 to generate Galpha i1/i2. Similarly, Galpha i2/i1 consists of the N-terminal BamHI fragment of Galpha i2 ligated to the C-terminal BamHI fragment of Galpha i1.

Site-directed Mutagenesis of G Protein alpha  Subunits-- Site-directed mutants of Galpha i1 and Galpha i2 were made using Stratagene QuickChange site-directed mutagenesis kit according to the manufacturer's protocols. Template pGBT9-Galpha i1 or pGBT9-Galpha i2 was amplified for 14 cycles of 12-min extensions, each using overlapping forward and reverse primers encoding the applicable mutation. All mutants were sequenced throughout the entire coding region to ensure desired mutagenesis as well as to screen against unwanted PCR-induced mutations.

Transformation of Competent Yeast-- Saccharomyces cerevisiae of strain HF7calpha were co-transformed with pGBT9 (containing Trp marker) and pGAD (containing Leu marker) vector constructions by standard lithium acetate procedures (CLONTECH Matchmaker two-hybrid system). Briefly, single yeast colonies were grown overnight at 30 °C with continuous shaking to an A600 of 0.6. Cells were harvested by centrifugation for 10 min at 3000 rpm, washed once in sterile H20, and resuspended in 2 ml of cold 100 mM lithium acetate. After shaking at 30 °C for 1 h, 100 µl of competent cells was added to 1-2 µg of transforming DNA in the presence of 5 µg of carrier salmon sperm DNA and 0.7 ml of 40% polyethylene glycol. Cells were heat shocked at 42 °C for 15 min, then collected with a quick spin, and plated on -Leu-Trp selective dropout agar medium to grow for 3 days at 30 °C. Four colonies of each construct were streaked on -Leu-Trp agar to propagate for assay.

Immunoblotting-- Yeast transformants were grown overnight to high density in 4-ml cultures, harvested, and resuspended in binding buffer (0.2 M Tris, pH 8.0, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, 20 µg/ml pepstatin A). Cells were lysed by vortexing three times for 1 min in the presence of glass beads at 4 °C and spun for 10 min at 12,000 × g to remove cell debris. 50 µg of lysate was loaded per lane onto SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with rabbit antibody common to G protein alpha  subunits (Calbiochem, La Jolla, CA) at 1:500 dilution in Tris-buffered saline/5% milk. Immunoreactivity was detected with horseradish peroxidase-conjugated donkey anti-rabbit secondary antibody (1:2000 dilution) and developed using ECL reagents according to the manufacturer's protocols (Amersham Pharmacia Biotech).

Liquid beta -Galactosidase Assays-- Single colonies of transformed cells were innoculated into 5 ml of SC-Leu-Trp agar and grown overnight to an A600 of 0.8. Cells were collected by centrifugation, washed once in Z buffer (60 mM Na2HPO4, 40 mM NaH2P04, 10 mM KCl, 1 mM MgSO4), resuspended in 300 µl of the same, and lysed by four freeze/thaw cycles. To start the assay, 100 µl of this cell lysate was suspended in 0.7 ml of Z buffer containing 0.27% beta -mercaptoethanol and then added to 0.16 ml of Z buffer containing 4 mg/ml o-nitrophenyl beta -D-galactopyranoside substrate. Suspensions were vortexed and incubated for 2 h at 30 °C. Color reactions were stopped with 0.4 ml of Na2CO3 and read at A420 after spinning out cell debris. beta -Galactosidase units (21) were calculated according to the manufacturer's protocols (CLONTECH), as follows: beta -galactosidase units = 1000 × A420/(t × v × A600), where t is 120 min of incubation, v is 0.1 ml of reaction volume·concentration factor, and A600 was 0.8 for the culture.

Histidine Growth Assays-- 5-ml cultures of yeast transformants were grown to an A600 of 1.0 and then 3 µl of 1:10 serial dilutions of confluent growths were spotted on either SC-Leu-Trp or SC-Leu-Trp-His agar plates and allowed to grow at 30 °C for 3 days.

Protein Expression and Purification-- Full-length G protein alpha  subunits Galpha i1, Galpha i2, Galpha i1(D229A), and Galpha i2(A230D) and full-length GAIP were expressed as GST fusion proteins by subcloning cDNAs downstream of the GST tag using EcoRI/SalI sites of the vector pGEX-6P-1 (Amersham Pharmacia Biotech). Each plasmid construct was transformed into bacterial strain BL21, grown overnight, and induced to express protein with 0.5 mM isopropyl-1-thio-beta -D-galactopyranoside. Cells were harvested by centrifugation, sonicated in TE containing 0.1 mM phenylmethylsulfonyl fluoride and 1 mM beta -mercaptoethanol, and solubilized with 1% Triton X-100. Lysates were cleared by centrifugation at 12,000 × g for 10 min, and supernatants were applied to pre-washed glutathione-Sepharose columns (Amersham Pharmacia Biotech). Columns were washed with TE containing phenylmethylsulfonyl fluoride and beta -mercaptoethanol and GST fusion proteins eluted with 10 mM glutathione. Purified proteins were buffer exchanged into TED buffer (20 mM Tris-HCl, pH 8, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol), concentrated to 1 mg/ml in Nanosep spin columns (Pall Filtron Corp.), and stored at -80 °C. Size and homogeneity of purified proteins were verified via Coomassie-stained SDS-polyacrylamide gel electrophoresis, and in-frame translation of G proteins was verified via immunoblot using a Galpha i1/Galpha i2-selective antibody (kind gift of Dr. David Manning, University of Pennsylvania, Philadelphia, PA).

GTPgamma S Competition Curves-- 100 nM purified GST-tagged G protein alpha  subunits were shaken for 4 h at 30 °C in the presence of 100 nM [35S]GTPgamma S and serial dilutions of 1-100 µM competing unlabeled GTPgamma S in 50 µl of binding buffer (50 mM HEPES, pH 8, 1 mM EDTA, 2 mM beta -mercaptoethanol, 10 mM MgSO4, 2 mM ATP, 30% glycerol, 1 mg/ml bovine serum albumin) (22). Reactions were filtered over BA85 nitrocellulose filters and washed three times with 2 ml of cold GTPgamma S STOP buffer (20 mM Tris-Cl, pH 8, 25 mM MgCl2, 100 mM NaCl). Filters were immersed overnight in scintillation fluid before counting to determine amount of [35S]GTPgamma S bound.

GTPase Assays-- 100 nM purified GST-tagged G protein alpha  subunits were loaded with 1 µM [gamma -32P]GTP (8000 cpm/pmol) for 20 min at 30 °C in 600 µl of GTPase buffer (0.1% lubrol PX, 50 mM HEPES, pH 7.5, 1 mM dithiothreitol, 5 mM EDTA). Reactions were chilled at 4 °C for 10 min, and assays were conducted at 6 °C. A 50-µl aliquot was removed immediately before initiating the reaction and quenched with 750 µl of 5% Norit activated charcoal in 50 mM NaPO4, pH 3. To initiate the reaction, 100 µM cold GTP and 15 mM MgSO4 (final concentrations) in the presence versus absence of 500 nM GST-tagged GAIP were added to reaction mixtures, and 50-µl aliquots were removed after 10 s, 20 s, 40 s, 1 min, 2 min, 3 min, 4 min, and 5 min and stopped as just described. Charcoal was precipitated by centrifugation for 15 min at 12,000 × g, and 400-µl free phosphate-containing supernatants were counted to determine the amount of Pi released per reaction.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Interactions of G Protein Fusions with GAIP-- To explore the structural basis for the differences in GAIP binding by the different members of the Galpha i family, we engineered a panel of Galpha protein chimeras and mutants. As a first step, native and engineered G proteins were assayed for the ability to bind GAIP using the yeast two-hybrid system. To make use of this system, Galpha protein cDNA constructs were subcloned downstream of a Gal 4-binding domain cDNA and coexpressed with a GAIP-Gal 4 activation domain fusion in the S. cerevisiae strain HF7Calpha . All fusions were immunoblotted to control for relative expression levels. An anti-G protein alpha  subunit antibody raised against the internal GTP-binding sequence common to all heterotrimeric G protein alpha  subunits recognized a protein of the appropriate molecular mass (about 65 kDa) for a G protein alpha  subunit fused to the Gal 4-binding domain in each of the clones transformed with a G protein fusion (data not shown). All of the clones expressed comparable levels of G protein fusion, and no protein of the same size was seen in clones transformed with pGBT9-binding domain alone.

Given such a similar background of G protein fusion expression, a measure of the strength of interaction between various G proteins and GAIP can be estimated from the relative activation of Gal 4-dependent reporters. The yeast strain HF7Calpha was stably transformed with cDNAs encoding both beta -galactosidase and histidine reporters downstream of a Gal 4 promotor. In this system, the promotor is activated in proportion to the degree of interaction between the Gal 4-binding domain and activation domain fusions (23). Thus, two different reporters were used to measure the relative strength of the interaction between the G protein-binding domain fusion and the GAIP activation domain fusion.

According to both histidine and beta -galactosidase reporter systems, robust interaction of GAIP was seen with Galpha i1, Galpha i3, and Galpha o, whereas the interaction with Galpha i2 was weak, and the interaction with Galpha s was undetectable (Fig. 1). These results are consistent with those obtained by DeVries et al. (6). Due to the quantitative nature of the assays, liquid beta -galactosidase assays were used for interaction comparisons henceforth. Because Galpha i1 gave a strong interaction with GAIP in its native conformation, which was statistically indistinguishable from that of Galpha i3 and Galpha o, and because this G protein was tested in every assay conducted, this level of interaction was designated as 100% for comparison with all other G protein constructs. 100% interaction in these assays corresponds to 1.4 beta -galactosidase units (21). The beta -galactosidase activity generated by GAIP co-transfected with pGBT9 vector alone (0.12 beta -galactosidase units) was considered background and was subtracted from all values for G protein-GAIP interactions before normalization.


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Fig. 1.   A, liquid beta -galactosidase assays of native G protein alpha  subunit interactions with GAIP. Yeast clones co-expressing indicated Gal 4-binding domain-Galpha fusions with activation domain-GAIP fusions were assayed for the interaction-dependent activation of a lacZ reporter. The amount of beta -galactosidase released was measured colorimetrically using the substrate ONPG. Two clones of each transformant were assayed and normalized to the interaction of GAIP with Galpha i1 defined as 100%. The results shown are the mean ± S.E. for n = 4-18 in triplicate. B, histidine-minus growth assays of native versus mutant G protein alpha  subunit interactions with GAIP. Yeast clones co-expressing indicated binding domain-Galpha fusions with activation domain-GAIP were assayed for their interaction-dependent activation of a histidine reporter. Each clone was grown and plated as detailed under "Experimental Procedures." Plates on the left show limiting dilutions of clones grown on tryptophan- and leucine-lacking agar medium to control for noninteraction dependent growth. Plates on the right show identical dilutions of clones grown on tryptophan-minus, leucine-minus, and histidine-lacking medium to assay for interaction-dependent histidine reporter expression. This assay has been performed twice with identical results.

Chimeras-- Because Galpha i3 interacted strongly with GAIP, whereas Galpha s did not interact at all, chimeras of Galpha i3 with Galpha s were generated in an attempt to localize the regions of Galpha i required for GAIP binding. A BamHI site that cuts both cDNAs roughly two-thirds into the length of the coding region was used to generate both chimeras (Fig. 2). This BamHI site conveniently separates all of the N-terminal alpha -helical domain from most of the GTPase domain (a small part of which is encoded at the very N terminus of the cDNA). The binding characteristics of these chimeras could thus substantiate the relative importance of these two domains in GAIP binding. However, neither chimera bound to GAIP (Fig. 2A). These results potentially indicate that both domains of Galpha i3 contribute important determinants for RGS binding, but the divergence in alpha s sequence from that of the alpha i family presents a number of other possible interpretations.


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Fig. 2.   Relative interaction of Galpha chimeras with GAIP. Liquid beta -galactosidase assays were conducted as described in the legend to Fig. 1A. Two clones of each transformant were assayed. The results shown are the means ± S.E. for n = 2-18 in triplicate. A, interaction-dependent release of beta -galactosidase from clones expressing chimeras of Galpha i3 and Galpha s. A schematic diagram of the chimeras is shown at the bottom. B, interaction-dependent release of beta -galactosidase from clones expressing chimeras of Galpha i1 and Galpha i2. A schematic diagram of these chimeras is shown at the bottom, where the asterisk indicates the position of Galpha i1 Asp229.

To discriminate among these possibilities, chimeras composed of the initial two-thirds of alpha i1 fused to the distal one-third of alpha i2 and the reciprocal Galpha i2/i1 chimera were prepared using an engineered BamHI site (Fig. 2B). Galpha i2 is highly homologous to Galpha i1, yet its interaction with GAIP is negligible compared with Galpha i1. The Galpha i2/i1 chimera interacted with GAIP just as strongly as native Galpha i1, whereas the reverse Galpha i1/i2 chimera, like wild type Galpha i2, showed little binding to GAIP (Fig. 2B). These results suggest that the Galpha i1 C terminus is required for GAIP interaction. The results may also imply that the determinants contributing to GAIP binding are entirely contained within the GTPase domain of the G protein, but there may be additional determinants that are conserved between the N termini of Galpha i1 and Galpha i2 that remain to be identified.

Site-directed Mutants-- As a next step, site-directed mutagenesis of Galpha i1 and Galpha i2 was used to further localize determinants contributing to the selectivity of GAIP interaction. Because Galpha i1 and Galpha i2 are 88% identical at the amino acid level but show vastly different GAIP binding capacities in the yeast two-hybrid system, the primary sequences of the two proteins were compared with identify candidate residues that might contribute to differential GAIP binding. Of the amino acids that differed between Galpha i1 and Galpha i2, reciprocal mutants were generated at eight different positions in the primary sequence based on the likelihood that a given position would affect RGS binding given its location in the three-dimensional crystal structure of Galpha i1 bound to RGS4 (11). The effects of C-terminal mutants were of particular interest due to the results of the chimeras, but a number of N-terminal mutants were also studied because they appeared to be close to potential RGS contact sites in the crystal structure (11). Of the five Galpha i1 mutants C-terminal to the BamHI site that were tested, several impaired binding to GAIP, but only D229A abolished it (Fig. 3A). Even more significantly, the reciprocal mutation in the corresponding residue in Galpha i2 (Galpha i2(A230D))2 produced a variant Galpha i2 that bound to GAIP as strongly as Galpha i1 (Fig. 3A). Thus, Galpha i1(D229) appears to be particularly important for GAIP interaction.


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Fig. 3.   A, relative interaction of C-terminal point mutants with GAIP. B, relative interaction of N-terminal point mutants with GAIP. Liquid beta -galactosidase assays were conducted as described in the legend to Fig. 1A. Two clones of each transformant were assayed. The results shown are the means ± S.E. for n = 2-18 in triplicate.

In addition to the C-terminal mutants shown in Fig. 3A, three N-terminal Galpha i1 mutants and the corresponding reciprocal Galpha i2 mutants were also assayed for beta -galactosidase activity. Consistent with the results of the Galpha i1/i2 chimeras, all of the N-terminal Galpha i1 mutants bound to GAIP, and none of the corresponding Galpha i2 mutants bound GAIP as strongly as Galpha i1 (Fig. 3B). Thus, none of these residues appears to be a necessary determinant for GAIP binding.

GTPase-deficient Mutants-- To determine whether different nucleotide-dependent conformations of these G proteins affected their relative GAIP affinities, GTPase-deficient mutants of Galpha i1 and Galpha i2 were generated to "trap" the alpha  subunits in their GTP-bound forms and assayed for binding to GAIP. In contrast to the wild type proteins, the "activated" forms of both Galpha i1 and Galpha i2 interacted at least as strongly with GAIP as wild type Galpha i1 (Fig. 4). Both Galpha i1(Q204L) and Galpha i2(Q205L) generated about a 4-fold increase in GAIP binding activity relative to that seen with wild type (nonactivated) Galpha i1, so that the selectivity of GAIP for Galpha i1 over Galpha i2 appears to be restricted to the interaction with their ground state (presumably GDP-bound) conformations. Two additional GTPase-deficient mutants, Galpha i1(R178C) and Galpha i2(R179C), were also tested and interacted very strongly with GAIP although less strongly than the Q204L/Q205L mutants.


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Fig. 4.   Relative interaction of GTPase-deficient mutant versus native Galpha subunits with GAIP. Liquid beta -galactosidase assays were conducted as described in the legend to Fig. 1A. Two clones of each transformant were assayed. The results shown are the means ± S.E. for n = 6-18 in triplicate.

Nucleotide Binding Affinity-- To explore the mechanism of the selectivity of GAIP for Galpha i1 over Galpha i2 in their GDP-bound states, the position of Galpha i1 aspartate 229 in relation to the bound RGS4 molecule in the published crystal structure was examined (Fig. 5). In the AlF4-activated state in which this G protein was crystallized, Asp229 appears closer to the nucleotide-binding site than to the RGS-binding site of this G protein. Therefore, we examined the relative GTPgamma S affinities of both Galpha i1 and Galpha i2 to determine whether there were differences in nucleotide binding affinity that in turn might affect their affinities for GAIP. Recombinant full-length Galpha i1, Galpha i2, Galpha i1(D229A), and Galpha i2(A230D) were GST-tagged, expressed in bacteria, and purified to homogeneity over glutathione affinity columns. The ability of unlabeled GTPgamma S to displace [35S]GTPgamma S from each of the proteins was measured over a range of GTPgamma S concentrations. The IC50 for [35S]GTPgamma S displacement was the same for all four proteins (Fig. 6), so differences in GAIP binding are not reflective of differences in nucleotide binding affinities.


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Fig. 5.   Position of Galpha i1 Asp229 in relation to bound RGS4 and GDP-Mg2+-AlF4 molecules. PDB 1AGR (2) showing the cocrystallization of Galpha i1 with RGS4 was downloaded from the Brookhaven National Labs Protein Data Bank and viewed using RasMol. The Galpha i1 subunit is shown in dark blue bound to a cyan RGS4 molecule. GAIP binding specificity determinant Galpha i1(Asp229) is pictured in yellow at the top of the pink Switch 3 region of Galpha i1. The bound GDP-AlF4 is the adjacent structure in green. Galpha i residues Arg178 and Gln204 are highlighted in red.


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Fig. 6.   Competition between GTPgamma S and 35S-GTPgamma S for binding to native and mutant G protein alpha  subunits. Purified GST-tagged G protein alpha  subunits (100 nM) were incubated with 100 nM [35S]GTPgamma S (200,000 cpm/50 µl assay volume) and indicated concentrations of unlabeled GTPgamma S and filtered as described under "Experimental Procedures." Each value is the mean ± S.E. of three experiments performed in triplicate.

GAP Activity-- Finally, to determine whether any functional differences might correlate with selective binding capacity, we tested the ability of GAIP to catalyze the GTPase activities of Galpha i1, Galpha i2, Galpha i1(D229A), and Galpha i2(A230D). GAIP catalyzed a 5-fold increase in the rate of GTP hydrolysis by Galpha i1 (Fig. 7A) but caused no increase in the GTPase rate of Galpha i2 (Fig. 7B) under the same conditions. In addition, GAIP only slightly increased the GTPase activity of Galpha i1(D229A) (from Kobs of 2.1 in the absence of GAIP to Kobs of 3.7 in the presence of GAIP) (Fig. 7C). Of particular interest, the rate of GTP hydrolysis seen for this mutant form of Galpha i1 in the presence of GAIP is similar to the GTPase rate of Galpha i2 in the presence of GAIP (Kobs = 4.2). Similarly, Galpha i2(A230D) now behaves more like Galpha i1 in that there is a significant increase in GAIP activation, and the GTPase rate seen in the presence of GAIP is similar to that seen for Galpha i1 in the presence of GAIP (Kobs = 5.2 for the former and 5.6 for the latter) (Fig. 7D). Therefore, the ability of GAIP to act as a GAP for these two Galpha i proteins and their reciprocal mutants correlates with its affinities for these proteins in their "ground states" as measured in the yeast two-hybrid assay.


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Fig. 7.   Effect of GAIP on single turnover GTPase activity of purified GST-tagged native and mutant Galpha subunits. Squares, G protein alone; triangles, G protein in the presence of GAIP. GST-GAIP (500 nM) was added to 100 nM [32P]GTP-loaded Galpha subunits in the presence of Mg2+ and excess unlabeled GTP to initiate reactions. A, Galpha i1; B, Galpha i2; C, Galpha i1(D229A); D, Galpha i2(A230D). Aliquots were removed at the indicated times, and free 32Pi released was measured. An average of 470 fmol 32Pi was released per assay, which was normalized to 100%. Values given are the means of four experiments for A, B, and C and the means of six experiments for D. The observed rate constants (Kobs) for each reaction were calculated based on an exponential association curve fit using GraphPad Prism.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

RGS proteins are a family of G protein regulators that down-regulate G protein-coupled responses by stimulating the GTPase activity of the Galpha subunits to which they bind (3, 5). Both the G protein binding and GAP activity of RGS molecules have been localized to a 130-amino acid domain (RGS domain) that is conserved among all RGS proteins (6, 10, 24). Within this domain, a number of residues have been shown to serve as contact points for Galpha protein binding (11, 25, 26).

Elucidation of the sites on G proteins with which RGS proteins interact and the selectivity of RGS proteins for different forms of Galpha have important implications for the mechanism by which RGSs stimulate alpha  subunit GTPase activity. The observation that RGS4 binds more strongly to the AlF4-GDP-Mg2+-bound state of Galpha i than to the GDP or GTP-bound states suggests that RGSs exhibit GAP activity by stabilizing the transition state for GTP hydrolysis by Galpha (7-9, 27, 28). The crystal structure of AlF4-GDP-Mg2+-Galpha i bound to RGS4 further reveals that the RGS interacts directly with the Switch regions of Galpha i, reducing their flexibility in this transition state mimic and thus further supporting this proposed GAP mechanism (11). It has also been observed that the sites on Galpha to which RGS proteins bind may interfere with the binding of the effector PLCbeta 1, suggesting another possible mechanism for Galpha i down-regulation by RGSs (13).

The sites on G protein alpha  subunits responsible for the selectivity with which RGS proteins bind have been less well studied. DeVries et al. (29) showed a significantly reduced GAIP interaction with a 10-amino acid truncation of Galpha i3, but a chimeric Galpha q containing the last 10 residues of Galpha i3 did not bind to GAIP, indicating that other determinants remain to be identified. More recently, Lan et al. (30) showed that a G184S mutation in Galpha o and the equivalent mutation in Galpha i1 prevents both binding to and activation by RGS4, extending the observation by DiBello et al. (31) that a mutant Gpa1 prevented a functional interaction with the yeast RGS sst2. However, because this glycine is a highly conserved Switch 1 residue, it appears to be required for all Galpha interactions with RGS molecules rather than a determinant for specificity. Finally, Natochin and Artemyev (32) showed that the interaction of Galpha t with human retinal RGS could be abolished by mutating serine 202 to the corresponding Galpha s aspartate, providing one candidate Galpha s site that might interfere with RGS binding. They recently extended this finding by showing that mutation of this Galpha s aspartate (Galpha s Asp229) to the serine which occurs in Galpha i family members at the corresponding Switch 1 position promotes binding to an RGS (33).

To extend the characterization of RGS/G protein specificities and their structure/function relationships, we sought to identify regions in the Galpha subunit that contributed to GAIP binding selectivity by testing the relative interaction strengths of GAIP with a number of native G protein alpha  subunits, mutants, and chimeras using the yeast two-hybrid system. In this system, GAIP interacts equally strongly with native forms of Galpha i1, Galpha i3, and Galpha o but very weakly with Galpha i2 and not at all with Galpha s. Both Galpha s/i3 and Galpha i3/s chimeras disrupted GAIP binding, indicating either that both the N and C termini of the Galpha i subunit contain determinants required for binding or that divergent sequences in the Galpha s protein relative to Galpha i may interfere with GAIP contact points. Galpha i1/i2 and Galpha i2/i1 mutants gave more interpretable results, indicating that the C-terminal domain of Galpha i1 is required for GAIP binding. This region constitutes most of the GTPase domain of the G protein, which is consistent with reports showing that GAIP binds in a groove within this domain (11). By comparison, the failure of either Galpha s chimera to bind may indicate that N-terminal inserts in the Galpha s sequence (such as amino acids 72-86) relative to Galpha i interfere with the RGS-Galpha binding surface or that other divergent residues in the Galpha s N-terminal portion interfere with RGS contact. The interfering aspartate (Galpha s residue 229) proposed by Natochin and Artemyev (32, 33) is in fact in the N-terminal portion of our chimeras, consistent with this possibility.

To further localize the region in the G protein C terminus responsible for GAIP selectivity, site-directed mutants were generated in which residues in Galpha i1 and Galpha i2 were swapped. Candidate residues were chosen on the basis of their conservation in Galpha i1 and Galpha i3 and divergence in Galpha i2. The mutation of aspartate 229 of Galpha i1 to the alanine present in Galpha i2 nearly abolished GAIP binding. Conversely, when aspartate was substituted for the alanine normally present at the same site in Galpha i2, the mutant Galpha i2 bound GAIP to the same extent as native Galpha i1. These results reveal the importance of aspartate 229 for the binding of Galpha i subunits in their native state to GAIP and potentially suggest a site of physical contact with GAIP. Yet, upon inspection of the Galpha i1-RGS4 crystal structure, this aspartate appears quite far from the sites of RGS4 interaction. Due to the location of Galpha i1 aspartate 229 at the far N terminus of Switch 3, it is possible that the position of this amino acid in the AlF4 transition state analogue in which it was co-crystallized with RGS4 differs from its position in the nonactivated state in which the Galpha is show selectivity for binding to GAIP. That is, it may be that in its GDP-bound (ground state) conformation, Galpha i1 Asp229 is in closer proximity to GAIP than in its AlF4-Mg2+-GDP-bound conformation.

Closer inspection of the RGS4-Galpha i1 crystal structure presents an alternative explanation. In this structure, aspartate 229 appears to be involved in a relay system that connects its carbonyl through a water molecule to lysine 270, which in turn maintains a hydrophobic interaction with GDP in the RGS4-Galpha i1 crystal structure. We hypothesized that removal of the carbonyl group at this position by mutation to an alanine might disrupt this relay system, destabilizing the binding of nucleotide and hence the binding of RGS, because its binding is dependent on the nucleotide-bound state of the G protein. To test this possibility, IC50 values for the ability of GTPgamma S to compete [35S]GTPgamma S binding by Galpha i1, Galpha i2, Galpha i1(D229A), and Galpha i2(A230D) were compared. The displacement curves were identical in all cases, implying that differences in nucleotide binding capacities do not account for RGS binding differences.

Finally, to determine whether there is also selectivity by GAIP for Galpha i1 versus Galpha i2 in their GTP-bound forms, GTPase-deficient mutants of both Galpha i1 and Galpha i2 were engineered and tested for GAIP binding in the yeast two-hybrid system. Interestingly, both Galpha i1(Q204L) and Galpha i2(Q205L) exhibited similarly high binding affinities to GAIP (about four times the native Galpha i1 interaction), consistent with an inability by GAIP to discriminate between the two proteins in their GTP-bound states. The Galpha i1(R178C) and Galpha i2(R179C) GTPase-deficient mutants interacted less strongly than the Q204L/Q205L mutants, although still more strongly than their native counterparts. This may reflect the ability of RGS proteins to partially restore the GTPase activity of R178C mutants, but not Q204L mutants (7), such that Q204L mutants remain in their GTP-bound states, but R178C mutants may reflect a mixture of conformations. These data also bring up an alternative explanation for the preferential binding of GAIP to nonmutated Galpha i1 over Galpha i2, namely that there is a greater population of GTP-bound Galpha i1 than GTP-bound Galpha i2 in the yeast cell. This could result from different rates of GTP/GDP exchange or GTP turnover by the two alpha  subunits. Formally, that remains a possibility. However, because mammalian Galpha proteins do not couple to yeast G protein-coupled receptors (34) and because G proteins remain GDP-bound in the absence of receptor stimulation (35), we find it more likely that there is a structural difference between the two Galpha is that is recognized by GAIP only in their nonactivated states.

To determine whether the ability of GAIP to discriminate between Galpha i1 and Galpha i2 only in their GDP-bound states has any functional significance, we measured the GAP activity of GAIP with each of these proteins and their mutants. GAIP enhanced the rate of GTP hydrolysis of Galpha i1 but not Galpha i2 under similar conditions. Furthermore, as predicted by the binding studies, Galpha i1(D229A) was a poor substrate for GAIP GAP activity compared with native Galpha i1, and Galpha i2(A230D) was comparable with Galpha i1 as a substrate for GAIP GAP activity. Although Berman et al. (7) showed GAIP-catalyzed increases in GTPase activity of both Galpha i1 and Galpha i2, Heximer et al. (17) also showed a greater enhancement by GAIP of Galpha i1 over Galpha i2 GTPase activity. Our results indicate that GAIP preferentially enhances Galpha i1 over Galpha i2 GTPase activity and that this activity correlates with the binding selectivity shown for Galpha is in their ground state conformations. In addition, because GTPase-deficient mutants of both alpha i1 and alpha i2 subunits bind tightly to GAIP, these results may imply that GAP binding is not sufficient for GAP catalytic activity. Indeed, differential effects on Galpha binding versus GAP activity were discerned by Chen et al. (25) using various RGS mutants, consistent with this idea. It may be that the difference in the binding affinities for GTP-bound versus GDP-bound Galpha conformations drives GTP hydrolysis, so that binding to the activated G protein conformation is not the only indicator of RGS functional selectivity.

The functional selectivity displayed by GAIP and other RGS proteins for G protein partners in vivo remains to be explored. The contributions of additional interacting partners, including C-terminal tails of GPCRs (36) and additional effector proteins (18-20), and post-translational modifications (37) will have to be considered to determine how individual RGS proteins modulate specific G protein signaling pathways.

    ACKNOWLEDGEMENTS

We sincerely thank Skip Brass for much help with the manuscript, Dave Manning and Eliot Ohlstein for encouragement and advice, Katie Freeman for yeast expertise and many helpful discussions, and Cathy Peishoff for help with structural interpretations.

    FOOTNOTES

* 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 correspondence should be addressed: University of Pennsylvania, 421 Curie Blvd., BRB-2, Rm. 913, Dept. Medicine, Philadelphia, PA 19104; E-mail: woulfe{at}pharm.med.upenn.edu.

dagger Deceased.

2 A one-residue insertion at amino acid 117 in the primary sequence of Galpha i2 with respect to Galpha i1 is responsible for the difference in numbering between these two alpha  subunits.

    ABBREVIATIONS

The abbreviations used are: GAP, GTPase-activating protein; PCR, polymerase chain reaction; bp, base pair; GST, glutathione S-transferase; GTPgamma S, guanosine 5'-O-(thiotriphosphate).

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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