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Originally published In Press as doi:10.1074/jbc.M702595200 on June 21, 2007

J. Biol. Chem., Vol. 282, Issue 33, 24231-24238, August 17, 2007
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Kelch Repeat Protein Interacts with the Yeast G{alpha} Subunit Gpa2p at a Site That Couples Receptor Binding to Guanine Nucleotide Exchange*

Thiruvur Niranjan1, Xuedong Guo, Jacob Victor, Ailan Lu2, and Jeanne P. Hirsch3

From the Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, New York 10029

Received for publication, March 26, 2007 , and in revised form, June 11, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The kelch repeat-containing proteins Krh1p and Krh2p are negative regulators of the Gpa2p signaling pathway that directly interact with the G protein {alpha}-subunit Gpa2p in the yeast Saccharomyces cerevisiae. A screen was carried out to identify Gpa2p variants that are defective in their ability to bind Krh1p but retain the ability to bind another Gpa2p-interacting protein, Ime2p. This screen identified amino acids Gln-419 and Asn-425 as being important for the interaction between Gpa2p and Krh1p. Gpa2p variants with changes at these positions are defective for Krh1p binding in vivo. Cells containing these forms of Gpa2p display decreased heat shock resistance and increased expression of a gene required for pseudohyphal growth. These findings indicate that the substitutions at positions 419 and 425 confer a degree of constitutive activity to the Gpa2p {alpha}-subunit. Residues Gln-419 and Asn-425 are located in the beta6-{alpha}5 loop and {alpha}5 helix of Gpa2p, which is the region that couples receptor binding to guanine nucleotide exchange. The results suggest that binding of Gpa2p to Krh1p does not resemble the binding of G{alpha} subunits to either Gbeta subunits or effectors, but it instead represents a novel type of functional interaction.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The yeast G protein {alpha}-subunit Gpa2p controls a signaling pathway that promotes rapid cell growth, inhibits stress responses, and induces filamentous growth under conditions of nutrient limitation. The effect of this pathway on cell growth appears to be due, at least in part, to an increase in the rates of ribosome biogenesis and protein translation that is mediated by Gpa2p (1, 2). Similarly, activation of Gpa2p causes an increase in invasive and pseudohyphal growth, as well as a decrease in stress responses such as heat shock resistance, sporulation efficiency, and accumulation of storage carbohydrates (3–5). Consistent with these findings, cells carrying a gpa2{Delta} mutation display a defect in filamentous growth and an increase in stress resistance (3, 6, 7). gpa2{Delta} mutants also display an increase in replicative life span, which is the number of mitotic divisions completed by a cell prior to senescence (8–10). The effect of the Gpa2p pathway on life span could be due either to its role in promoting cell growth or to its role in inhibiting stress responses.

The way in which signals are transmitted through the Gpa2p pathway is not completely understood. The most well established component of this pathway is the receptor that couples to Gpa2p, which is called Gpr1p (4, 11, 12). The Gpr1p receptor is also required for pseudohyphal and invasive growth, and this requirement can be overcome by constitutive activation of Gpa2p, as would be expected for a G protein that acts downstream of its coupled receptor (13, 14). Gpr1p is present on the cell surface, indicating that it detects an extracellular signal (4). Recent evidence indicates that Gpr1p is a low affinity glucose receptor that responds to high concentrations of glucose in the extracellular environment (15, 16).

Other components of the Gpa2p pathway are still in the process of being characterized. A functional survey of the yeast genome has failed to reveal candidate genes encoding beta- and {gamma}-subunits that could form a classical G protein heterotrimer with the Gpa2p {alpha}-subunit (3). However, some progress has been made in identifying potential downstream effectors by characterizing proteins that display genetic or physical interactions with Gpa2p.

At least one component of Gpa2p signaling appears to involve stimulation of adenylyl cyclase by Gpa2p, resulting in production of cAMP and activation of protein kinase A (PKA).4 Although it was originally reported that a gpa2{Delta} mutation does not affect the increase in cAMP that occurs upon addition of glucose to starved cells (17), subsequent studies have shown that under certain conditions a gpa2{Delta} mutation does cause a decrease in the glucose-induced cAMP spike (7, 12, 14). Moreover, recent evidence indicates that adenylyl cyclase binds to Gpa2p in a manner that is highly specific for the GTP-bound form of the {alpha}-subunit (5). Therefore, it is likely that adenylyl cyclase functions as a downstream effector of Gpa2p.

Stimulation of adenylyl cyclase activates the cAMP-dependent kinase PKA, and genetic evidence indicates that Gpa2p functions as a positive regulator of PKA signaling. Deletion of TPK2, which encodes the PKA catalytic subunit required for filamentous growth, eliminates the effect of constitutively active Gpa2p on expression of FLO11, which encodes a cell surface flocculin required for filamentous growth (18). Similarly, the effects of Gpa2p activation on its transcriptional targets are eliminated in a strain containing a variant form of PKA that has a very low level of unregulated kinase activity (1).

Some effects of Gpa2p activation cannot be explained by stimulation of adenylyl cyclase. Therefore, a number of studies have attempted to identify additional effectors of Gpa2p. Other potential effectors that could transmit signals from Gpa2p include phospholipase C (19), the Ras GTPase-activating proteins Ira1p and Ira2p (20), and the kelch repeat proteins Krh1p and Krh2p (also called Gpb2p and Gpb1p, respectively) (5, 18, 21).

The KRH1 (for kelch repeat homolog) gene was identified in a two-hybrid screen for proteins that interact with Gpa2p; KRH2 encodes a protein that is 35% identical to Krh1p (5, 18, 21). Krh1p and Krh2p do not display any obvious sequence identity to other known proteins, but they do contain seven kelch repeats, segments of about 50 amino acids that contain a characteristic double glycine motif (22). Cells containing krh1{Delta} krh2{Delta} mutations display phenotypes indicative of a high level of PKA activity, including increased pseudohyphal growth, increased FLO11 expression, decreased heat shock resistance, and decreased storage of reserve carbohydrates (5, 18, 21). These results demonstrate that Krh1p and Krh2p function as negative regulators of PKA signaling. Moreover, deletion of KRH1 and KRH2 causes increased phosphorylation of PKA substrates (23), confirming that Krh1p and Krh2p negatively regulate PKA kinase activity. Some evidence indicates that the effect of Krh1p and Krh2p on PKA is not through the regulation of cAMP levels. For example, in cells that lack all adenylyl cyclase function, deletion of KRH1 and KRH2 lowers the concentration of cAMP required to maintain viability (5, 23). Moreover, krh1{Delta} krh2{Delta} mutations have effects in cells that lack either the high affinity or low affinity cAMP phosphodiesterases (23). Given that adenylyl cyclase and the cAMP phosphodiesterases control the synthesis and degradation of cAMP, these results appear to indicate that Krh1p and Krh2p inhibit PKA by a process that does not involve regulating the intracellular cAMP concentration. Consistent with this idea, two-hybrid assays performed in krh1{Delta} krh2{Delta} strains reveal a decrease in binding between the catalytic and regulatory subunits of PKA, which would be expected to produce an increase in PKA activity (5). However, it has also been shown that krh1{Delta} krh2{Delta} strains are defective in their ability to generate a glucose-induced cAMP spike (5, 21). This result can be interpreted to mean either that the kelch repeat proteins act upstream of adenylyl cyclase or that the high level of PKA activity in these strains results in feedback inhibition of cAMP synthesis (24). Therefore, it is still not clear precisely how Krh1p and Krh2p function in the Gpa2p signaling pathway.

Here we show that substitution of certain amino acids in the region of Gpa2p that couples receptor binding to guanine nucleotide exchange eliminates its ability to bind Krh1p. Changes at these positions resulted in phenotypes associated with an increase in the amount of activated Gpa2p. These results indicate that the kelch repeat proteins bind to Gpa2p in a manner that does not resemble that of other G{alpha}-binding proteins. Instead, this study suggests that the interaction between the kelch repeat proteins and the G protein {alpha}-subunit represents a novel functional relationship.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Media—Yeast cells were grown on YEPD (2% glucose), and strains under selection were grown on synthetic dropout media, as described (4). Strains used for two-hybrid assays were PJ69-4A (25) and HF7c (Clontech). Strain SKY762 has the genotype MATa ura3-52 trp1::hisG leu2::hisG his3::hisG, and strain HS188-5D is a derivative of SKY762 that contains a gpa2::TRP1 allele, which was constructed as described (4).

Plasmid Construction—Site-directed mutagenesis was performed on plasmid pT7-LG-GPA2, which consists of a 1.6-kb BamHI fragment containing the GPA2 gene cloned into the BamHI site of pT7-blue (4). A to G substitutions were introduced at positions 1273, 1274, and 1256 (numbers refer to nucleotide positions in the GPA2 coding sequence) to produce plasmids pG21273-T7, pG21274-T7, and pG21256-T7, respectively. Construction of plasmid pGBT9-GPA2 has been described previously (4). To construct plasmids pG21273G-T9.1, pG21274G-T9.1, and pG21256G-T9.1, the KpnI-PstI fragment in pGBT9-GPA2 was replaced with the corresponding KpnI-PstI fragments from pG21273-T7, pG21274-T7, and pG21256-T7, respectively.

To construct a version of GPA2 containing a nuclear localization sequence at the N terminus, first an NcoI site was introduced at the start codon of GPA2 by performing site-directed mutagenesis on pT7-LG-GPA2 to produce pGPA2NC-T7.1. Then oligonucleotides NLS1F (5'-CATGCCAAAGAAGAAGAGAAAGGTCATGCATGC-3') and NLS2R (5'-CATGGCATGCATGACCTTTCTCTTCTTCTTTGG-3') were annealed, and the double-stranded fragment was cloned into the NcoI site of pGPA2NC-T7.1 to produce pNLSGPA2-T7.2. The 1.6-kb BamHI fragment from pNLSGPA2-T7.2 was cloned into the BamHI site of YEp352 to produce pNLSGPA2-352.1. To construct plasmids pNLSG2ND-352.11, pNLSG2NS-352.10, and pNLSG2QR-33.26, the 0.35-kb BssHII-SacI fragment in pNLSGPA2–352.1 was replaced with the corresponding 0.35-kb BssHII-SacI fragments containing A to G substitutions at positions 1273, 1274, and 1256, respectively.

Construction of plasmid pGPA2-33.1 has been described previously (4). To construct plasmids pG2ND-33.11, pG2NS-33.10, and pG2QR-33.9, the 1.6-kb BamHI fragments from pG21273-T7, pG21274-T7, and pG21256-T7, respectively, were cloned into the BamHI site of YCplac33.

Construction of plasmid YEp181-FLKRH1 has been described previously (18). A NotI site was introduced after the start codon of the KRH1 gene by site-directed mutagenesis, and a 0.7-kb NotI fragment containing the green fluorescent gene was cloned into this NotI site to produce YEp181-GFP.FLKRH1.

Mutagenic PCR and Two-hybrid Screen—Error-prone PCR was performed as described previously (26), using pGBT9-GPA2 as a template in 30 cycles of PCR with oligonucleotide primers T91F (5'-CCTCGAGAAGACCTTGACATGATT-3') and T92R (5'-GGTAGAGGTGTGGTCAATAAGAGC-3'). Yeast strain PJ69-4A (25) carrying plasmid TH14 (18) was cotransformed with the pool of PCR-generated mutagenized fragments and linearized vector pGBT9, which had been digested with BamHI and PstI, allowing recombination to occur in vivo. Transformants were replica-plated to synthetic medium plates containing 3-amino-1,2,4-triazole (10 mM) without histidine, and colonies that did not grow were identified. Mutated versions of pGBT9-GPA2 from colonies that did not grow on selective medium were isolated, amplified in Escherichia coli, and transformed into strain PJ69-4A carrying either plasmid TH14 (KRH1) or TH1-1 (IME2). Versions of pGBT9-GPA2 that scored negatively for interaction with TH14 but positively for interaction with TH1-1 were sequenced. Nucleotide changes identified in the plasmids of interest were individually introduced into pGBT9-GPA2 by site-directed mutagenesis to identify the change that was responsible for the phenotype.

RNA Isolation and Real Time RT-PCR—Yeast RNA extraction was performed as described previously (4), with the following modifications. After ethanol precipitation, the RNA sample was incubated with 50 units of DNase I (10 units/µl; Roche Applied Science) in Buffer H (Roche Applied Science) at 30 °C for 60 min. Purified RNA was resuspended in 30 µl of TE; the A260 was measured, and the concentration was adjusted to 0.5 µg/µl.

For real time RT-PCR, primers oFLO11.9 (5'-GTTATTACCACTGAGTCATCTGTTG-3') and oFLO51 (5'-TGTTGTAGCTAGTTGGGATGTAG-3') were used to amplify a 193-bp segment of FLO11. Primers oActin1 (5'-TCGTGCTGTCTTCCCATCTATC-3') and oActin2 (5'-GTAGAAGGTATGATGCCAGATC-3') were used to amplify a 191-bp segment of ACT1, and the relative RNA levels derived from this reaction were used to standardize the amount of mRNA in the samples. Standard curves were produced by serially diluting RNA samples. The reactions were performed using a LightCycler-RNA Master SYBR Green I kit (Roche Applied Science) as described by the manufacturer with the following modifications: a 20-µl standard reaction contained 1.3 µl of 50 mM manganese acetate, 2 µl of primer mix (5 mM each), 7.5 µl of RNA Master SYBR Green 1 reaction mixture, and 1 µl of RNA sample (0.5 µg/µl). The cycling program consisted of 95 °C for 5 s, 55 °C for 5 s, and 72 °C for 12 s, for 45 cycles. Real time RT-PCR was performed on a LightCycler version 3.5 instrument (Roche Applied Science), and the results were analyzed using LightCycler software.

Yeast Methods—Yeast transformation and heat shock assays were performed as described previously (4) except that cells were incubated for 45 min at 50 °C for the heat shock assay. beta-Galactosidase assays were performed using 30 ml of pelleted log phase cells resuspended in 200 µl of lysis buffer (0.1 M Tris-HCl, pH 7.5, 0.05% Triton X-100). Cells were disrupted by freezing and thawing, and reaction mixtures contained 1.3 mg of 2-nitrophenyl-beta-D-galactopyranoside. Units were calculated as 1000 x A420/time(min) x protein concentration (mg).

Microscopy—Microscopy was performed with live cells in culture medium at room temperature. Fluorescence microscopy was performed using a Zeiss Axioplan 2 microscope with a x63 objective and an fluorescein isothiocyanate filter set (Chroma Technology).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Screen for Gpa2p Variants Defective for Krh1p Binding Identifies Two Critical Residues—The kelch repeat-containing proteins Krh1p and Krh2p (also called Gbp2p and Gbp1p, respectively) are negative regulators of yeast PKA signaling that bind to the G protein {alpha}-subunit Gpa2p (18, 21). To explore further the functional relationship between the kelch repeat proteins and the G{alpha} subunit, a two-hybrid screen was performed to isolate alleles of GPA2 that encode products that are defective for Krh1p binding. The strategy for this screen was to isolate GPA2 alleles that produce a negative result in a two-hybrid assay with a KRH1 plasmid as well as a positive result with a plasmid encoding another Gpa2p-interacting protein. This strategy ensured that the isolated GPA2 mutant alleles encode stable proteins that retain at least one function.

Mutant alleles of GPA2 were generated by error-prone PCR followed by gap repair using a plasmid with GPA2 fused to the GAL4 DNA binding domain (4). Mutagenized GPA2 plasmids were tested in a two-hybrid reporter strain containing a plasmid with a portion of KRH1 fused to the GAL4 activation domain (18). GPA2 plasmids that tested negative in this assay (His-, Ade-) were isolated and re-transformed into strains containing either the KRH1 construct or another GAL4-AD fusion plasmid that encodes a different Gpa2p-interacting protein, Ime2p (27). The GPA2 alleles of interest score negatively for interacting with Krh1p but positively for interacting with Ime2p. Several GPA2 alleles fulfilling these criteria were isolated. Mutant GPA2 alleles specifically defective for the interaction with Krh1p were sequenced to identify the changes that cause decreased binding. In general, these alleles contained multiple mutations. Therefore, individual mutations in GPA2 were made by site-directed mutagenesis and tested for their contribution to the phenotype by growth on medium lacking histidine (Fig. 1A). This procedure identified changes at positions 1256, 1273, and 1274 in the GPA2 coding sequence as being responsible for the defect in Krh1p binding. The change at position 1256 converts the amino acid at position 419 from a glutamine to an arginine. The changes at positions 1273 and 1274 are independent mutations that affect the same codon. The change at position 1273 converts the amino acid at position 425 from an asparagine to an aspartic acid, and the change at position 1274 converts the same amino acid to a serine.

To quantify the degree of interaction observed in the two-hybrid assays, beta-galactosidase liquid assays were performed. The level of interaction of wild type Gpa2p with Krh1p was approximately equal to its level of interaction with Ime2p (Fig. 1B). The Gpa2p variants Gpa2pQ419R, Gpa2pN425D, and Gpa2pN425S retained about 60% of their ability to interact with Ime2p but displayed no detectable interaction with Krh1p. These results indicate that, in a two-hybrid assay, alterations at residues 419 and 425 specifically affect the interaction of Gpa2p with Krh1p.

Gpa2p Variants with Changes at Amino Acids 419 and 425 Are Defective for Krh1p Binding in Vivo—To assess whether the changes in Gpa2p identified by the two-hybrid screen affect its binding to full-length wild type Krh1p, an assay that determines binding in vivo was used. It was shown previously that overexpression of a version of Gpa2p that contains a nuclear localization sequence (NLS) causes a change in the subcellular distribution of Krh1p from the cytoplasm to the nucleus (28). This change in localization is thought to be due to direct binding of Krh1p to nuclear Gpa2p. Therefore, this assay was used to study the interaction of Krh1p with the Gpa2p variants identified in the two-hybrid screen. As described previously (28), GFP-Krh1p localizes to the cytoplasm in cells overexpressing empty vector, but redistributes to the nucleus in cells overexpressing NLS-Gpa2p (Fig. 2, top panels). However, in cells overexpressing the N425D, N425S, and Q419R versions of NLS-Gpa2p, GFP-Krh1p localizes to the cytoplasm (Fig. 2, middle and bottom panels). These results demonstrate that the identified changes at residues 419 and 425 in Gpa2p cause a defect in its ability to bind wild type Krh1p in live cells.


Figure 1
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  		FIGURE 1.
Two-hybrid assays to identify mutations in GPA2 that confer a defect in Krh1p binding. A, strain HF7c carrying either plasmid TH1-1, which encodes a fusion to IME2, or TH14, which encodes a fusion to KRH1, and carrying one of the following plasmids was plated to synthetic complete medium or selective medium lacking histidine: pGBT9-GPA2 (WT), pG21197G-T9.1 (I399M), pG21256G-T9.1 (Q419R), pG21273G-T9.1 (N425D), or pG21274G-T9.1 (N425S). B, beta-galactosidase assays were performed using extracts from strain HF7c carrying either plasmid TH1-1 (gray bars) or TH14 (black bars) and carrying one of the following plasmids: pGBT9 (-), pGBT9-GPA2 (WT), pG21273G-T9.1 (N425D), pG21274G-T9.1 (N425S), or pG21256G-T9.1 (Q419R). Values are the mean and standard deviation from three independent experiments.

 
Gpa2p Variants That Do Not Bind Krh1p Are Constitutively Active—Activation of Gpa2p results in phenotypes associated with an increase in intracellular cAMP, including decreased heat shock resistance, decreased sporulation efficiency, and increased invasive and pseudohyphal growth (3, 4, 6, 7). To determine whether changes in Gpa2p that impair Krh1p binding have an effect on Gpa2p function, low copy plasmids carrying the GPA2 mutant alleles under the GPA2 promoter were transformed into a gpa2{Delta} strain. These strains were then tested for phenotypes known to be affected by Gpa2p function.


Figure 2
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  		FIGURE 2.
Localization of GFP-Krh1p in cells containing NLS-Gpa2p variants defective for Krh1p binding. Strain SKY762 carrying plasmid YEp181-GFP.FLKRH1 and one of the following plasmids was grown to log phase and viewed by fluorescence microscopy with a fluorescein isothiocyanate filter set: YEp352 (vector), pNLSGPA2–352.1 (NLS-Gpa2), pNLSG2ND-352.11 (NLS-Gpa2N425D), pNLSG2NS-352.10 (NLS-Gpa2N425S), or pNLSG2QR-33.26 (NLS-Gpa2Q419R).

 
Cells carrying either empty vector or a wild type GPA2 plasmid displayed a survival rate of ~15% after heat shock (Fig. 3A). Expression of wild type Gpa2p has no effect on heat shock resistance because the activity of the normal protein is likely to be at a low level in stationary phase, the condition under which these assays are carried out. In contrast, cells carrying a GPA2N425S plasmid displayed a 2-fold decrease in survival rate, and cells carrying a GPA2Q419R plasmid displayed a 3.6-fold decrease in survival rate. The effect of GPA2N425D on the heat shock survival rate was not significantly different from that of wild type GPA2. A decrease in heat shock resistance is associated with mutational activation of the Gpa2p {alpha}-subunit. For example, an alteration that impairs GTPase activity causes decreased heat shock resistance (4). The finding that expression of the Gpa2pN425S and Gpa2pQ419R variants results in a decrease in heat shock resistance indicates that the amino acid changes in these variants cause some degree of constitutive activation of the G protein.


Figure 3
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  		FIGURE 3.
Heat shock sensitivity and FLO11 RNA abundance in cells containing Gpa2p variants defective for Krh1p binding. A, percent survival after heat shock was determined for strain HS188-5D (gpa2{Delta}) transformed with each of the following plasmids: YCplac33 (-), pGPA2–33.1 (WT), pG2ND-33.11 (N425D), pG2NS-33.10 (N425S), and pG2QR-33.9 (Q419R). Values plotted are the mean ± S.D. from three independent experiments; *, p < 0.02; **, p < 0.01. B, RNA was isolated from the strains described in A and assayed for the relative amount of FLO11 and ACT1 RNA by real time RT-PCR. Values are plotted as the relative amount of FLO11 RNA normalized to ACT1 RNA and are the mean ± S.D. from three independent experiments; *, p < 0.02; **, p < 0.003.

 
Another phenotype associated with Gpa2p activation is an increase in the degree of pseudohyphal and invasive growth in strains that are competent for these processes (3, 6). The degree of pseudohyphal and invasive growth correlates with induction of the FLO11 gene, which encodes a cell surface flocculin that is involved in these types of filamentous growth (29). To assess the effect of the Gpa2p variants on FLO11 expression, real time RT-PCR assays were performed on RNA from strains expressing wild type and mutated forms of GPA2.

Deletion of GPA2 caused a significant decrease in FLO11 RNA abundance (Fig. 3B), as described previously (14, 18). Expression of the Gpa2pN425S and Gpa2pQ419R variants caused a small but significant increase in FLO11 RNA levels when compared with expression of the wild type protein. The effect of the Gpa2pN425D variant on FLO11 expression was essentially the same as that of wild type Gpa2p. These results support the idea that the N425S and Q419R changes result in an increase in the amount of activated Gpa2p.

Altered Amino Acids of Gpa2p Are in the beta6-{alpha}5 Region of the {alpha}-Subunit—High resolution crystal structures of heterotrimeric G proteins were used to locate the positions of the altered amino acids in the three-dimensional structure (30, 31). The positions of these residues were mapped onto the {alpha}-subunit structure in the mammalian heterotrimer Gi{alpha}1/Gbeta1/G{gamma}2 (30). Positions 419 and 425 are closely opposed and are present on a region of the {alpha}-subunit that is on the opposite side of the surface that binds to the beta-subunit in a heterotrimeric G protein (Fig. 4A). Residue 419 is in the loop between the beta6 strand and the {alpha}5 helix (Fig. 4B), at the second position of the 4-amino acid guanine nucleotide-binding motif that is present in all G{alpha} subunits (32). Residue 425 is at the N-terminal end of the {alpha}5 helix close to the guanine nucleotide-binding pocket (Fig. 4B). Substitutions at these sites or at adjacent sites in mammalian G{alpha} subunits have been shown to result in increased guanine nucleotide exchange (Fig. 4C; see "Discussion").


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Gpa2p variants defective in their ability to bind Krh1p were identified by a method that did not make any assumptions about the Krh1p-binding site, other than that it would not be identical to that of Ime2p. This screen identified amino acids in the beta6-{alpha}5 region of the {alpha}-subunit as being involved in this function. The changes present in these variants probably cause an increase in the proportion of Gpa2p that is bound to GTP. However, this property cannot account for their effect on Krh1p binding, because a form of Gpa2p that is predominantly in the GTP-bound state retains the ability to bind Krh1p in a two-hybrid assay (18).

The importance of the beta6-{alpha}5 region was demonstrated recently by the finding that the coupling of receptor binding to guanine nucleotide exchange requires movement of the {alpha}5 helix of the G{alpha} subunit (33). This study showed that binding of activated receptor at the {alpha}-subunit C terminus causes the {alpha}5 helix to rotate and move toward the beta6 strand. Movement of the {alpha}5 helix alters the conformation of the beta6-{alpha}5 loop, which results in an increased rate of GDP dissociation. In our study, binding of Gpa2p to Krh1p was shown to be affected by altering amino acids in the {alpha}5 helix and the beta6-{alpha}5 loop (see Fig. 4B), a region that is critical for transmitting the structural change that couples receptor binding to GDP release. Because the kelch repeat proteins are negative regulators of signaling, it is possible that they function by either reducing the level of activated Gpa2p or preventing activated Gpa2p from transmitting a signal to downstream components of the pathway. Therefore, one possibility raised by these findings is that binding of Krh1p to Gpa2p constrains the movement of the beta6-{alpha}5 region, thereby limiting the ability of liganded Gpr1p to induce activation of the G protein. Alternatively, it is possible that binding of Krh1p to Gpa2p reduces its rate of spontaneous guanine nucleotide exchange. However, it should be noted that two different groups have reported previously that Krh1p and Krh2p do not function as guanine nucleotide dissociation inhibitors for Gpa2p when assayed in standard in vitro reactions (5, 28).

The guanine nucleotide binding pocket of G{alpha} subunits includes the conserved amino acid motif TCAT (TQAT in Gpa2p) in the beta6-{alpha}5 loop, which directly contacts the guanine ring of GDP (30, 31). The effect of substitutions in this region of mammalian {alpha}-subunits on their guanine nucleotide binding properties has been described in previous reports (see Fig. 4C). For example, a change in Go at the position equivalent to Gpa2p glutamine 419 (Go-C325A) causes a 10-fold decrease in its affinity for GDP but does not affect its affinity for GTP (34). A number of studies have shown that changes in the adjacent {alpha}5 helix also affect this process. In Gt, a change at amino acid 327 (Gt-N327A), which is equivalent to Gpa2p asparagine 425, causes a substantial increase in the basal guanine nucleotide exchange rate (35).


Figure 4
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  		FIGURE 4.
A, amino acid changes in Gpa2p variants mapped onto a space-filling representation of mammalian heterotrimeric G protein Gi{alpha}1/Gbeta1/G{gamma}2 (Protein Data Bank entry 1GP2) using Cn3D (30). The {alpha}-subunit is shown in green; the beta-subunit is shown in blue; the {gamma}-subunit is shown in yellow. Positions equivalent to 419 and 425 in Gpa2p are shown in purple. B, amino acid changes in Gpa2p variants mapped onto a tube representation of mammalian {alpha}-subunit Gi{alpha}1. The beta6 strand, beta6-{alpha}5 loop, and {alpha}5 helix regions are shown in magenta. Carbon atoms at positions 419 and 425 are shown as spheres. GDP is shown in blue as a ball-and-stick representation. Outline of the receptor binding surface is based on Oldham et al. (33). C, analogous changes in mammalian G{alpha} subunits. Amino acids in the beta6-{alpha}5 loop (green) and the {alpha}5 helix (cyan) that, when altered, affect guanine nucleotide exchange (33–36, 39). Amino acids in Gpa2p (red) that, when altered, affect Krh1p binding.

 
In agreement with results obtained with mammalian G{alpha} subunits, we have found that Gpa2p variants containing changes in the beta6-{alpha}5 loop and {alpha}5 helix display some degree of constitutive activity, consistent with the idea that they undergo increased guanine nucleotide exchange. This conclusion is based on the observation that cells containing these variants are more heat shock-sensitive and express an increased level of FLO11 RNA, phenotypes known to be associated with increased activation of Gpa2p. Because purified mammalian {alpha}-subunits containing changes in the beta6-{alpha}5 region are altered in their ability to undergo guanine nucleotide exchange, it is likely that the increased activity of Gpa2p variants containing changes in this region is due, at least in part, to intrinsic changes in the guanine nucleotide binding properties of Gpa2p. Therefore, further studies will be necessary to evaluate the consequences of eliminating the ability of Gpa2p to associate with Krh1p without affecting its ability to bind guanine nucleotides.

Another study that addressed which part of Gpa2p is involved in binding the kelch repeat proteins was based on the idea that these proteins mimic G protein beta-subunits because of their potential to form a seven-bladed beta-propeller structure that is very similar to the structure of WD40 repeat-containing beta-subunits (21). Because beta-subunits are known to interact with the N-terminal {alpha}-helix of {alpha}-subunits, a series of Gpa2p N-terminal deletion constructs was tested for their ability to interact with Krh1p (also called Gbp2p) (28). This approach identified sites important for the Gpa2p-Krh1p interaction in regions encompassing amino acids 1–14 and 31–46 of Gpa2p. The fact that our study did not identify any residues in the N-terminal region of Gpa2p as being important for Krh1p binding could be due to the constraint placed on our screen, which required that altered forms of Gpa2p retain an interaction with Ime2p. If Ime2p binding requires an intact N-terminal region of Gpa2p, our approach would not have identified mutations encoding changes in this region. The previous study also reported that N-terminally deleted forms of Gpa2p that were defective for both Krh1p and receptor binding conferred wild type signaling activity (28). The interpretation of this finding was that decreased signaling mediated by deleted forms of Gpa2p because of their defective interaction with the receptor could be precisely offset by increased signaling because of their defective interaction with the kelch repeat proteins. However, our results raise the possibility that N-terminally deleted forms of Gpa2p retain the ability to bind Krh1p, perhaps with a lower affinity, through a binding site in the beta6-{alpha}5 region. Therefore, the consequences of completely disrupting the interaction between Gpa2p and the kelch repeat proteins remains an open question.

Given that three independent mutations from an unbiased screen were found to alter amino acids in or near the guanine nucleotide binding pocket of Gpa2p, the most straightforward interpretation of these results is that Krh1p binds to Gpa2p in this region. However, we cannot rule out the possibility that the changes we have identified alter Krh1p binding by causing large scale conformational changes of Gpa2p. Although the overall structural effects of these particular changes in Gpa2p are not known, a similar change in the beta6-{alpha}5 loop of another G protein {alpha}-subunit had no effect on global protein structure as determined by solving its crystal structure (36). Moreover, a variant of the Go {alpha}-subunit that contains a substitution at the position equivalent to Gpa2p glutamine 419 retains the ability to be cross-linked to a beta-subunit (34). Therefore, it seems likely that the changes at amino acids 419 and 425 of Gpa2p do not cause a global conformational change but rather directly modify a binding site for Krh1p. If this were the case, it would not be consistent with the idea that the kelch repeat proteins bind to Gpa2p in a manner that mimics G protein beta-subunits, as proposed by others (21). The beta-subunit binding regions of mammalian {alpha}-subunits include the N-terminal {alpha}-helix ({alpha}N helix), which is not conserved in Gpa2p, and the switch interface regions (beta2-strand/beta3-strand/{alpha}2-helix). The {alpha}N helix and switch interface regions are on the opposite side of the {alpha}-subunit from the guanine nucleotide binding pocket (30, 31). The idea that the kelch repeat proteins do not function as beta-subunit mimics is also supported by results concerning the differential affinity of Krh1p for Gpa2p bound to either GDP or GTP. In one study, the binding of Krh1p and Krh2p to Gpa2p-GDP was reported to be 2–8-fold higher than their binding to Gpa2p-GTP (21). In a two-hybrid assay, binding of the C-terminal region of Krh1p to a form of Gpa2p that is predominantly bound to GTP was 2–3-fold higher than its binding to wild type Gpa2p (18). Another study reported essentially equal binding of Krh1p to Gpa2p-GDP and Gpa2p-GTP (5). In contrast, the affinity of beta-subunits for G{alpha}-GDP is more than 100-fold higher than their affinity for G{alpha}-GTP (37). If there is only a small difference in the affinity of Krh1p and Krh2p for Gpa2p-GDP compared with Gpa2p-GTP, it is not likely to result in the type of molecular switch that occurs when G{alpha} subunits bind to GTP and dissociate from Gbeta subunits.

Genetic experiments combining mutant alleles of GPA2 and KRH1/2 have been used to try to order these signaling components in a genetic pathway. The results of these studies are consistent with the kelch repeat proteins acting either at the level of Gpa2p or downstream of Gpa2p (5, 18, 21). These results have led us and others to propose that Krh1p and Krh2p function as effectors of the {alpha}-subunit that inhibit a downstream signaling component when Gpa2p is present in the GDP-bound form. In this model, inhibition by Krh1p and Krh2p is relieved by their binding to Gpa2p-GTP, resulting in an increase in PKA activity. Alternatively, it has been proposed that Krh1p and Krh2p function similarly to G protein beta-subunits by binding to and stabilizing Gpa2p-GDP, thus reducing the level of signal transmitted by Gpa2p (21). The results presented here do not provide further support for either of these models. If Krh1p and Krh2p function as effectors, they would be expected to interact with the G{alpha} Switch II region ({alpha}2-helix), as well as with the {alpha}3-beta5 and {alpha}4-beta6 loops (38). Similarly, if Krh1p and Krh2p function as beta-subunit mimics, they would be expected to interact with the G{alpha} N-terminal {alpha}-helix and switch interface regions, as described above. Instead, it appears that the association between the kelch repeat proteins and Gpa2p is a novel type of interaction that could represent a unique functional relationship. Given that many kelch repeat proteins of unknown function are present in the sequenced mammalian genomes, it is possible that homologues of Krh1p and Krh2p play a novel role in G protein-mediated pathways in other organisms as well.


   FOOTNOTES
 
* This work was supported by Grant-in-aid 9951029T from the American Heart Association, Heritage Affiliate, and by Grant GM074242 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Present address: Dept. of Medicine, Nephrology Division, Albert Einstein College of Medicine, Bronx, NY 10461. Back

2 Present address: Dept. of Pharmacology, Weill Medical College of Cornell University, New York, NY 10021. Back

3 To whom correspondence should be addressed: Dept. of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, Box 1603, 1 Gustave L. Levy Place, New York, NY 10029. Tel.: 212-241-0224; Fax: 212-996-7214; E-mail: jeanne.hirsch{at}mssm.edu.

4 The abbreviations used are: PKA, protein kinase A; NLS, nuclear localization sequence; RT, reverse transcription. Back


   ACKNOWLEDGMENTS
 
We thank S. Palecek and S. Kron for strains used in this work. We also thank T. Peeters, J. Thevelein, and M. Versele (Katholieke Universiteit Leuven, Leuven-Heverlee, Belgium) for sharing results before publication. We also thank Anny Cheng for excellent technical assistance.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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