©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Differential Ability to Form the G Protein Complex among Members of the and Subunit Families (*)

(Received for publication, November 13, 1995; and in revised form, January 10, 1996)

Kang Yan (1) Vani Kalyanaraman (1) Narasimhan Gautam (1) (2)(§)

From the  (1)Departments of Anesthesiology and (2)Genetics, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have determined the relative abilities of several members of the G protein beta and subunit families to associate with each other using the yeast two-hybrid system. We show first that the mammalian beta1 and 3 fusion proteins form a complex in yeast and that formation of the complex activates the reporter gene for beta-galactosidase. Second, the magnitude of reporter activity stimulated by various combinations of beta and subunit types varies widely. Third, the reporter activity evoked by a particular combination of beta and subunit types is not correlated with the expression levels of these subunit types in the yeast cells. Finally, the reporter activity shows a direct relationship with the amount of hybrid beta complex formed in the cell as determined by immunoprecipitation. These results suggest that different beta and subunit types interact with each other with widely varying abilities, and this in combination with the level of expression of a subunit type in a mammalian cell determines which G protein will be active in that cell. The strong preference of all subunit types for the beta1 subunit type explains the preponderence of this subunit type in most G proteins.


INTRODUCTION

Most of the neurohormonal signaling pathways in mammals are mediated by heterotrimeric G proteins(1, 2) . Most cells contain many different G protein subunit types, and yet particular agonists evoke a highly specific response in a cell by activating a defined G protein-mediated signaling pathway(3) . Various mechanisms can contribute to this specificity. For instance, in a cell that contains many different alpha, beta, and subunit types, only certain types may be capable of forming a heterotrimeric complex because of the differences in the intrinsic affinity of these subunit types for one another. It has been shown that interactions between two different subunit types (1 and 2) and three different beta subunit types (beta1-beta3) are selective, indicating that this is indeed a mechanism for achieving specificity(4, 5) . However, these experiments were performed using in vivo or in vitro systems that could detect differences in the ability of these subunit types to interact but were not sufficiently sensitive to detect low level interaction between some of the subunit types. To obtain a more sensitive measure of the interaction between various members of the beta and subunit families, we used the yeast two-hybrid system. In this system, the proteins with the potential to interact are expressed as hybrids of two different domains of a transcription factor. If the proteins interact with each other, the transcription factor domains are in proximity and capable of activating a promoter that controls reporter activity(6) . We chose this system because it measures protein-protein interaction in a yeast cell and is therefore a reasonably accurate reflection of the ability of the interacting proteins to form a complex in a cell. Furthermore, it is highly sensitive in comparison with the methods used before to measure protein-protein interaction. For instance, in an assay of interaction between p53 and T antigen, it was shown that p53 mutants, whose interaction with T antigen could not be detected in an immunoprecipitation assay, were actually capable of interaction with T antigen since co-expression of the mutant p53 and T antigens as hybrids resulted in measurable reporter activity in the two-hybrid system(6) . In addition, the same study showed that in the case of mutant p53 proteins, whose interaction with T antigen could be detected by immunoprecipitation, there was a direct relationship between the amount of protein complex immunoprecipitated and the reporter activity detected when the same proteins were co-expressed in the two-hybrid system. This raised the possibility of determining the efficacy of complex formation between different beta and subunit types based on the magnitude of reporter activity. Using this system, we tested interaction between the five beta subunit types (beta1-beta5) that have been identified so far and six subunit types (1-5 and 7) with different primary structures and tissue-specific expression (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) . The interaction between the beta and subunits is of special interest because these two subunits are tightly bound as a complex and are thought to act inside the cells in this form(2) . The transcripts for beta1-beta4 subunits are expressed ubiquitously, while beta5 is expressed only in the brain(7, 9, 11, 12, 13) . The beta3 protein has been inferred to be the beta subunit associated with a cone photoreceptor-specific subunit type(22, 23) . The subunits show more variation than the beta subunits in their tissue-specific distribution. 1 is specific to rod photoreceptors. (^1)2 is present in several tissues(16) . 3 and 4 are brain-specific(18, 21) . 5 is ubiquitous(19) . 7 is present in several tissues but similar to 2 is enriched in brain(20) . By testing the interaction between a variety of beta and subunit types, we attempted to determine whether interactions to form the beta complex were highly selective (resulting in the formation of complexes between some subunit types but not others) or variable over a continuous spectrum of interacting abilities.

Functionally, the beta complex has been known for a long time to be required for interaction of a G protein with various receptors (24, 25, 26) . Recent results indicate that the subunit directly and specifically interacts with the receptor, raising the possibility that different subunit types could direct association of G proteins with various receptors(27, 28) . It is also known that the beta complex can directly regulate effector function(2) . If beta complexes made up of different beta and subunit types had different properties at each of these two points in a G protein-mediated signaling pathway, the mechanisms that control the formation of specific forms of the beta complex in a cell will also regulate the signaling properties of a cell.

The ability to assay interactions between two families of proteins with diverse structures also allows us to examine the molecular basis of interactions between protein families that form a multimeric complex.


MATERIALS AND METHODS

Strains

Yeast strain Y190 (MATa gal4 gal80 his3 trp1-901 ade2-101 ura3-52 leu2-3,-112 +URA3::-GAL->lacZ, LYS2::-GAL->HIS3 cyh^r) (29) was kindly provided by Dr. K. Blumer (Washington University Medical School, St. Louis, MO). Y190 was grown in YPD and co-transformed with plasmids containing TRP1 and LEU2 markers using the lithium acetate method(30) . The transformants were selected by culture on a synthetic medium lacking Trp and Leu.

cDNAs and Constructs

Plasmids pAS1 and pACTII were kind gifts from F. L. Li (Washington University Medical School, St. Louis, MO). pAS1 is a 2-µm TRP1 plasmid with the ADH1 promoter driving expression of the Gal4 binding domain (amino acids 1-147). pACTII is a 2-µm LEU2 plasmid with the ADH1 promoter driving expression of the Gal4 activation domain (amino acids 768-881). Both plasmids contain the sequence encoding nuclear targeting signals fused in frame to the transcription factor domains. cDNAs for beta4 and beta5 were obtained from Dr. M. I. Simon, California Institute of Technology, Pasadena, CA. 5 was obtained from Dr. N. N. Aronson, University of South Alabama, Mobile, AL. The coding portion of the 7 cDNA was synthesized by using a set of overlapping oligonucleotides spanning the known sequence (20) . cDNAs for the 1, 2, 3, 4, 5, and 7 subunits were amplified by polymerase chain reaction with specific primers and subcloned in-frame downstream of the Gal4 binding domain in pAS1. The polymerase chain reaction fragments for 1, 2, and 3 were digested with NcoI, which had a site at the 5` end of each of these fragments. The cut fragments were inserted into NcoI/SmaI cut pAS1. The polymerase chain reaction fragment for 4 was digested with HindIII, which had a site at the 5` end, filled with Klenow fragment of DNA polymerase I and inserted into pAS1, which was cut with NdeI and filled with Klenow fragment. The 5 fragment was digested with NcoI/SalI and inserted into pAS1 cut with NcoI/SalI. The polymerase chain reaction fragment for 7 was digested with XhoI/EcoRI, filled with Klenow fragment and inserted at the SmaI site of pAS1. Sequences and reading frame of each construct were confirmed by determining the nucleotide sequence. cDNAs coding for the mammalian G protein beta1, beta2, beta3, beta4, and beta5 subunits were cut from other vectors and subcloned in-frame downstream of the Gal4 activation domain in pACTII. beta1 cDNA was digested with EcoRI, filled with Klenow fragment, and inserted into the NcoI site of pACTII after filling with Klenow fragment. beta2 cDNA was digested with HindIII, filled with Klenow fragment, cut again with BamHI, and inserted at the SmaI/BamHI site of pACTII. To facilitate subcloning of the beta3 cDNA in frame, a synthetic linker was designed with an NcoI site at the 5` end and a HindIII site at the 3` end (5`-CATGGCAGCAGCAA-3`). beta3 cDNA digested with HindIII/BamHI was inserted along with the linker into NcoI/BamHI-digested pACTII. The linker joins the NcoI site of pACTII and the HindIII site in the beta3 cDNA. To facilitate subcloning of the beta4 cDNA in frame, a synthetic linker containing a NcoI site at the 5` end and a PvuII site at the 3` end (5`-CATGGCAATGAGCCAG-3`) was inserted upstream of the beta4 cDNA. The beta4 cDNA was digested with PvuII/BamHI and inserted along with the linker into the NcoI/BamHI-cut pACTII. The linker joins the NcoI site of pACTII with the PvuII site of the beta4 cDNA. beta5 cDNA was digested with BamHI/XbaI, where the XbaI site was filled with Klenow fragment, and the fragment inserted into the BamHI/XhoI site of pACTII, where the XhoI site was filled with Klenow fragment. Correct reading frame of each beta constructs was confirmed by determining the nucleotide sequences. The control plasmids expressing BD(^2)-Tat, BD-STE4, AD-GPA1, and AD-GRR1 were kind gifts from F. L. Li.

Assay of beta-Galactosidase Activity

Yeast transformants were grown overnight in synthetic medium lacking Trp and Leu, diluted in the medium next morning, and grown to mid-log phase. The cells were harvested and assayed for beta-galactosidase activity as described previously(31) .

Immunoblot Analysis

Yeast transformants were grown in the synthetic medium lacking Trp and Leu to the mid-log phase. 5 A units of yeast cells were harvested and lysed by vortexing in 0.2 ml of buffer containing 50 mM Tris, pH 8.0, 0.5% SDS, 0.5% Triton X-100, 10 µg/ml phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, with half the volume of glass beads. The lysate was centrifuged at 14,000 rpm at 4 °C for 10 min to recover the supernatant. 1 µg of total protein from each extract was electrophoresed in a 12% SDS-polyacrylamide gel and blotted onto a membrane (Immobilon-P, Millipore). The expression of subunits was detected with peptide-specific antibodies, BG specific to 2, and CG specific to 3. These antibodies were used after appropriate dilution (1:600)(32) . Antibodies were purified from antisera using the appropriate peptides on affinity columns(32) . The expression of beta subunits was detected with the antibody GAL4 AD mAb against the Gal4 activation domain (amino acids 768-881)(33) .

Immunoprecipitation

Yeast transformants were grown as described before. 5 A units of yeast cells were harvested and lysed by vortexing in 0.2 ml of buffer containing 20 mM Tris, pH 7.5, 1 mM EDTA, 0.2% Triton X-100, 0.01% SDS, 10 µg/ml phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, with half the volume of glass beads. The lysate was centrifuged at 14,000 rpm at 4 °C for 10 min to recover the supernatant. NaCl concentration in the supernatant was brought up to 137 mM. 25 µg of total protein in 100 µl were incubated with 10 µl of the antibody specific to 3 on ice for 60 min. The immunocomplex was mixed with 30 µl of pre-equilibrated protein A-Sepharose by rocking at 4 °C for 60 min. The protein A-Sepharose was washed with 3 times 0.5 ml TBST (20 mm Tris, pH 7.6, 137 mM NaCl, 0.1% Tween-20) and boiled in 50 µl of sample buffer for 5 min. The supernatant was recovered by centrifugation, and the protein A-Sepharose was discarded. 20 µl of the sample were fractionated by electrophoresis on a 12% SDS-polyacrylamide gel and immunoblotting was performed. The 3 fusion protein was detected with the CG antibody (1:600). The protein co-immunoprecipitated with 3 was detected with the GAL4 AD mAb (1:100).


RESULTS

A Mammalian G Protein beta Subunit and Subunit Interact in the Yeast Two-hybrid System

The beta1 subunit type fused to the AD of the Gal4 transcription factor and the 3 subunit type fused to the binding domain (BD) of the Gal4 transcription factor were expressed together in a yeast strain containing the gene for the reporter, beta-galactosidase, under the control of the GAL promoter. beta-Galactosidase activity in these cells was compared with a positive control, cells co-expressing a yeast G protein alpha subunit type, Gpa1, and a yeast beta subunit type, Ste4. These two proteins have been shown to interact strongly in the two-hybrid system(34) . Co-expression of BD-3+AD-beta1 resulted in significant stimulation of beta-galactosidase activity comparable with the positive control (Fig. 1A). beta-Galactosidase activity in the cells expressing BD-3+AD-beta1 was also compared with two negative controls, cells expressing BD-3+Grr1 (a protein involved in glucose metabolism(35) ) or AD-beta1+Tat (a protein from the human immunodeficiency virus(36) ) (Fig. 1A). Neither BD-3 nor AD-beta1 activated reporter activity significantly in the presence of these proteins, indicating that the reporter activity stimulated by BD-3+AD-beta1 is most likely due to specific interaction between these hybrid proteins.


Figure 1: beta1-3 interaction in the two-hybrid system. beta1, 3, Gpa1, Ste4, Grr1, and Tat were expressed as fusion proteins (see ``Materials and Methods''). A, beta-galactosidase activity from cells of different genotypes (described under ``Materials and Methods''). Gpa1, a yeast G protein alpha subunit, and Ste4, a yeast beta subunit, served as positive controls (34) . As unrelated proteins, Grr1 and Tat served as negative controls. The bars represent the mean value of beta-galactosidase activity determined from three different transformants. B, formation of the beta13 complex in yeast. Yeast extracts from the equivalent of 1 A unit of cells were immunoprecipated with the 3-specific CG antibody. The proteins immunoprecipitated were detected with same antibody on the immunoblot shown. AD-beta1 that co-immunoprecipitated with the CG antibody was detected with an antibody specific to the Gal4 activation domain (GAL4 AD mAb). Y190, untransformed control yeast cells. Only portions of blots containing the hybrid proteins identified based on reactivity and mobility are shown.



To examine whether the reporter activation resulted directly from complex formation between the hybrid beta and subunits, transformants expressing the same combinations of hybrids as those mentioned above (excepting Gpa1+Ste4) were lysed, and the proteins in the cell extract were immunoprecipitated with a 3-specific antibody, CG, which is directed at the N-terminal 14 amino acids of 3(32) . We have shown that the epitope for this antibody is available in the beta13 complex so that it is capable of immunoprecipitating 3 when bound to the beta subunit and also co-immunoprecipitating beta1(37) . Immunoprecipitates from different transformants were electrophoresed and immunoblotted with specific antibodies. The results of immunoblotting are shown in Fig. 1B. The CG antibody immunoprecipitated the BD-3 hybrid protein of expected molecular weight which is 30 kDa (7-kDa 3+23-kDa BD-nuclear targeting domain fusion) (second lane from left), but an unrelated protein Grr1 was not co-immunoprecipitated with 3, indicating that these two proteins did not form a complex in the yeast cells. No protein was specifically immunoprecipitated from untransformed Y190 cells or cells expressing Tat+beta1, confirming the specificity of the CG antibody for 3. Immunoprecipitation of the BD-3 hybrid from cells co-expressing AD-beta1 resulted in the co-immunoprecipitation of a protein with the expected molecular weight of the AD-beta1 hybrid, which is 60 kDa (36-kDa beta subunit+24-kDa AD-nuclear targeting domain fusion), demonstrating complex formation inside the cells between BD-3 and AD-beta1 (last lane from left). This result showed that the stimulation of reporter activity in beta1+3 cells (Fig. 1A), is due to complex formation between these hybrids.

These results also indicated that the mammalian beta and subunits formed a complex inside the yeast cells, even though they were fused to the Gal4 transcription factor domains. Moreover, the complex was capable of activating the GAL1 promoter, resulting in significantly enhanced reporter activity.

Interaction of Different Subunit Types with Various beta Subunit Types

Since the results above indicated that reporter activity was an indicator of complex formation between the beta and subunits, we tested the ability of different members of each of the beta and subunit families to interact with each other by measuring the beta-galactosidase activity elicited by each combination. The cDNA for each subunit type was subcloned downstream of the appropriate transcription factor domain ``in-frame,'' using different methods as described under ``Materials and Methods.'' All of the beta subunits were expressed fused to the activation domain, and all of the subunits were expressed fused to the binding domain. Transformant colonies containing various combinations of beta and subunit types were grown and assayed for beta-galactosidase activity. In Fig. 2, the beta-galactosidase activity from cells expressing different combinations of beta and subunit types are shown. beta-Galactosidase activity varied significantly among cells expressing the same subunit with different beta subunits. Apart from the negative control shown in each panel of Fig. 2(BD- subunit type + AD-Grr1), we examined other genotypes also as controls: (i) cells co-expressing each of the beta subunit types as AD-beta plus the binding domain alone (pACTII containing a beta subunit type cDNA+pAS1); (ii) cells expressing each of the subunit types as BD- plus the activation domain alone (pAS1 containing a subunit type cDNA+pACTII); and (iii) cells co-expressing BD-Tat with each of the beta subunit types as AD-beta. None of the cells co-expressing these proteins activated the reporter significantly (data not shown).


Figure 2: Differential interactions among the G protein beta and subunit types. Bars in each panel show the amounts of beta-galactosidase activity detected from cells containing specific combinations of beta and subunit types. The genotype of the cell is denoted below each bar. Reporter activity estimated from cells co-expressing the particular BD- subunit type with AD-GRR1 was one of the negative controls and is shown in each panel. Other negative controls are described in the text. The beta-galactosidase activity form all control cells was lower than 0.05 units (data not shown). The bars represent the mean (± S.E.) derived from six different transformants.



Expression Levels of the beta and Subunit Type Hybrids in Yeast Cells

To ensure that the differences in reporter activity among particular combinations of beta subunit type were not a reflection of differential expression of the hybrid proteins, we tested cells co-expressing the 2 subunit type with various beta subunit types for the level of expression of each hybrid protein. Reporter activity from cells of each genotype was then compared with the hybrid protein levels to determine if there was any correlation. Fig. 3A shows the reporter activity from cells co-expressing 2 with different beta subunit types. The results of immunoblotting cell extracts from the same cells with an antibody specific to the activation domain are shown in Fig. 3B, bottom. The bars above the immunoblot in panel B are estimates of the intensity of individual bands obtained by densitometry. Since the amount of protein loaded from cell extracts of each genotype is the same, the bars indicate the relative amounts of each beta subunit type expressed. Comparison of Fig. 3A and B shows that there is no correlation between the reporter activity and the expression levels of the beta subunit types. Fig. 3C is a similar comparison of the amount of the 2 hybrid protein expressed in cells of different genotypes determined by immunoblotting with the BG antibody specific to 2(32) . Again there is no correlation between the 2 expression levels and the reporter activity.


Figure 3: Comparison of reporter activity with the expression level of hybrid proteins in cells co-expressing BD-2 and different beta subunits. A, mean value of beta-galactosidase activity obtained from cells of each genotype. Activity from cells of each genotype is expressed as a percentile of activity from 2+beta1 (same data as in Fig. 2). B, immunoblot probed with GAL4 AD mAb, showing the relative amounts of AD-beta subunit types in 1 µg of total proteins in the cell extract from various genotypes. Protein levels based on densitometry of the bands are expressed for each genotype as a percentile of the level in AD-beta1. C, immunoblot probed with a 2-specific antibody, BG, showing the relative amounts of BD- subunit types in 1 µg of total proteins in the cell extract from various genotypes. Protein levels based on densitometry of the bands are expressed for each genotype as a percentile of the level in BD-2. Only portions of blots containing the hybrid proteins identified based on reactivity and mobility are shown. The immunoblots from B and C were scanned for quantitation using a laser densitometer and the Image Quant program (Molecular Dynamics). Results shown are representative of two independent experiments.



Complex Formation Is Correlated with Reporter Activity

The following experiments were performed to determine if the amount of complex formed between two beta and hybrids was correlated with the amount of reporter activity elicited by the same combination. Cells expressing the 3 subunit type with the beta1 or beta3 subunit types were lysed, and the hybrid beta complex in the cell extract was immunoprecipitated with the 3-specific antibody, CG. We chose these two genotypes because the reporter activity from these cells showed a distinct difference (2.5-fold) (Fig. 4A) and also because we possessed a 3 subunit-specific antibody that could co-immunoprecipitate any beta subunit bound to the 3 subunit (37) . The amount of AD-beta subunit type present in the immunoprecipitates from cells co-expressing beta1+3 were compared with cells co-expressing beta3+3. Immunoblotting was performed with the antibody to the activation domain that does not discriminate between the two beta subunits as in the experiments above. Fig. 4B shows the results of comparing the amount of beta subunit hybrid from these cells by immunoblotting and the relative amounts of protein determined by densitometry. Since the beta hybrids in this panel have been co-immunoprecipitated with 3, the relative amounts of the beta hybrids in the immunoprecipitate indicate the relative levels of the two complexes, beta13 and beta33, present in the cells. There was a positive correlation between the reporter activity and the amount of beta complex in cells of each genotype (Fig. 4, A and B). To ensure that the difference in the level of complex formation was not due to the expression levels of these subunit types, we examined equal amounts of cell extracts from each genotype by immunoblotting with antibodies to 3 or the activation domain. Results shown in Fig. 4, C and D, indicate that the expression levels of both hybrids, AD-beta and BD-3 were approximately the same in the cells containing beta1+3 and beta3+3. Differences in expression levels cannot therefore be the cause for differential complex formation between 3+beta1 as compared with 3+beta3.


Figure 4: Relationship between reporter activity and complex formation. A, mean value of beta-galactosidase activity from cells containing 3+beta1 and 3+beta3 (same data as in Fig. 2) expressed as relative percentiles. B, immunoblot with bands corresponding to the beta subunit hybrids and the relative amounts of proteins in these bands as determined by densitometry. The immunoblot contains the immunoprecipitate obtained with a 3 antibody, CG, from 25 µg of total proteins extracted from each of the transformants, 3+beta1 and 3+beta3. Conditions used for immunoprecipitation from cell extracts of both genotypes were identical to allow quantitative comparison of the amount of beta complex present. The proteins co-immunoprecipitated with BD-3 were blotted and probed with GAL4 AD mAb. The BD-3 immunoprecipitated with 3 antibody is not shown since a proportion of it may not be bound to beta. Bars above the bands show the relative amount of AD-beta1 and AD-beta3 co-immunoprcipitated with BD-3 as determined by densitometry. The amount of AD-beta3 is expressed as a percentage of AD-beta1. C, immunoblot probed with GAL4 AD mAb, showing the relative amounts of AD-beta1 and AD-beta3 in 1 µg of total proteins obtained from cells containing 3+beta1 and 3+beta3. The amount of AD-beta3 is expressed as a percentage of AD-beta1. D, immunoblot probed with 3 antibody, CG, showing the relative amount of BD-3 in 1 µg of total proteins obtained from cells containing 3+beta1 and 3+beta3. The amount of BD-3 from cells containing 3+beta3 is expressed as a percentage of BD-3 from cells containing 3+beta1. Only portions of blots containing the hybrid proteins identified based on reactivity and mobility are shown. Quantitation of protein bands on the immunoblots in B, C, and D were performed as described in the legend to Fig. 3. Results shown are representative of two independent experiments.




DISCUSSION

The reporter activity resulting from interaction between two hybrids may be affected by several different factors: efficiency of complex formation, expression levels of the hybrids, and accessibility of the activation domain to the transcription machinery. It is also possible that the beta and subunit types influence the localization of individual hybrids to the nucleus, although both transcription factors possess nuclear targeting signals upstream. Results shown in Fig. 3indicate that the differential reporter activity from cells containing various combinations of beta and subunit types is not related to the expression levels of the hybrid proteins, implying that it results from the different levels of beta complex formation. The results presented in Fig. 4support this implication. These results show that there is a direct correlation between the reporter activity and the amount of beta complex formed in the cells. The differential reporter activity noted among the different genotypes is therefore mainly due to different levels of hybrid beta complexes formed in these cells and not any of the other reasons mentioned above. This confirms a previous report of a positive correlation between the amount of immunoprecipitated complex of two hybrids and reporter activity stimulated by those hybrids(6) . Many combinations of beta and subunit types have significant reporter activity in comparison with the negative control, subunit type+Grr1 (Fig. 2). This indicates that complex formation does occur between many of the beta and subunit types examined here. However, the extent of complex formation differs considerably among the different combinations of beta and subunit types (Fig. 2).

The G protein subunits are post-translationally modified by the attachment of a prenyl group to the C terminus(38) . There is evidence that yeast cells possess the same enzymes as mammalian cells that modify various proteins with the two types of prenyl groups, farnesyl and geranygeranyl(39) . It is therefore most likely that all of the mammalian subunits expressed in yeast are prenylated appropriately. However, since it is known that prenylation of the subunits is not required for interaction with the beta subunit(4, 40) , even if a mammalian subunit type was inefficiently prenylated it would not affect beta complex formation in yeast cells. The results from the immunoprecipitation of the beta complex in Fig. 1and Fig. 4support this directly.

Interaction of a Particular Subunit Type with Each Member of the Family of beta Subunit Types

The results from examining the interaction of the 1 and 2 subunit types with beta1, beta2, and beta3, using other methods has shown that (i) 1 interacts poorly with beta2 and beta3 and (ii) 2 interacts poorly with beta3(4, 5) . The results in Fig. 2confirm these previous results. The ability of 1 to interact better with beta1 but not beta2 or beta3 is consistent with the presence of beta1 in rods and the absence of beta2 or beta3 in rods (22, 23) . As seen in Fig. 2, 1 also fails to interact with beta4 and beta5. However, the relatively small difference in the interaction capabilities of 1-beta1 and 1-beta2 is unexpected. One explanation is that the sensitivity of the two hybrid assay allows for the detection of weak interactions that would not have been detected in the previous assays that have been used. It is unlikely to be due to the beta11 combination, in comparison with beta21, being deleterious to yeast cells, since there was no significant difference in the growth rate of cells expressing various combinations of 1 with beta subunit types. In contrast to 1, the 2 subunit is expressed in several different tissues and at especially high levels in brain(16) . Its higher level of interaction with beta1 and beta2 is thus consistent with the ubiquitous distribution of these two beta subunit types. 3 is a subunit type that is mainly present in brain and is expressed at very high levels(18) . Immunohistochemistry with antibodies specific to this subunit show highly restricted expression of the protein in brain and retina.^1 3 interacts well with all the beta subunits. However, unlike 2, it interacts especially well with beta5 in addition to beta1 and beta2. This interaction is significant in view of the restricted expression of beta5, which is present only in brain(13) . 4 is a subunit type that is specific to brain based on the expression of its RNA(21) . (^3)Consistent with this distribution, it interacts especially well with beta5. Although the pattern of specificity of 4 for beta subunits is broadly similar to 3, 4 shows more preferential interaction with beta1, beta2, and beta5 (Fig. 2). 5 and 7 are present in a variety of tissues, although 7 is especially abundant in brain. Both subunit types favor interactions with the beta1 and beta2 subunit types considerably compared with the others.

In comparison with the other beta subunit types, the beta3 subunit type is consistently poorer at interacting with all of the subunit types tested. This would indicate that differentially high levels of expression of this subunit type would be required to ensure its appropriate interaction with a subunit type. This may be the reason for its restricted expression in cone photoreceptors compared with rods (22, 23) . There is also the possibility that beta3 selectively interacts much better with a subunit specific to cones, c, compared with other subunit types. The predominant beta subunit types in the G protein beta complexes purified from a variety of mammalian tissues are beta1 and beta2, although G(t) contains only beta1(41) . This preference for the beta1 and beta2 subunits is explained by the data in Fig. 2; all of the subunit types, other than 1, have a strong preference for these subunit types.

Recent evidence indicates that the subunit interacts directly with the receptor(27, 28) . If different subunit types specifically interacted with various receptors, this intrinsic ability of beta and subunit types to favor complex formation with certain types and not others would be a primary level of control over which G protein would be active in a cell.

Relationship between Structural Homology and Efficiency of Complex Formation

Table 1and Table 2show the extent of homology between the different beta and subunit types. Comparison of the structural homology of subunit types in each family with the pattern of interaction with each other (Fig. 2) shows that they are not directly related. For instance, although 3 is much more homologous to 2 (80%), it shows a pattern of interaction with members of the beta subunit family that is distinct from 2 but similar to 4, with which it shares only 62% homology by identity. Similarly, beta5 interacts with 4 almost as well as beta1 and beta2, despite the primary structure of beta5 being distinctly different from beta1 and beta2 (50% identity). beta4, which is highly homologous to beta1 and beta2 (90% identity), is much less effective at interacting with the 4 subunit type (Fig. 2). These results raise questions about the molecular basis of the interactions between members of the beta and subunit families. One possibility is that a few critical residues provide most of the binding energy between the interacting proteins. In this scenario, residues that are involved in interactions between beta1 and 4 are conserved in beta5, although most of the remaining primary structure of beta5 is divergent from beta1. These critical residues in beta1 are, however, not conserved in beta4, although most of the remaining primary structure of beta4 is identical to beta1. Inspection of the amino acid sequences of the beta and subunit types examined here shows that there is only one residue, Ala-193 in beta1, that is conserved in beta5 and not in beta4, where it is substituted with Ser. Similarly there is only one residue, Ile-55 in 3 that is conserved between 3 and 4 but not in 2, where it is substituted with another hydrophobic residue Leu. It is unclear whether these minimal alterations can result in the striking differences in the ability of these subunit types to form complexes. It seems likely that there are other mechanisms at the basis of discriminatory interactions between members of these two G protein subunit families. Some studies have attempted to identify the domains and the amino acids involved in conferring selectivity to interactions between beta11 compared with beta21(4, 42, 43, 44) . These studies have identified a C-terminal domain in beta1 (amino acids 215-293) and a 14-residue stretch in 1 (amino acids 33-49) to be important for this selectivity. However, it is unclear whether the residues involved in interaction between various beta and subunit types are in all cases located at homologous positions on the primary structure of these subunit types or if these residues are situated in distinctly different domains. The ability to obtain a sensitive quantitative measure of the interactions between several beta and subunit types in the two-hybrid system allows us to identify the roles of individual amino acids of each subunit in these interactions in vivo through extensive mutagenesis. This will also help elucidate some of the rules at the basis of interaction between protein families that form multimeric complexes.






FOOTNOTES

*
This work was supported by a grant 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 by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Established Investigator of American Heart Association. To whom correspondence should be addressed. Tel.: 314-362-8568; Fax: 314-362-8571.

(^1)
H. K. W. Fong and N. Gautam, unpublished results.

(^2)
The abbreviations used are: BD, binding domain; AD, activation domain.

(^3)
It has been reported recently that in human tissues the transcript for this subunit is expressed almost ubiquitously (Ray, K., Kunsch, C., Bonner, L. M., and Robishaw, J. D. (1995) J. Biol. Chem.270, 21765-21771). The [Abstract/Full Text] reason for this difference in expression pattern noted between mouse and human tissues is unclear.


ACKNOWLEDGEMENTS

We thank Frank L. Li for materials and valuable discussions.


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