COMMUNICATION:
Structural Determinants for Interaction with Three Different Effectors on the G Protein beta  Subunit*

(Received for publication, October 30, 1996, and in revised form, November 19, 1996)

Kang Yan Dagger and Narasimhan Gautam Dagger §

From the Department of Dagger  Anesthesiology and § Genetics, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

In the yeast two-hybrid system, a 100-residue fragment (beta 1A) from the N terminus of the beta 1 subunit interacts with domains specific to adenylyl cyclase 2 (AC2), the muscarinic atrial potassium channel (GIRK1), and phospholipase C-beta 2 (PLC-beta 2). Based on the crystal structure of the G protein, beta 1A is composed of an N-terminal alpha  helix, a loop, and five beta  strands in which the C-terminal four beta  strands form a beta  sheet, the first of seven sheets that make up the propeller structure of the beta  subunit. A mutant of beta 1A (L4P, L7P, and L14P), in which the alpha  helix was potentially destroyed, interacted poorly with the G protein gamma  subunit but effectively with domains of AC2, GIRK1, and PLC-beta 2. In contrast, another mutant of beta 1A (S72A, D76A, and W82A), in which a network of hydrogen bonds was disrupted, interacted poorly with GIRK1 and PLC-beta 2 domains, but effectively with the gamma  subunit and the AC2 domain. These results suggest that the proper folding of the first five beta  strands in the G protein beta  subunit is a requirement for appropriately positioning residues that interact with GIRK1 and PLC-beta 2. Furthermore, since mutations that potentially disrupted the folding of these beta  strands did not affect interaction with AC2, the structural determinants on the G protein beta  subunit for interaction with various effectors may be different.


INTRODUCTION

The G protein beta gamma complex plays an important role in modulating the function of a variety of effectors in cellular signaling. The effectors regulated by the beta gamma complex include adenylyl cyclases, phospholipase C-beta 2, and potassium channels (1-5). The beta gamma complex directly interacts with several effectors, and binding domains for the beta gamma complex have been identified in these effectors (6-11). Since the beta  and gamma  subunits form a tight complex in mammalian cells, individual roles of the beta  and gamma  subunits in effector regulation are unclear. Using the yeast two-hybrid system, we have been investigating interaction of the beta  and gamma  subunits with effectors. We previously demonstrated that it was the beta  subunit that interacts with domains specific to adenylyl cyclase type 2 (AC2)1 and the muscarinic atrial potassium channel (GIRK1) and that a 100-residue fragment (beta 1A) from the N terminus of the beta 1 subunit interacted with these effector domains as effectively as the whole beta  subunit (12). These results imply that the beta  subunit is an important element in regulating effector activity. The identification of a subdomain within the N-terminal 100-residue fragment of the beta 1 subunit will provide further insight into the mechanisms by which the beta gamma complex regulates effector function.

To examine interaction between the beta gamma complex and effectors, we have specifically used the yeast two-hybrid system because it allows us to directly measure the effect of mutations in a protein on its ability to bind another protein in vivo. Biologically relevant information about the structural basis of interactions in multiprotein complexes has been obtained through deletion and mutational analysis in the two-hybrid system (e.g. Refs. 13-15). We have also successfully used the yeast two-hybrid system to demonstrate that different members of the beta  and gamma  subunit families have differential abilities to form a complex and also to show that an N-terminal domain on the beta  subunit interacts with domains specific to two effectors (12, 16). We showed that the formation of the beta gamma complex as fusion proteins in yeast cells activated a reporter gene, and that the reporter activity was directly related to the amount of the beta gamma complex formed in yeast cells (16). Our finding using the two-hybrid system that the first 100 residues are important for interaction with AC2 have been supported by recent results obtained from cross-linking and modeling experiments with the beta gamma complex and the AC2 domain (17). In this report, we have used the two-hybrid system to analyze interaction of the beta gamma subunits with AC2 and GIRK1 as well as phospholipase C-beta 2 (PLC-beta 2). A domain (residues 580-641) of PLC-beta 2 has recently been shown to directly bind to the G protein beta gamma complex, and a fragment containing this domain has been shown to inhibit the beta gamma complex-mediated activation of phospholipase C-beta 2 (9).

The crystal structures for the G protein heterotrimer and the beta gamma complex indicate that the N-terminal 100 residues of the beta  subunit (beta 1A) that interacts with AC2 and GIRK1 is made up of distinct domains in terms of secondary structure (18-20). It consists of an N-terminal alpha  helix, a loop, and five beta  strands. The first of these five strands is the outermost strand of the seventh beta sheet in the beta  subunit. The C-terminal four beta  strands of beta 1A form the first beta  sheet. Based on the crystal structure, hydrogen bonding is suggested between residues in the loops that connect particular beta  strands and residues within specific beta  strands. These hydrogen bonds are important for stabilizing the folded structure of the multiple beta  strands of the beta  subunit. We have introduced mutations into the first 100 residues of the beta  subunit that potentially disrupt either the alpha  helix or the hydrogen bonds that are important for stabilizing the folding of these five beta  strands. The mutants have been examined for interaction with both the gamma  subunit and the effector domains.


MATERIALS AND METHODS

Strains

Yeast strain Y190 and the plasmids pAS1 and pACTII have been described before (12, 16).

Constructs

pAS1 contains the Gal4 binding domain, and pACTII contains the Gal4 activation domain. cDNAs encoding a protein or a protein domain were subcloned downstream of either the Gal4 binding domain in pAS1 or the activation domain in pACTII. cDNAs for the gamma  subunits were subcloned in pAS1 and cDNAs for the beta  subunits, and beta 1A-beta 1D were subcloned in pACTII, as described previously (12, 16). A synthetic oligonucliotide cassette encoding AC2Q (residues 956-982 of adenylyl cyclase 2) was subcloned into NcoI/BamHI cut pAS1 (12). DNA encoding GKN (residues 1-83 of GIRK1) was amplified by polymerase chain reaction using specific primers and subcloned into NcoI/BamHI cut pAS1 (12). DNA encoding the PLC62 domain (residues 580-641 of PLC-beta 2) was amplified by polymerase chain reaction using specific primers and subcloned into both pAS1 and pACTII which were cut with NcoI/BamHI. DNAs encoding two mutants of beta 1A fragment, beta 1Am1 (L4P, L7P, and L14P) and beta 1Am2 (S72A, D76A, and W82A), were amplified by polymerase chain reaction using specific primers and subcloned into NcoI/BamHI cut pACTII. Correct reading frames and nucleotide sequence after polymerase chain reaction were confirmed by nucleotide sequencing. Control plasmids expressing BD-Tat and AD-Grrl have been described (16).

Assay of beta -Galactosidase Activity

The liquid culture assay of beta -galactosidase activity has been described (16).


RESULTS AND DISCUSSION

Since the G protein beta  subunits were targets for domains specific to two effectors (12), we wanted to know whether the beta subunits were also able to bind to a domain specific to another effector, phospholipase C-beta 2. PLC62 is a domain with 62 residues specific to phospholipase C-beta 2. This domain was able to bind to the beta gamma complex directly as a fusion protein (9). We first examined whether the PLC62 domain was able to interact with the G protein beta  subunits. In our study, PLC62 was a hybrid with the DNA binding domain of the Gal4 transcription factor, and the beta  subunits were hybrids with the activation domain of Gal4. These hybrids were co-expressed in a yeast strain containing a reporter gene that encodes beta -galactosidase. If PLC62 interacts with the beta  subunits, the Gal4 transcription factor domains will be brought together to activate the expression of the reporter gene. When the PLC62 domain was co-expressed with the beta  subunits in yeast cells, the reporter gene was significantly activated, with higher reporter activities in cells expressing beta 1, beta 2, and beta 5 subunits (Fig. 1A). This is consistent with reports that a beta gamma complex containing either beta 1 or beta 5 was able to activate phospholipase C-beta 2 (4, 21). The different reporter activities elicited by PLC62 with different beta  subunit types may be due to differential interaction between PLC62 and the beta  subunit types. Co-expression of PLC62 with different gamma  subunit types did not significantly activate the reporter gene (Fig. 1B). We then tried to identify a region of the beta  subunit that interacts with PLC62 by co-expression of this effector domain with various regions of the beta 1 subunit. The beta 1 subunit was split into four different fragments using an appropriate restriction site as described before (12). Fragments included the following regions of the protein. beta 1A, residues 1-100; beta 1B, residues 101-187; beta 1C, residues 188-261; and beta 1D, residues 262-340. The N-terminal fragment (beta 1A, residues 1-100) of the beta 1 subunit interacted with PLC62 as effectively as the whole beta  subunit (Fig. 1C). beta 1A also interacted more effectively with gamma  subunits than the entire beta 1 subunit (Ref. 12 and data not shown). Although PLC62 did not interact with other regions of the beta 1 subunit, this does not rule out the possibility that these fragments may be folded improperly in yeast. Together with our previous finding (12), these results strongly indicate that the N-terminal 100 residues from the beta 1 subunit contain subdomains that interact with three different effectors. Are these putative subdomains in beta 1A distinct structural determinants or overlapping regions?


Fig. 1. Interaction of PLC62 with various beta gamma subunit types and various regions of the beta 1 subunit. A-C, beta -galactosidase activity from cells of different genotypes was estimated as described under "Materials and Methods." Fragments include the following residues. beta 1A, residues 1-100; beta 1B, residues 101-187; beta 1C, residues 188-261; and beta 1D, residues 262-340 (12). Unrelated proteins, Grr1 and Tat, served as negative controls (16). The bars represent mean value of beta -galactosidase activity from three different transformants examined in each of two independent experiments. In several cases where activity was very low, error bars are too small to be shown. beta 1 activity from panel A is shown again in panel C as reference.
[View Larger Version of this Image (19K GIF file)]


Based on the crystal structure of the G protein, the beta  subunit is a propeller composed of seven beta  sheets with an N-terminal alpha  helix. beta 1A, a 100-residue fragment from the N terminus of the beta 1 subunit, encompasses an N-terminal alpha  helix, five beta  strands, and a loop connecting the alpha  helix and the beta  strands (18, 20). The N-terminal alpha  helix of the beta 1 subunit forms the beginnings of a parallel coiled-coil with the N-terminal alpha  helix of the gamma subunit. The first of the beta  strands is the outer strand of the seventh beta  sheet in the propeller structure of the beta 1 subunit. The next four beta  strands form the first beta  sheet. Both the alpha  helix and/or the beta  strands could be involved in interaction with these effector domains.

To test if the N-terminal alpha  helix is involved in interaction with effectors, we introduced mutations into beta 1A that have the potential to destroy the alpha  helix. The alpha  helix is stabilized by hydrogen bonds between the NH and CO groups of the main chain. The proline side chain is different from others in that its side chain is bonded to both the nitrogen and alpha -carbon atoms to form a ring structure. This prevents the N atom of proline from participating in hydrogen bonding and should form steric obstacles to the formation of an alpha -helix. The alpha  helix situated at the N terminus of the beta subunit is amphipathic and forms part of a coiled coil with a similar amphipathic helix from the N terminus of the gamma  subunit. In the first mutant of beta 1A, beta 1Am1, we therefore chose to substitute three leucines in the alpha  helix along the hydrophobic surface of the helix (Fig. 2A). These leucines (Leu-4, Leu-7, and Leu-14) in beta 1A were substituted with prolines. We then co-expressed beta 1Am1 with the gamma  subunit and each of the effector domains in yeast cells. The reporter activities induced by beta 1Am1 in combination with the gamma  subunit or the effector domains are shown in Fig. 2, B-E. As expected based on the three-dimensional structure for the beta gamma complex, the ability of beta 1Am1 to interact with the gamma  subunit was dramatically decreased (Fig. 2B). The reporter activity stimulated by co-expression of beta 1Am1 and gamma 5 is about one-tenth of that seen in cells co-expressing beta 1A and gamma 5. It is highly unlikely that this result is due to altered expression of the mutant protein since we have repeatedly examined the expression of a variety of hybrids and found them to be expressed at similar levels (12, 16). The reduced interaction could result from both the loss of leucines at the binding surface and the disruption of the helix due to proline substitutions. We do not have direct biophysical results that show disruption of the N-terminal alpha  helix of the beta  subunit, but, based on the effect of proline substitutions on the formation of an alpha  helix, it is most likely that the helix is disrupted. Although the N-terminal alpha  helix that is required for interaction with the gamma  subunit was potentially destroyed by the proline substitutions, the ability of beta 1Am1 to interact with AC2Q, GKN, and PLC62 was decreased less than 20% (Fig. 2, C-E). This indicates that the regions of beta 1A that interact with the effector domains were not significantly affected by potential disruption of the amphipathic alpha  helix. The ability of the mutations in beta 1Am1 to affect interaction with the gamma  subunit is consistent with previous results from mutant analysis of beta -gamma interaction (22).


Fig. 2. Interaction of beta 1Am1 with AC2Q, GKN, and PLC62. A, diagrammatic representation of the N-terminal 14 residues of beta 1Am1 fragment as a helical wheel. Circles represent individual residues. Residues are numbered based on position in the primary structure. Substituted residues are indicated in solid circles. B-E, beta -galactosidase activity from cells of different genotypes estimated as described under "Materials and Methods." The bars represent mean value of beta -galactosidase activity from three different transformants examined in each of two independent experiments. In panel B, beta 1Am1, the error bar was too small to be shown.
[View Larger Version of this Image (21K GIF file)]


Since the results above indicated that residues in the N-terminal alpha  helix of beta 1A were unlikely to be involved in the interaction with AC2Q, GKN, and PLC62, we investigated whether the appropriate folding of the beta  strands in beta 1A was a requirement for interaction with the effector domains. To address this question, we made the second mutant of beta 1A-beta 1Am2. The beta  subunit is made up of seven WD repeats of about 40 amino acids each (23). Based on the crystal structure of the beta  subunit, four residues that are largely conserved within each of the seven WD repeats have been implicated in forming hydrogen bonds that are important for stabilizing the characteristic folded structure of beta  strands (18, 20). In the mutant beta 1Am2, three of these residues, Ser-72, Asp-76, and Trp-82, which form hydrogen bonds were replaced with alanines. In the crystal structure of the beta gamma complex, the beta  strands 2-5 of the beta  subunit form the first beta  sheet in the G protein beta  subunit. Ser-72 is within the third beta  strand, Trp-82 is within the fourth beta  strand, and Asp-76 is within the loop connecting the third and the fourth beta  strand (Fig. 3A). Hydrogen bonding occurs between Asp-76 and His-54 which is located in the loop connecting the first and second beta  strands (Fig. 3A). Thus, a turn in the first beta  sheet is coupled to the first beta  strand. Ser-72 is hydrogen-bonded with Trp-82 as well as His-54. This bonding couples the third with the fourth beta  strand as well as the loop downstream of the first strand. Trp-82 is also in the hydrophobic core of the first beta  sheet and in contact with several residues. The folded structure formed by the five beta  strands in beta 1A is therefore likely to be disturbed significantly by altering Ser-72, Asp-76, and Trp-82 to alanines in the mutant beta 1Am2.


Fig. 3. Interaction of beta 1Am2 with AC2Q, GKN, and PLC62. A, diagrammatic representation of the "first" beta  sheet in the beta 1 subunit based on crystal structure (19). Arrows represent the four beta strands that form the first beta  sheet. Lines represent loops connecting these beta  strands. Solid circles represent the residues important in forming hydrogen bonds among different beta  strands and in stabilizing the folding of the beta  strands. Dotted lines represent hydrogen bonds between these residues. Substitutions are shown. B-E, beta -galactosidase activity from cells of different genotypes estimated as described under "Materials and Methods." The bars represent mean value of beta -galactosidase activity from three different transformants examined in each of two independent experiments.
[View Larger Version of this Image (18K GIF file)]


We first tested whether the beta 1Am2 mutant was able to interact with the gamma  subunit by co-expressing beta 1Am2 with gamma 5 in yeast cells. beta 1Am2 interacted with gamma 5 as effectively as beta 1A (Fig. 3B), indicating that the mutations in the region of the beta  strands do not affect complex formation with the gamma  subunit. Combined with the results from the beta 1Am1 mutant, this implies that residues in the alpha  helical region of beta 1A are major determinants for interaction with the gamma  subunit. Wild type beta 1A elicited significant reporter activity with the three effector domains, AC2Q, GKN, and PLC62 (Fig. 3, C, D, and E). The magnitude of activity induced by different combinations was very different, consistent with our previous results (12). The higher activity induced by beta 1A with AC2Q compared to the other two domains is most likely due to differential binding. This is based on our previous results that show direct relationship between reporter activity elicited by a particular combination of hybrids and efficiency of complex formation between the same hybrids as determined by immunoprecipitation (16). Furthermore, the lower reporter activity elicited by beta 1A with GKN and PLC62 should result from significant protein-protein interaction because these effector domains do not show significant reporter activity when co-expressed with a variety of control proteins, unrelated proteins, gamma  subunit types, or the beta 1B-D fragments. The differences in reporter activities induced by the GKN and PLC62 domains with beta 1A in comparison with a variety of controls are clearly apparent in filter assays where yeast cells have been examined for beta -galactosidase activity (Ref. 12 and data not shown). When the mutant, beta 1Am2, was co-expressed with GKN or PLC62 in yeast cells, beta 1Am2 was significantly affected in its interaction with these two effector domains, and the efficiency of interaction was decreased 3-4-fold (Fig. 3, D and E). As in the case of beta 1Am1, it is highly unlikely that this result is due to altered expression of the mutant protein since we have examined the expression of a variety of hybrids and found them to be expressed approximately at similar levels (12, 16). It is possible that the residues (Ser-72, Asp-76, and Trp-82) themselves are directly involved in binding to both GKN and PLC62. Altering these residues in beta 1A would therefore result in defective interaction with the effector domains. However, we think it is more likely that interaction of beta 1Am2 with GKN and PLC62 is affected because the folding of the beta  strands in beta 1A is disrupted. This inference is based on the following. (i) Ser-72 and Trp-82 are located within the beta sheet formed by beta  strands 2-5 where they are unlikely to be accessible to an effector domain. (ii) It seems unlikely that we could have at random mutated precisely those residues that are critical for binding with GKN and PLC62. Moreover, the insignificant effect of the L4P, L7P, and L14P mutations on beta 1Am1 binding with GKN and PLC62 show that mutational alterations of beta 1A do not in general lead to gross misfolding that renders the protein incapable of interaction with other proteins. The effective interaction of beta 1Am2 with AC2Q (Fig. 3C) further emphasizes the specificity of the effect of mutations in beta 1A on effector domain interactions.

Thus, the results from the analysis of the beta 1Am2 mutant imply that residues in beta 1A that interact with GKN and PLC62 require appropriate folding of the first five beta  strands of the beta  subunit to be able to bind these effector domains. Based on the ability of the alpha  subunit to disrupt binding of an effector with the beta gamma complex, candidates for such residues are those that are positioned in the bends between the beta  strands 1-2, 3-4, and 5-6 as well as the exposed portions of the beta  strands located at the same surface of the beta gamma complex which interacts with the alpha  subunit.

We have identified an N-terminal fragment of 100 residues from the G protein beta  subunit that is able to interact with domains specific to three different effectors, AC2, GIRK1, and PLC-beta 2. Within this fragment, the structural determinant for interaction with AC2 seems to be different from that for interaction with GIRK1 and PLC-beta 2 because of the following reason. Potential disruption of the folded structure of the beta  strands that should occur in the beta 1A fragment of the beta  subunit has no significant effect on the interaction of the mutant beta 1A with AC2Q. But potential disruption of the folded structure of the beta  strands in the mutant beta 1Am2 significantly affects interaction of this mutant beta 1A with GKN and PLC62. It is still likely that the structural determinant(s) for interaction with AC2Q are in the exposed surfaces formed by the folding of the five beta  strands. A domain (residues 52-100) of the beta 1 subunit that contains beta  strands 2-5 (which form the first beta  sheet) was able to interact with AC2Q in the two-hybrid system, although not as effectively as beta 1A (data not shown). Cross-linking and modeling implicate residues 75-165 of the beta  subunit in binding with the AC2Q peptide (17). A peptide specific to residues 86-105 of the beta 1 subunit has also been shown to inhibit interaction of the beta gamma complex with adenylyl cyclase 2 in a sequence-specific manner.2 These results further confirm a role for residues in the structure formed by the first five beta  strands in interaction with AC2. The simplest interpretation of our results is that these residues do not require the formation of the folded structure of interacting beta  strands to be able to bind AC2Q.

The presence of different structural determinants for AC2Q versus GKN/PLC62 in beta 1A suggests that this may be one mechanism that the beta gamma complex uses to discriminate between structurally distinct effectors. Similarly, mutations at the C terminus of the beta  subunit affected PLCbeta 2 activation but not activation of the mitogen-activated protein kinase pathway (24). The specificity of interaction of the beta gamma complex with GIRK1 and PLC-beta 2 could be affected by domains other than those identified here. It was reported that two domains of GIRK1 were able to bind to the beta gamma complex, an N-terminal domain and a C-terminal domain (7, 8). GKN is the N-terminal domain of GIRK1 that is able to bind to the N-terminal fragment (beta 1A) of the beta 1 subunit. The C-terminal domain of GIRK1 could bind to another site on the beta gamma complex that has not been identified yet. This second site on the beta gamma complex may be important for specific interaction between the beta gamma complex and GIRK1.

We have begun to understand the individual roles of the G protein beta  and gamma  subunits in cellular signaling. It is likely that the gamma  subunit plays an important role in receptor interaction (25, 26). The results here together with our previous findings (12) indicate an important role for the beta  subunits in effector interaction. Results from other laboratories also support this role. The AC2Q peptide was chemically cross-linked to the beta  subunit (17), and the extreme C-terminal domain of the beta  subunit was important for activation of phospholipase C-beta 2 in COS cells (24). Since prenylation of the gamma  subunit is essential for appropriate interaction with effectors (e.g. Refs. 4 and 27), the lipid group at the C terminus of the gamma  subunits could also directly interact with a site on the effector molecules.


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.
   Established Investigator of American Heart Association. To whom correspondence should be addressed. Tel.: 314-362-8568; Fax: 314-362-8571; E-mail: gautam{at}morpheus.wustl.edu.
1    The abbreviations used are: AC2, adenylyl cyclase 2; GIRK1, muscarinic atrial potassium channel; PLC-beta 2, phospholipase C-beta 2.
2    R. Iyengar, personal communication.

Acknowledgments

We thank Dr. P. Gierschik for the PLC-beta 2 cDNA and Dr. R. Iyengar for communicating results prior to publication.


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