©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of a Discrete Region of the G Protein Subunit Conferring Selectivity in Complex Formation (*)

Chunghee Lee (§) , Takeshi Murakami (¶) , William F. Simonds (**)

From the (1) Metabolic Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The identification of multiple G protein and subunit subtypes suggests a potential diversity of heterodimers, which may contribute to the specificity of signal transduction between receptors and effectors. The assembly of and subtypes is selective. For example, can assemble with but not with , whereas assembles with both isoforms. To identify the structural features of the and subunits governing selectivity in heterodimer assembly, a series of nonisoprenylated chimeras of and was constructed, and their interaction with and was assessed by their ability to direct expression to the cytosol in cotransfected COS cells. All of the /chimeras were capable of interacting with as judged by the cotransfection assay. Chimeras containing sequence near the middle of the molecule between two conserved sequence motifs were capable of interacting as well with . Among 12 divergent residues in this region, it was found that replacement of three consecutive amino acids in , Glu-Glu-Phe (residues 38-40), with the three corresponding amino acids of , Ala-Asp-Leu (residues 35-37), conferred the ability to assemble with . The reciprocal chimera containing Glu-Glu-Phe in the context of failed to assemble with . The last residue of this triplet is occupied by a leucine in all known mammalian subunits except and appears to be a key determinant of the ability of a subunit to assemble with . This locus maps to a region of predicted -helical structure in the subunit, likely to represent a point of physical contact with the subunit.


INTRODUCTION

GTP binding regulatory proteins (G proteins) are heterotrimers consisting of three subunits, , , and (1) . The and subunits exist as a tightly bound complex that can only be dissociated under denaturing conditions and that functions as an entity throughout the signaling cycle (2, 3) . The formation of heterodimers from particular combinations of and subtypes may contribute to signaling specificity as the complex interacts with G, effectors, and receptors (4, 5) . We developed a transient transfection paradigm that allows us to assess the ability of different and subunits to dimerize by monitoring the cytosolic expression of subunits upon cotransfection with nonisoprenylated constructs (6) . This assay was recently used to study truncated and point-mutated subunits (7) , leading to the development of a model for interaction in which the N terminus of forms a coiled coil-like structure with . Pronin and Gautam (8) used a similar paradigm to study subunit specificity in heterodimer formation. Using this and other approaches, several laboratories have demonstrated that interacts with but not with , whereas both subunits interact with and (8, 9, 10) . This paradigm was recently used to map the domains of the subunit responsible for discrimination between and using a series of /chimeras (11) . With respect to the subunits, there is only 38% sequence identity at the amino acid level between (12) and (13, 14) , and little is known about the domain or domains that are responsible for their differential ability to assemble with . We report here the construction of a series of /chimeras and point mutants that map the region responsible for discrimination to a short stretch of three amino acids in a predicted -helical domain. This putative -contact site lies just C-terminal to a proposed coiled coil region of the subunit.


EXPERIMENTAL PROCEDURES

Construction of Expression Vectors

Chimeric and mutant cDNA constructs were made by the polymerase chain reaction (15) using Pyrococcus furiosus DNA polymerase (Stratagene, La Jolla, CA) and wild-type bovine (16) , human (17) , bovine (12) , or (13) as the original templates. The 3` primers for the constructs contained mutations in the codon for C AAX() domain cysteine resulting in prenylation-deficient mutants (C71S in and C68S in (6) ). The mutagenic oligos used to generate chimeric components created junctions as follows: ``QLK'' junctions, after Lys codon AAG (residue 23 in , 20 in ); ``EDPL'' junctions, after Leu codon TTA (residue 53 in , 50 in ) in chimeras 112*, 212*, and 2 (21) 2*; ``EDPL'' junctions, before Glu codon GAG (residue 50 in , 47 in ) in chimeras 121*, 221*, and 1 (12) 1*; ``VSK'' junctions, before Val codon GTG (residue 33 in , 30 in ). Cluster and point mutants of subunits utilized the corresponding codons of and vice versa. Polymerase chain reaction amplification of the cDNAs was used to eliminate the untranslated flanking sequences. Polymerase chain reaction products were purified, digested, and ligated into pCDM8.1 (18) according to standard procedures (19) . Plasmids were amplified in Escherichia coli MC1061/P3 (InVitrogen, San Diego, CA) and purified by a commercially available column adsorption method (Qiagen, Chatsworth, CA). The DNA sequences of the inserts were verified by the method of chain termination (20) using Sequenase 2.0 (U. S. Biochemical Corp.).

Cell Culture and Transfection

COS-7 cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 100 units/ml penicillin and 100 units/ml streptomycin. Cells were transfected at approximately 80% confluency with DEAE-dextran (500 µg/ml) in phosphate-buffered saline (PBS) without Caand Mg(Biofluids, Rockville) according to standard procedures (21) . Typically 5 µg of each construct and 15 µg of each construct was used per 75-cmculture flask except where indicated. The total amount of DNA added was kept constant by the addition of vector DNA as needed. Cells were harvested after 2 days, washed in phosphate-buffered saline, pelleted, and kept at -80 °C prior to analysis.

Protein Analysis

Cells were lysed and fractionated into a particulate and soluble fraction as described (6) . Protein concentrations were determined according to Lowry with bovine serum albumin as standard (22) . Limited tryptic digestion of soluble proteins was accomplished using L1-tosylamido-2-phenylethyl chloromethylketone-treated trypsin (Sigma T-8642) at a ratio of 1:20 or 1:25 (w/w) by incubation at 37 °C for 30 min, at which time the reaction was terminated by the addition of denaturing sample buffer and boiling (23, 24, 25) . Proteins were separated on polyacrylamide gels under denaturing conditions and transferred to polyvinylidene difluoride membranes by electrotransfer for 12 h at 150 mA in Dunn's buffer (26) . Analysis of subunits was performed on 10 or 11% polyacrylamide gels (27) , whereas analysis of subunits employed tricine gels according to the method of Schägger and von Jagow (28) . Immunoblotting was performed as described previously (29) . The primary antibodies used were directed against residues 330-340 of (the sequence is identical in ) (SW), residues 58-68 of (PE), residues 53-70 of (LVKG), or residues 47-64 of (EDPL). Primary antibodies were detected by autoradiography of blots following incubation with I-labeled protein A as described (7) .


RESULTS

Findings from several laboratories have demonstrated selectivity in heterodimer assembly so that while both and can assemble with or -C68S (2*), but not can form heterodimers with or -C71S (1*) (8, 9, 10) . To determine which residues in were responsible for its inability to interact with , we made chimeras between 1* and 2* with the aim of identifying the minimal sequence of , which might confer interaction in the context of a 1* polypeptide. For convenience in this paper, chimeras are identified by subscripts in a three- or four-digit binary code in which 1 indicates that sequence was used, 2 that sequence was used. A schematic overview of the chimeras employed in this study is presented in Fig. 1, A and B. Three-part chimeras were initially constructed using the conserved sequence motifs Gln-Leu-Lys (QLK, residues 21-23 in and 18-20 in ) and Glu-Asp-Pro-Leu (EDPL, residues 50-53 in and 47-50 in ) as junctions between chimeric components (Fig. 1 A). Thus chimera 121* refers to the chimera containing sequence from the N terminus to the QLK motif and from the EDPL motif to the C terminus, with the sequence in between. A second set of chimeras further divided the middle segment into two regions with a junction at the Val-Ser-Lys sequence motif (VSK, residues 33-35 in and 30-32 in ) (Fig. 1, B and C). These chimeras are described by a four-digit subscript such that chimera 2 (12) 2* refers to the nonisoprenylated chimera containing sequence from the N terminus to the QLK motif and from the VSK motif to the C terminus, with the sequence between.


Figure 1: Schematic overview of the chimeric constructs employed in this study and comparison of and sequence homology around a putative contact site. A and B, schema depicting the structure of the /chimeras employed in this study. Open bars indicate polypeptide sequence derived from , and closed bars indicate that derived from . Junctions between chimeric segments indicated by vertical lines correspond to sequence motifs conserved in both and as follows: Gln-Leu-Lys (QLK, residues 21-23 in and 18-20 in ), Glu-Asp-Pro-Leu (EDPL, residues 50-53 in and 47-50 in ), and Val-Ser-Lys (VSK, residues 33-35 in and 30-32 in ). Numbers to the right of each construct correspond to its descriptive subscript, employing a 3- or 4-digit binary code as explained in the text. Before C-terminal processing, contains 74 and contains 71 residues. C, sequence homologies between the conserved VSK and EDPL motifs of and as indicated by the algorithm GAP of the Genetics Computer Group (39). Boxed residues represent the region containing a putative contact site identified in this study. Numbers in the margins indicate residue position.



To study the subunit selectivity of the chimeras, they were cotransfected into COS-7 cells with either or . assembly was determined by assessing the ability of these nonisoprenylated mutant subunits to direct expression of subunit to the soluble protein fraction (6, 7) . Recombinant and nonisoprenylated subunits associate in a stable soluble complex (10, 30) , and trypsin treatment of a COS cell cytosolic fraction containing * produces a stable 26-27-kDa C-terminal fragment characteristic of native complexes (Ref. 31 and see below). Transfection of or cDNA alone produced an increase in immunoreactivity over vector-transfected controls in the crude particulate fraction (Fig. 2, A and B, lanes 3 and 5, cf. lane 1) but not in the soluble fraction (Fig. 2, A and B, lanes 4 and 6). The increase in immunoreactivity in the crude membrane fraction due to transfection of cDNA alone was more marked than observed previously (6) but is consistent with work from other laboratories (31) . While these differences in the crude membrane fraction may result from variations in the efficiency of separation of the low speed nuclear pellet from the postnuclear fractions (6) , they were not explored further in this study.


Figure 2: Selectivity in assembly demonstrated by the cytosolic shift assay. COS cells were transfected with vector alone ( Con) or with or cDNAs alone or in combination with -C71S (1*) ( A) or -C68S (2*) ( B) cDNAs as indicated. After fractionation into particulate ( P) and soluble ( S) components, aliquots of 50 µg were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting with the /C-terminal antibody SW ( upper panels, on 10% polyacrylamide gels) or the -directed C-terminal antibodies PE (C-terminal) or EDPL (C-terminal) ( lower panels, on tricine gels (28)) as described under ``Experimental Procedures.'' Shown are 24-h autoradiograms after incubation with I-labeled protein A. To the left, the relative mobility of marker proteins is shown in kDa (36/35 kDa, the SW-reactive doublet from bovine brain; 6.5 kDa, aprotinin). The data shown are representative of five such experiments with similar results.



Cotransfection in COS cells of with either 1* or 2* resulted in the expression of both and subunits in the cytosol (Fig. 2, A and B, lanes 8). In contrast, cotransfection with 2* but not 1* resulted in the cytosolic expression of both subunits (Fig. 2, A and B, lanes 10), confirming the previously described incompatibility of with and 1* (8, 9, 10) . It should be noted that in cotransfected cells, the appearance of significant immunoreactivity in the cytosol correlates with the presence of a signal there (Fig. 2, A and B, cf. lanes 8 and 10). This results presumably from subunit stabilization engendered by cotransfection of compatible and subunits as recently described (32) . Subsequent screening of nonprenylated chimeras for their ability to assemble with or was therefore performed by analysis of cytosolic fractions only.

Soluble heterodimers containing nonisoprenylated subunits show no functional interaction with effector molecules such as adenylyl cyclase (10) and phospholipase C- (33, 34) nor with G (10) , although reports to the contrary have appeared (35) . To verify that the global folding of the subunits in the soluble nonprenylated complex mimics that of functional complexes, analysis by limited tryptic digestion was performed (Fig. 3) (23, 24, 25) . A 26-kDa C-terminal fragment was protected from trypsin in the cytosolic fraction of COS cells cotransfected with 1*, 2*, and 2*, which comigrated with the fragment generated by identical treatment of authentic brain (Fig. 3) consistent with previous reports (31) . Prior heat denaturation abolished the trypsin resistance of the C-terminal fragments (Fig. 3). These findings support the contention that soluble, nonprenylated complexes resemble native heterodimers in the global conformation of their subunits.


Figure 3: Limited tryptic digestion of COS cell cytosolic fractions. Soluble fractions (25 µg of protein) of COS cells transfected with vector alone (-) or with cDNAs for or in combination with -C71S (1*) or -C68S (2*) were either left on ice or incubated with trypsin (1 µg) for 37 °C for 30 min either without or with prior heat treatment at 80 °C (5 min) as indicated ( lanes 1-10). Samples were subsequently analyzed by SDS-polyacrylamide gel electrophoresis (11% polyacrylamide) and immunoblotting with the C-terminal antibody SW as described under ``Experimental Procedures.'' Lanes 11-13 show the results of an identical analysis of purified bovine brain ( ) performed in parallel (200 ng combined with 25 µg of control COS cytosol/lane). Autoradiograms were exposed for 14 h. To the left are indicated the relative mobility of untreated subunit and the major 26 kDa tryptic fragment. The experiment was repeated three times with similar results.



Because our strategy was to estimate the ability of chimeras to assemble with subunits by monitoring cytosolic immunoreactivity, we sought experimental conditions that would minimize variability because of unequal distribution of and cDNAs during cotransfection or due to small variations in the quality of chimeric cDNA plasmid preparations. We wanted conditions therefore in which the amount of but not cDNA would be limiting with respect to the expression of cytosolic subunit. Under these conditions, any observed differences in soluble expression among cotransfectants employing different mutant constructs would be more likely to reflect differences in subunit expression and protein-protein interaction. To establish these conditions, a constant amount of cDNA was cotransfected with increasing amounts of 2* cDNA and the level of cytosolic expression monitored. was chosen because the gain of interaction by 1* mutants substituted with -specific residues was the principal end point of mutagenesis. The signal generated from 5 µg of cDNA became saturated at 10-15 µg of 2* cDNA (not shown). For subsequent screening of /chimeras, therefore, a 3:1 ratio of to cDNA (w/w) was employed.

The first set of nonisoprenylated chimeras was constructed using the conserved QLK and EDPL sequence motifs as junctions dividing the subunit into three regions (Fig. 1 A). Transfection of vector or 1 cDNA alone produced no immunoreactivity in the cytosol (Fig. 4, A, lanes 1 and 2) as previously shown. In contrast cotransfection of 1 with 1* (111*), 2* (222*), and each of six three-part chimeras, 121*, 211*, 221*, 112*, 122*, and 212*, all promoted the appearance of cytosolic -immunoreactivity (Fig. 4 A, lanes 3-10 upper panel). Immunoblotting with -specific C-terminal antibodies confirmed the expression of all of the chimeric constructs (Fig. 4 A, lanes 3-10, lower panels). In contrast to the results with , only chimeric constructs 121*, 221*, and 122* along with 2* (222*) supported cytosolic expression when was cotransfected (Fig. 4 B, lanes 4, 6, 8, and 10). These results focussed our attention on the region of the subunit between the QLK and EDPL sequence motifs as chimeras containing but not sequence here proved capable of assembling with .


Figure 4: Selective cytosolic expression of subunit isoforms cotransfected with different 1*/2* chimeras. COS cells were transfected with vector ( Con) or cDNA alone or cDNA in combination with 1* ( -111*), one of six 1*/2* chimeras, or 2* ( -222*). The nomenclature and composition of the 1*/2* chimeras are explained in the text and the legend to Fig. 1 A. Shown are 24-h autoradiograms of immunoblots of the cytosolic fractions of experiments employing ( A) and ( B) cDNAs. The antibodies employed are as explained in the legend to Fig. 2. Three other experiments produced an identical expression pattern.



A second set of chimeras derived from 121* and 212* was created by subdividing the middle region around a conserved Val-Ser-Lys (VSK) sequence motif (Fig. 1 B). Like the parental chimera 121* (1 (22) 1*), chimera 1 (12) 1* was capable of directing the expression of both and to the cytosol (Fig. 5 A, upper panel, lanes 5, 6, 8, and 9). Similarly, expression of both chimeras 1 (22) 1* and 1 (12) 1* was demonstrable in immunoblots of cells cotransfected with either or (Fig. 5 A, lower panel, lanes 5, 6, 8, and 9). In contrast chimera 1 (21) 1* displayed a 1*-like phenotype, interacting with but not with (Fig. 5 A, lanes 11 and 12). These results suggested that residues within the region between the VSK and EDPL motifs of and determine the ability to dimerize with (Fig. 1 C). Consistent with this interpretation was the finding that chimera 2 (12) 2* but not 2 (21) 2* (Fig. 1 B) interacted with in the cytosolic shift assay (data not shown). These results confirm a recent report by Spring and Neer (36) who utilized subunit chimeras synthesized by in vitro translation to map the discrimination region to this same 14-amino acid region (Fig. 1 C). As noted previously with nonchimeric * subunits (7, 11) , when transfected alone, none of the three * chimeras produced significant cytosolic or immunoreactivity (Fig. 5 A, lanes 4, 7, and 10).


Figure 5: Selective cytosolic expression of subunit isoforms cotransfected with different */* chimeras or *-derived cluster mutants. COS cells were transfected with vector (-), , , or cDNA alone or the combination of or cDNA with 121* (1(22)1) or one of the 1*/2* chimeras 1(12)1* or 1(21)1* ( A) or one of the 1*-derived cluster mutants 1*-ADL, 1*-MAYC, or 1*-AHAK ( B) as indicated. The nomenclature and composition of the 1*/2* chimeras and cluster mutants is explained in the text. Shown are 24-h ( A) or 14-h ( B) autoradiograms of immunoblots of cytosolic fractions (50 µg/ lane) employing antibodies SW () or LVKG (C-terminal) as indicated. Similar results were obtained in three additional experiments.



Among the 14 amino acids that lie between the conserved VSK and EDPL sequence motifs, 12 residues differ between and (Fig. 1 C). A pair of cysteine residues at positions 36 and 37 in , represented by twin alanine residues in , can be linked by disulfide bond formation to Cysof the subunit in the presence of cupric phenanthroline (37) . This pair of cysteines lies closely apposed to the N-terminal -helical region of in a recent model of interaction based on a coiled coil interaction (7) . In a study of the effect on assembly of point mutations in the putative coiled coil region of , it was found that mutation of both Cysand Cysto Ala failed to confer the ability to assemble with .() The 10 remaining divergent residues in were therefore replaced with sequence in three mutant constructs, each containing a cluster of three or four point mutations. In mutant 2*-ADL, residues 38-40 of 1*, Glu-Glu-Phe, were replaced with Ala-Asp-Leu. Mutant 1*-MAYC was made by replacing residues 41-44 of 1* with the sequence Met-Ala-Tyr-Cys (the Tyr is conserved in both and ). In mutant 1*-AHAK residues 46-49 were replaced by the sequence Ala-His-Ala-Lys. Fig. 5 B shows the results of cotransfection of the three 2* cluster mutants with and . When transfected alone, none of the three 2* cluster mutants produced significant cytosolic or immunoreactivity (Fig. 5 B, lanes 4, 7, and 10). While cotransfection of all three cluster mutants with resulted in the cytosolic expression of immunoreactivity, only 2*-ADL produced a significant cytosolic signal when the cotransfection employed (Fig. 5 B, upper panel, lane 6, cf. lanes 9 and 12). As expected, steady-state chimera immunoreactivity in the cytosol of -cotransfected cells correlated with the strength of the signal and was greatest in the case of 2*-ADL (Fig. 5 B, lower panel, lane 6, cf. lanes 9 and 12). These observations identify this triplet of amino acids as the dominant locus between the conserved VSK and EDPL motifs of and determining the ability to interact with (Fig. 1 C). This region forms part of an -helix in both and according to secondary structure predictive algorithms (38, 39) .

In order to analyze the individual contribution of residues within this triplet of amino acids, a series of point mutants were made exchanging individual residues in this region between 1* and 2*. Point mutants E38A, E39D, and F40L of 1* were made and transfected alone or in combination with either or (Fig. 6 A). When transfected alone, none of the three point mutants of 1* produced significant or immunoreactivity in the cytosol (Fig. 6 A, lanes 4, 7, and 10). Cotransfection of all three 1*-derived point mutants with , however, resulted in a significant cytosolic shift of (Fig. 6 A, upper panel, lanes 5, 8, and 11) and definite -immunoreactivity (Fig. 6 A, lower panel, lanes 5, 8, and 11). Upon cotransfection with , only 1*-F40L interacted significantly as seen in both and blots (Fig. 6 A, lane 12, cf. lanes 6 and 9). When residues from this region of were substituted into 2* to create 2*-EEF and 2*-L37F, these mutants, like the parental construct 2*, were expressed and directed -immunoreactivity to the cytosol when cotransfected with but not when transfected alone (Fig. 6 B, lanes 4, 7, and 10 cf. lanes 5, 8, and 11). Both 2*-EEF and 2*-L37F, however, demonstrated a greatly reduced ability to interact with by comparison with parental 2* (Fig. 6 B, lanes 6 and 9 cf. lane 12). Construct 2*-EEF was somewhat more selective against than the L37F point mutant as evidenced by both and immunoblots (Fig. 6 B, lane 6 cf. lane 9). The 2*-derived point mutants A35E and D36E interacted significantly with in similar experiments (not shown).


Figure 6: Selective cytosolic expression of subunit isoforms cotransfected with point mutants of 1* or 2*. COS cells were transfected with vector (-), , , or cDNA alone or the combination of or cDNA with the 1*-derived point mutants E38A, E39D, or F40L ( A) or the 2*-derived mutants 2*-EEF or L37F or the parental construct 2* ( B) as indicated. Shown are 7-h ( A) or 17-h ( B) autoradiograms of SW (), LVKG (C-terminal) or EDPL (C-terminal) immunoblots of cytosolic fractions (50 µg/ lane) as indicated. Two additional experiments yielded essentially the same results.



The iterative process of 1* mutagenesis employed in this study identified the chimera or mutant best able to interact with at any given stage by comparison with ``sibling'' mutants containing similar amounts of sequence. A side-by-side longitudinal comparison of the entire series of -interacting 1* chimeras and mutants revealed however that the 1*-F40L point mutant did not consistently support the cytosolic expression of to the same extent as 2*, 121*, 1 (12) 1*, or 1*-ADL (Fig. 7 A, lanes 3, 5, 7, and 9 cf. lane 11). To exclude the possibility that the chimeric and mutant derivatives of 1*, which directed expression to the cytosol, did so by virtue of an anomalous interaction with the polypeptide, a limited trypsin digestion of cytosolic fractions from cells cotransfected with and 121*, 1 (12) 1*, 1*-ADL, or 1*-F40L was performed (Fig. 7 A). In all cases, a 26 kDa C-terminal fragment of was protected from further degradation, which comigrated with the C-terminal fragments derived from coexpressed 2* treated in parallel (Fig. 7 A, lanes 6, 8, 10, and 12 cf. lane 4). Tryptic analysis of cytosolic fractions from cells cotransfected with and 2*-EEF or 2*-L37F similarly demonstrated preservation of a 26-kDa C-terminal fragment (Fig. 7 B). These results confirm the native folding of the heterodimers formed between subunits and these mutant derivatives of 1* or 2*.


Figure 7: Limited tryptic digestion of COS cell cytosolic fractions cotransfected with or and mutant derivatives of 1* or 2*. COS cells were transfected with vector alone (-) or with cDNAs for or in combination with 1*, 2*, 121*, 1(12)1*, 1*-ADL, or 1*-F40L ( A) or 2*, 2*-EEF, or 2*-L37F ( B) as indicated. Soluble fractions (25 µg of protein) were either left on ice or incubated with trypsin (1 µg) at 37 °C for 30 min where indicated. Shown are 12-h ( A) or 17-h ( B) autoradiograms of SW ( C-terminal) immunoblots employing different batches of [I]-protein A. Similar results were obtained in two other experiments.




DISCUSSION

The substitution of progressively smaller linear segments of into the context of a nonprenylated mutant of has allowed the identification of a short segment of predicted -helix three residues in length, which confers the ability to form heterodimers with . This locus is contained within the 14-amino acid region of the subunit between the conserved VSK and EDPL sequence motifs first identified as a discrimination region in a report by Spring and Neer (36) , which appeared while this manuscript was in preparation. The present study identifies a conserved hydrophobic residue, Phein and Leuin , which appears to play a major role in determining the phenotype with respect to selectivity. Interestingly, all known mammalian subunits except contain a leucine in this position (), and , , , and can all form stable heterodimers with (8, 9, 10, 40, 41, 42, 43) . Because the leucine-containing is currently available only as a partial cDNA clone (44) , its ability to assemble with remains an open question. Since the interaction of the 1*-F40L and 1*-ADL mutants was weaker than that of chimeras containing additional sequence, it is quite likely that residues neighboring the conserved hydrophobic amino acid contribute to the proper conformation of this locus. The domain of identified here may not be the only region determining selectivity in assembly, however, since neither , , nor assemble with (8, 9) . The novel component of dimers purified from retina has not yet been identified (45) , and whether its sequence in this region resembles the known subunits is unknown.

While it seems likely that this region of may represent a point of physical contact with the subunit, it is also possible that the effect of the mutations described here results from a more general perturbation of the conformation of the chain affecting a distant contact site (or sites). The ability of the mutants employed in this study to protect the 26-kDa C-terminal fragment of from further tryptic digestion would make a grossly abnormal conformation unlikely, however. In either case, the deleterious effect of Phe on assembly relative to Leu might result from steric hindrance: the inability of to accommodate the aromatic side chain at a point of contact or an unfavorable effect of the bulky Phe side chain imparted to a remote contact site on . Because the discrete domain of described here was identified by screening for selectivity in /interaction, these results in no way exclude the possibility of other nonselective contact sites between and ( e.g. through possible coiled coil formation (7, 46) , see below).

A model of interaction involving the parallel arrangement of -helical segments in a coiled coil was recently proposed based on mutagenesis of the subunit (7) and molecular modelling (7, 46) . This model accounted for the proximity of residues Cysin and Cysin inferred from earlier cross-linking studies (37) . The favored model (of two that differed in the domain of the subunit involved) would position the three-amino acid region of identified in the current study just C-terminal to the putative coiled coil domain (7) ().

The three-residue domain of identified in this study also lies immediately C-terminal to the pair of cysteine residues implicated in the cross-linking study (37) , placing residue Pheapproximately 4.5-6 Å in axial distance away from Cysif this region of is indeed -helical. One might therefore expect the corresponding domain in , which contacts and discriminates between and , to map close to residue Cys. To the contrary, results from two laboratories exclude the N-terminal region of as the site of discrimination (8, 11) . A recent study of /chimeras that mapped the domains responsible for selective assembly with found that multiple residues dispersed in the linear sequence contributed to this function (11) . In simple two-component chimeras, the shortest linear segment of or that conferred the parental phenotype spanned residues 215 to the C terminus (11) . This region of the subunit consists of internally homologous repeat segments of approximately 40 amino acids in length, termed GH-WD repeats, or WD-40 repeats, which have been also identified in a phylogenetically and functionally diverse array of proteins (47, 48, 49) . A concentration of residues in the subunit important for discrimination between 1* and 2* was found in the variable connecting segments joining conserved portions of the 5th and 6th GH-WD repeats (11) .

Because this study of /chimeras and the preceding one employing /chimeras (11) used the same paradigm to address directly complementary questions, the most economical interpretation would be that the discrete domain on identified here interacts with multiple residues of the subunit, including most likely residues in the variable connecting segments within and adjacent to the 5th and 6th GH-WD repeats (47) . Predictive algorithms suggest the secondary structure of these connecting segments may include turns or coils (38, 39) . Thus the compact folding of the subunit inferred from previous biochemical studies (25) might position the residues of subserving discrimination into a single hydrophobic surface or pocket sufficient to accommodate the residue corresponding to Phein .

  
Table: Homologies among G protein subunits around a putative contact site identified in and

An alignment of the amino acid sequences predicted by cDNAs for bovine (12), (13), (44), murine (44), bovine (50), and (51) flanking a putative contact site is shown. Residues aligning with the three-amino acid locus identified in this study are shown in boldface. An overlying ``c'' indicates involvement in a putative coiled coil structure according to model 1 of Ref. 7. A similar coiled coil model of by Lupas et al. (46) suggests that Sermay be the last involved residue. An asterisk identifies a conserved hydrophobic residue identified in the current study, which helps define the specificity of this proposed contact site. The two Cys residues immediately preceding this region in (Cys) are those that can be cross-linked to Cysof by cupric phenanthroline (37). Numbers indicate the residue positions predicted by the cDNAs, except in the case of the sequence shown for , which derives from a partial clone (44). It should be noted that human differs from the bovine sequence by the substitution of Val for Phe at residue 40 (52).



FOOTNOTES

*
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.

§
Present address: NHLBI, Laboratory of Cellular Signaling, Bldg. 3, Rm. B-102, Bethesda, MD 20892.

Present address: Dept. of Cardiovascular Medicine, Hokkaido University School of Medicine, Kita-15 Nishi-7 Kita-ku, Sapporo 060, Japan.

**
To whom correspondence should be addressed: NIDDK, Metabolic Diseases Branch, Bldg. 10, Rm. 8C-101, 10 Center Dr., MSC 1752, Bethesda, MD 20892-1752. Tel.: 301-496-9299; Fax: 301-402-0374; E-mail: wfs@helix.nih.gov.

The abbreviation used is: C AAX, Cys-aliphatic-aliphatic-Ser/Leu.

A. Garritsen, R. Collins, and W. F. Simonds, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. J. Hurley for the generous gifts of and cDNAs and Dr. N. Gautam for the generous gifts of and cDNAs. We also thank Dr. Regina Collins for help with cell culture, George Poy for oligonucleotide synthesis, and Dr. Andrew Shenker for critical review of the manuscript. Finally, we thank Dr. Allen Spiegel for continued support and encouragement.


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