©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Bovine Brain G Isoforms Have Distinct Subunit Compositions (*)

(Received for publication, November 23, 1994; and in revised form, January 13, 1995)

Michael D. Wilcox Jane Dingus Eric A. Balcueva (1) William E. McIntire Nitin D. Mehta Kevin L. Schey Janet D. Robishaw (1) John D. Hildebrandt (§)

From the Department of Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29464 and Geisinger Clinic, Weis Center for Research, Danville, Pennsylvania 17822

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The subunit composition of the major bovine brain G(O) and G(i) proteins (G, G, G, G, and G) was characterized using antibodies against specific isoforms. Each of the purified G protein heterotrimers contained a heterogeneous population of subunits, and the profiles of the subunits found with G, G, and G were similar. In contrast, each G(O) isoform had a distinct pattern of associated subunits. These differences were surprising given that all three alpha(O) isoforms are thought to share a common amino-terminal sequence important for the binding of beta dimers and that the alpha and alpha proteins may come from the same alpha mRNA. The free alpha and alpha subunits had unique elution behaviors during MonoQ chromatography, compatible with differences in their post-translational processing. These results indicate that both the alpha and subunit compositions of heterotrimers define the structure of an intact G protein. Furthermore, the exact subunit composition of G protein heterotrimers may depend upon regulated expression of different subunit isoforms or upon cellular processing of alpha subunits.


INTRODUCTION

Heterotrimeric G proteins mediate transmission of extracellular signals from cell-surface receptors to intracellular effectors(1, 2, 3, 4, 5) . They are a diverse family of proteins with a common structure composed of alpha, beta, and subunits(6, 7, 8) . Historically, they are named for their alpha subunit. Receptors catalyze exchange of bound GDP for GTP on the alpha subunit and promote dissociation of the G protein into a separate alpha subunit and a beta dimer(1) . This suggests simultaneous regulation of multiple cellular responses by G proteins since both components independently regulate intracellular effectors(9, 10, 11) .

G protein subunits exist in a variety of isoforms. To date, 20 alpha, 5 beta, and 6 subunits have been described(1, 2, 3, 4, 5, 12, 13, 14) . (^1)Random association of these subunits would generate literally hundreds of different heterotrimeric proteins. Whether preferred combinations of isoforms combine to form a more limited number of distinct complexes is not clear(15, 16) . Regardless, G protein-coupled receptors seem to require heterotrimers containing specific alpha, beta, and subunit isoforms(17, 18, 19) . Thus, receptor regulation of G protein function must be strongly influenced by how heterotrimer composition is determined.

We have separated bovine brain G protein heterotrimers based upon their constituent alpha subunits and then determined their subunit composition using isoform-specific antibodies(12, 20) . All of the purified G proteins contained a heterogeneous mixture of subunits, suggesting a great diversity of the number of possible heterotrimer combinations. However, for the major brain G protein, G(O)(21, 22) , the subunit composition varied between alpha(O) splice variants and between alpha(O) subunits thought to differ because of post-translational modifications. These results support the idea of specific alpha and subunit associations (23) and indicate that both subunits are important to define a heterotrimer. Furthermore, the exact subunit composition of G protein heterotrimers may depend both upon regulated expression of different subunit isoforms and upon cellular processing of alpha subunits.


EXPERIMENTAL PROCEDURES

Materials and General Procedures

Affi-Gel-5 and Affi-Gel-102 were from Bio-Rad, and sulfo-MBS (^2)was from Pierce. The subunits were resolved on a 10-20% gradient gel using a Tricine buffer system(24) . Protein bands were visualized by either Coomassie Blue or silver staining(25) . The staining intensities of protein bands on SDS-polyacrylamide gels were measured as described (26) using an Omnimedia XRS scanner with Image analysis software (National Institutes of Health). Protein concentrations were determined either by the bicinchoninic acid procedure (Pierce) or by scanning densitometry of protein bands on Coomassie Blue-stained SDS-polyacrylamide gels.

Preparation of Bovine Brain G Protein Isoforms and Subunits

Bovine brain G proteins were prepared according to Sternweis and Robishaw(21) , with modifications by Kohnken and Hildebrandt(27) . This mixture of heterotrimers was separated on an HR 5/5 MonoQ FPLC column (Pharmacia Biotech Inc.). G protein (10 mg) was loaded onto the MonoQ column and eluted over 60 min at 1.0 ml/min with a linear gradient of 100-200 mM NaCl in 20 mM Tris, 1 mM EDTA, 1 mM dithiothreitol, 0.1% Thesit, 0.7% CHAPS, pH 8.0. Peak fractions of each G protein heterotrimer were pooled and passed one or more (for G) times over the MonoQ column. G and G were resolved into their alpha subunits and beta dimers using previously described procedures implemented on an FPLC column(26, 27) . Pooled purified proteins were concentrated and exchanged into 20 mM HEPES, 1 mM EDTA, 1 mM dithiothreitol, 0.1% Thesit, 100 mM NaCl using an Amicon concentrator with a YM-10 membrane.

Preparation of Antisera and Immunoblotting

Immunoblotting for subunit isoforms was carried out following a high temperature transfer procedure (28) and isotype-specific antisera (20) as described previously. Bound antibodies on immunoblots for the subunits were visualized using a I-labeled goat anti-rabbit F(ab`)(2) fragment.

Isoform-specific alpha subunit antisera were raised to the sequences NTYEDAAAY(C) (antiserum AO1, residues 295-303 of alpha), KNNLKDCGLF (antiserum AI12, COOH-terminal residues of alpha and alpha), and GNLQIDFADPQR (antiserum AI2L, residues 89-100 of alpha). Peptides AI12 and AI2L were coupled to keyhole limpet hemocyanin with glutaraldehyde(29) , and peptide AO1 was coupled to keyhole limpet hemocyanin using sulfo-MBS(30) . Antisera to the coupled peptides were raised in rabbits according to Green et al.(30) . The alpha(O) antibody, recognizing the sequence NLKEDGISAAKDVK (residues 22-35) common to all alpha(O) isoforms(31) , was a generous gift of Dr. Ravi Iyengar. Anti-alpha antibodies were affinity-purified using peptide immobilized on Affi-Gel-15 (AI12 and AI2L) or on Affi-Gel-102 treated with sulfo-MBS (AO1). Bound antibodies were eluted either with 0.1 M glycine, pH 2.5, 0.5 M NaCl neutralized with 2 M ammonium bicarbonate (AO1 and AI12) or with ActiSep elution medium (AI2L) from Sterogene (Arcadia, CA). Immunoblotting for alpha subunits followed the methods of Towbin et al.(32) . Bound antibody was visualized using the ECL reagents from Amersham Corp.


RESULTS AND DISCUSSION

Isolation of G Protein Heterotrimers Based upon Their alpha Subunit Composition

Chromatography of mixed G protein preparations on a MonoQ FPLC column (Fig. 1B) resolved heterotrimers containing three distinct alpha subunits of 39, 41, and 40 kDa (Fig. 1A), corresponding to the alpha subunits of G(O), G, and G, respectively (33, 34, 35, 36) . When the fractionated G proteins were analyzed by SDS-polyacrylamide gel electrophoresis, scanning densitometry of the alpha subunit bands showed that G(O)alpha could be resolved into three distinct peaks (Fig. 1C). The first two peaks were the G and G isoforms(15, 33, 34, 36) , respectively, whose alpha subunits are products of two splice variants of a single alpha(O) gene(37, 38) . (^3)The third G(O) peak was apparently the same heterotrimer referred to as G by Katada and co-workers(15, 34) . For consistency with past publications(35, 36) , it is referred to here as G.


Figure 1: Separation of bovine brain G protein heterotrimers. A, 10-µl aliquots of 1-ml fractions were analyzed on a 13% acrylamide, 0.4% bisacrylamide SDS-polyacrylamide gel and stained with Coomassie Blue. B, the elution of the G protein isoforms was continuously monitored by changes in the absorbance at 280 nm. The NaCl gradient is indicated (- - -). C, the intensity of each alpha subunit band in the gel in A was analyzed by scanning densitometry to determine the elution profiles of specific alpha subunit isoforms. The elution positions of five G protein isoform peaks (based upon alpha subunits) are indicated: circle, G(O) isoforms (39 kDa alpha) including G, G, and G; bullet, G (41 kDa alpha); , G (40 kDa alpha).



One or more additional FPLC steps were required to isolate each of five distinct G protein heterotrimers corresponding to G, G, G, G, and G (Fig. 2A). These assignments were confirmed by immunoblotting with alpha subunit-specific antibodies (Fig. 3). Galpha and Galpha were both recognized by antibodies specific for an alpha sequence (39) . This agrees with the conclusion of Shibasaki et al.(38) that Galpha and Galpha are both translated from the same alpha splice variant. Silver-stained gels (Fig. 2A) and immunoblots (Fig. 2B) revealed minor cross-contamination of the G protein heterotrimers, particularly G and G. Nevertheless, each G protein was at least 80% pure. G, G, and G were nearly homogeneous. The stoichiometry of GTPS binding to the purified heterotrimers was 0.9:1 (assuming an M(r) of 80,000) for all of the isoforms except G, which was 0.8:1. These data suggest that these proteins are purified as intact viable heterotrimers.


Figure 2: A, analysis of the alpha and beta subunit composition of the purified G protein isoforms by gel electrophoresis. Purified G protein isoforms (1 µg) were loaded in each lane of a 10-20% gradient SDS-polyacrylamide gel. The resolved alpha and beta subunits were visualized by silver staining. B, analysis of the subunit composition of the purified G protein isoforms by gel electrophoresis. The gel represents a typical analysis of three sets of G protein heterotrimers. Purified G protein isoforms (1 µg) were resolved on a 10-20% gradient acrylamide, 0.8% bisacrylamide gel using a Tricine buffer system. The protein bands were visualized by silver staining after first staining with Coomassie Blue.




Figure 3: Analysis of G isoform preparations using antibodies against specific alpha isoforms. Purified G protein isoforms (1 µg) were separated on 13% acrylamide, 0.4% bisacrylamide gels, transferred to nitrocellulose, and immunoblotted with antibodies with the indicated specificity for alpha isoforms (see ``Experimental Procedures'').



Subunit Association with G Protein Heterotrimers

The G protein heterotrimers described above were separated based upon their alpha subunit composition. Their associated subunit isoforms were analyzed using two different techniques. The first was analysis by electrophoresis on Tricine gels(24) , which resolved the subunits into three discrete bands (Fig. 2B). These three bands, from top to bottom, are thought to correspond to (3), (2), and a combination of (7) and (5)(40) . The second technique was analysis by immunoblotting (Fig. 4) using isoform-specific antibodies generated to unique sequences in four subunits thought to be present in brain(12, 20) . Analysis of these data is complicated by the fact that there are several additional incompletely characterized subunits.^1 Nevertheless, from these data, we can draw important conclusions that are only reinforced by the recognition of additional G protein subunit heterogeneity.


Figure 4: Immunoblot analysis of subunit distribution among G isoforms. Purified G protein isoforms (1 µg) were resolved on 15% polyacrylamide gels and immunoblotted with antisera of the indicated subunit specificity as described under ``Experimental Procedures.'' The immunoblots shown are typical of the results of the analysis of three sets of heterotrimers, with the exception of 3, as described in the text.



Both techniques verified the observation that G protein heterotrimers, defined by their alpha subunits, contain multiple subunit isoforms and hence multiple combinations of beta dimers(15, 16) . Furthermore, G and G had very similar subunit compositions that were not substantially different from that of G. Although interesting and potentially important, this conclusion may have to be refined as subunit heterogeneity is further characterized. In addition, such nonspecific association of subunits could have resulted from random recombination of the subunits during the isolation of the heterotrimers. Surprisingly, the three G(O) isoforms had different subunit profiles (Fig. 4). For example, G had considerably more (2) than did G or G. Even more striking was the minimal amount of (7) in G and the virtual absence of (5) in G. In contrast, the contribution of beta(1) and beta(2) to each of the different heterotrimers was the same (data not shown). These results cannot be explained by random association of alpha subunits with beta dimers, either in vivo or during G protein purification.

Variability of (3) Results

The alpha/ associations reported above were highly reproducible, and identical results were seen in three separate heterotrimer preparations. The only exception to this was the distribution of (3) among the G proteins. In particular, the amount of (3) in G could vary a great deal from that shown in Fig. 4, where there is less (3) in G than in G. In another preparation, G had much more (3) than any of the other heterotrimers. Two previous studies using gel electrophoretic behavior (15, 16) appear to report conflicting results for the amounts of (3) associated with G. The difference between those two studies is similar to the variability seen by us here (Fig. 4). The significance of these observations is not entirely clear, but is likely related to the fact that free (3) subunits can be found dissociated from beta subunits(41) . A reasonable explanation for our results and those of others (15, 16) is that the association of 3 with beta is labile and results in its variable recovery with different G proteins, particularly G.

Origin and Significance of Different Subunits Associated with G(O)alpha Isoforms

Galpha and Galpha are thought to be products of two splice variants of a single G(O)alpha gene(37, 38) , referred to as alpha and alpha, respectively(42, 43) . The different subunit combinations observed with these two alpha isoforms could result from coordinated expression of alpha and in different tissues or cells or from preferences of alpha for particular beta isoforms during heterotrimer formation. In either case, both the alpha and subunit compositions of heterotrimers define the intact G protein. Recent evidence for direct binding of the subunit to alpha (23) is consistent with the idea that heterotrimers are made up of specific subunit combinations. Since alpha and alpha are thought to have identical NH(2)-terminal sequences likely to be involved in alpha binding to beta(44) , their different subunit preferences could suggest an additional region of subunit interactions near the COOH terminus. Alternatively, different post-translational modifications of alpha(O) splice variants at their NH(2) termini could also explain their different subunit preferences.

G and G were separated by us (Fig. 1) and by others (15, 34) based upon their differing behavior on a MonoQ column. Antibody and proteolytic peptide mapping data suggest that Galpha and Galpha come from the same alpha mRNA(15, 38) , and our results agree with this conclusion (Fig. 3). However, since G and G differ in their subunit composition (Fig. 4), it is possible that their alpha subunits are in fact identical and that their different elution during MonoQ chromatography is due solely to their different beta dimer compositions. To test this possibility, alpha and alpha were separated from their beta dimers and individually examined on a MonoQ column (Fig. 5). The position of the protein in each case was determined by GTPS binding activity and immunoreactivity with an alpha-specific antibody (AO1). alpha eluted as a single major peak with a shoulder probably indicative of some contamination with alpha. Interestingly, alpha eluted as two fairly sharp peaks in two of three preparations, suggesting additional alpha heterogeneity. Regardless, the two isolated alpha subunits, alpha and alpha, still eluted differently on the MonoQ column. Thus, not only are the beta dimers of G and G different, but their alpha subunits differ as well. Although these results do not exclude the possibility that alpha and alpha are internal splice variants of the alpha(O) gene, existing evidence suggests that they have the same coding sequence. Four mRNAs have been shown to be generated from the bovine alpha(O) gene(45) . One corresponds to alpha and would generate alpha. The other three differ in their 3`-untranslated regions, but code for identical alpha proteins. Presumably, alpha and alpha are derived from these three mRNAs. This suggests that they are proteins with the same sequence, but different post-translational modifications.


Figure 5: Comparison of the elution profiles of alpha and alpha on a MonoQ FPLC column. The alpha (circle) and alpha (up triangle) isoforms (100 µg each) were loaded in separate experiments onto a MonoQ column and eluted at 1 ml/min using a linear NaCl concentration gradient as indicated(- - - ). The elution profiles of the alpha isoforms were determined by GTPS binding activity in the fractions indicated and by immunoblotting of sample aliquots using the alpha-specific antibody characterized in Fig. 3.



As discussed above, the different patterns of subunit expression observed with G and G could result from different preferences of alpha subunits for specific beta dimers. The variable interaction of isoforms with alpha subunits (46) might support such a conclusion. Alternatively, our results could be explained by the regulated coexpression of alpha and subunit isoforms in different cells, or perhaps even the same cell. This could be related to the multiple transcripts coding for the alpha protein(45) . Future studies will be required to define the relative contributions of these two possibilities. Immunocytochemistry with site-specific antibodies and in situ hybridization determining the expression of variable alpha and subunit proteins and transcripts will be important to resolve the possible colocalization of these proteins. Complementary biochemical studies of the G(O) isoforms will help determine the preferential interaction of alpha subunits with beta dimers and the functional significance of these unique heterotrimer combinations.

Muscarinic and somatostatin receptors are thought to couple specifically to the alphabeta(3)(4) and alphabeta(1)(3) heterotrimers, respectively, in their regulation of Ca channels in GH(3) cells(17, 18, 19) . These results suggest exquisite specificity in the recognition of G protein heterotrimers by receptors. It remains unclear whether specific G protein subunit combinations occur randomly or if there are mechanisms that dictate the composition of heterotrimers. Such mechanisms would have great significance for the regulation of cellular function by G protein-coupled receptors. More important, in addition to the previously reported heterogeneity in alpha/ subunit associations(15, 16) , our results indicate that these subunits do exist in preferred combinations. This is not only true for the association of isoforms with splice variants of a single alpha subunit, such as alpha and alpha, but also for alpha subunits that may differ only by their post-translational modifications, which may be the case for alpha and alpha. The nature of the putative modifications that distinguish these proteins is not yet known; however, many potential modifications, such as palmitoylation, carboxymethylation, and phosphorylation, are reversible and regulatable. Thus, it may be possible that cellular regulation of the modification of G protein subunits could ultimately affect the composition of G protein heterotrimers and direct their subsequent receptor and effector interactions.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants DK37219 (to J. D. H.) and GM39867 (to J. D. R.) and by the Medical University of South Carolina University Research Committee. 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.

§
To whom correspondence should be addressed: Dept. of Pharmacology, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29464. Tel.: 803-792-3209; Fax: 803-792-2475.

(^1)
There are at least four additional subunit isoforms based upon cDNA cloning (J. Robishaw, unpublished observations).

(^2)
The abbreviations used are: sulfo-MBS, m-maleimidobenzoylN-hydroxysulfosuccinimide ester; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; FPLC, fast protein liquid chromatography; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GTPS, guanosine 5`-O-(thiotriphosphate).

(^3)
All mammals are thought to express at least two alpha(O) proteins derived from alternatively spliced mRNAs from a single gene(37, 47, 48) . These mRNAs are often referred to as alpha and alpha. The two alpha(O) proteins are identical in their first 240 residues and 80% homologous in their COOH-terminal 114 residues. Complete cDNA sequences have been reported for both transcripts in many species, but a bovine sequence has been reported only for alpha(39, 49) . Sequencing of peptides from bovine alpha indicates that it is homologous to the alpha transcript found in other species(38) . Nevertheless, antibodies specific for the most unique sequence in rat alpha do not recognize bovine alpha(50, 51) , suggesting species variation in this site. Thus, although it is clear that bovine alpha and alpha are splice variants compatible with the alpha and alpha variants in other species, their exact relationship has not yet been determined.


ACKNOWLEDGEMENTS

We thank Bronwyn Tatum for expert assistance in the preparation of the G proteins used in this study.


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