©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Intersubunit Surfaces in G Protein Heterotrimers
ANALYSIS BY CROSS-LINKING AND MUTAGENESIS OF beta (*)

(Received for publication, August 24, 1995; and in revised form, October 23, 1995)

Irene Garcia-Higuera (§) Thomas C. Thomas (¶) Fei Yi Eva J. Neer

From the Department of Medicine, Cardiovascular Division, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Heterotrimeric guanine nucleotide binding proteins (G proteins) are made up of alpha, beta, and subunits, the last two forming a very tight complex. Stimulation of cell surface receptors promotes dissociation of alpha from the beta dimer, which, in turn, allows both components to interact with intracellular enzymes or ion channels and modulate their activity. At present, little is known about the conformation of the beta dimer or about the areas of beta that interact with alpha. Direct information on the orientation of protein surfaces can be obtained from analysis of chemically cross-linked products. Previous work in this laboratory showed that 1,6-bismaleimidohexane, which reacts with cysteine residues, specifically cross-links alpha to beta and beta to (Yi, F., Denker, B. M., and Neer, E. J.(1991) J. Biol. Chem. 266, 3900-3906). To identify the residues in beta and involved in cross-linking to each other or to alpha, we have mutated the cysteines in beta(1), (2), and (3) and analyzed the mutated proteins by in vitro translation in a rabbit reticulocyte lysate. All the mutants were able to form beta dimers that could interact with the alpha subunit. We found that 1,6-bismaleimidohexane can cross-link beta(1) to (3) but not to (2). The cross-link goes from Cys in beta(1) to Cys in (3). This cysteine is absent from any of the other known isoforms and therefore confers a distinctive property to (3). The beta subunit in the beta(1)(2) dimer can be cross-linked to an unidentified protein in the rabbit reticulocyte lysate, generating a product slightly larger than cross-linked beta(1)(3). The beta subunit can also be cross-linked to alpha, giving rise to two products on SDS-polyacrylamide gel electrophoresis, both of which were previously shown to be formed by cross-linking beta to Cys in alpha(o) (Thomas, T. C., Schmidt, C. J., and Neer, E. J.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10295-10299). Mutation of Cys in beta(1) abolished one of these two products, whereas mutation of Cys abolished the other. Because both alpha-beta cross-linked products are formed in approximately equal amounts, Cys and Cys in beta are equally accessible from Cys in alpha(o). Our findings begin to define intersubunit surfaces, and they pose structural constraints upon any model of the beta dimer.


INTRODUCTION

Heterotrimeric guanine nucleotide binding proteins (G proteins) (^1)are key components of the transmembrane signaling machinery. They link cell surface receptors to intracellular enzymes or ion channels whose modulated activity ultimately leads to the cellular response. G proteins are made up of three different polypeptides, alpha, beta, and : the alpha subunit binds and hydrolyzes GTP; the beta and subunits form a very tight complex and can therefore be considered as a functional monomer. In the inactive state, GDP is bound to the heterotrimer. However, upon receptor stimulation, GDP is replaced by GTP, and the alpha subunit becomes activated and dissociates from the beta dimer. The free subunits, both alpha and beta, can then modulate the activity of target effectors (reviewed by Neer(1995)).

The structural basis for the alpha subunit function is now better understood because of the recent solution of the crystal structure of GTP- and GDP-liganded forms of two alpha subunits: transducin and alpha (Noel et al., 1993; Lambright et al., 1994; Coleman et al., 1994). In contrast, little is known about the structure of beta. The beta subunit is predicted to contain an amino-terminal amphipatic alpha helix that may be involved in coiled-coil interactions with (Lupas et al., 1992). The remaining sequence is made up of seven repeating units of approximately 43 amino acids each (Fong et al., 1986). These repeating units, also found in many other proteins, consist of a conserved core, usually starting with the sequence Gly-His (GH) and ending with Trp-Asp (WD) and are therefore referred to as WD repeats. Each core is predicted to form a beta strand-turn-beta strand-turn-beta strand structure, followed by a loop of variable length leading to the next core (Neer et al., 1994). The subunit is predicted to be largely alpha helical (Lupas et al., 1992). In solution it behaves as an asymmetrical, extended molecule (Mende et al., 1995). A cysteine residue near its carboxyl terminus is prenylated, and this lipid modification has been shown to be essential for membrane attachment of the beta dimer (reviewed by Casey(1994) and Wedegaertner et al.(1995)). There is, however, little information about the spatial organization of the beta repeats and about the overall configuration of the dimer. The parts of beta that interact with alpha or with effectors have not been identified, although genetic analysis in yeast has suggested some important residues (Leberer et al., 1992; Whiteway et al., 1994). However, because the surface of the beta dimer facing the alpha subunit will presumably be exposed after dissociation of the subunits, it is likely that this area would be important for interaction with effectors. Therefore, to understand how signaling by beta is regulated, we need to define the regions of the subunits that face each other and eventually to locate the contact points.

Analysis of mutated and chimeric molecules can help identify regions important for protein-protein interactions. However, such an approach cannot discriminate regions that are near each other from regions that maintain the conformation necessary for the interaction. More direct information on the orientation of protein surfaces can be obtained from analysis of chemically cross-linked products. This laboratory has previously shown that purified G protein subunits can be cross-linked using 1,6-bismaleimidohexane (BMH), a homobifunctional cross-linker reacting with sulfhydryl groups of cysteine residues. BMH specifically cross-links alpha to beta and beta to . Hydrodynamic analysis demonstrates that the cross-linked product containing alpha and beta is composed of one alpha and one beta subunit and likewise, the cross-linked beta product is composed of only one beta and one subunit (Yi et al., 1991; Thomas et al., 1993b). However, as is common for cross-linked proteins, these products migrate anomalously in SDS-PAGE. (^2)

We have previously established that BMH cross-links alpha(o) to beta through cysteine 215 in alpha(o) (Thomas et al., 1993a). This cysteine residue is found in the alpha(2) helix, a region shown to have a different conformation in alpha-GDP compared with alpha-GTP (Lambright et al., 1994). We now report the identification of the sites in beta and involved in the formation of the alphabeta and beta cross-links. These studies begin to map the regions of beta near the alphabeta contact surface, and they set physical limits to the distance between residues in alpha, beta, and .


MATERIALS AND METHODS

Site-directed Mutagenesis

Cysteine residues in beta(1) and (2) were mutated to alanines using the Altered Sites in vitro Mutagenesis System (Promega). This system uses a dual primer method to introduce a site-directed mutation in the cloned sequence and, simultaneously, antibiotic resistance in the vector, thus allowing a straightforward procedure for selection of mutants. For this purpose, rat beta(1) and bovine (2) cDNAs (generous gifts of Dr. R. R. Reed and Dr. M. I. Simon) were subcloned into the pAlter vector, and single stranded DNA was obtained following the manufacturer's instructions and used as template to anneal the mutagenic oligonucleotide and the ampicillin repair oligonucleotide. The presence of mutations was confirmed by sequencing (U. S. Biochemical Corp. Sequenase 2.0 kit). The mutants that did not behave like the wild type on cross-linking assays (beta(1) C25A, beta(1) C204A, and beta(1) C271A) were completely sequenced to rule out the presence of additional mutations.

Cysteine residues in position 30 and 50 in (3) (cDNA kindly provided by Dr. M. I. Simon) were individually mutated by polymerase chain reaction using primers that included conveniently located restriction sites at the 3` end of the mutated residue (StyI and BamHI, respectively). The detailed sequences of the oligonucleotides used as primers are TGCTGCCTTGGACACCTTTATCCGGGCCAA for mutation of Cys and GAGGGGATCCTCAGCGGCG for mutation of Cys. The inserts were completely sequenced to ensure that no additional mutations were introduced during the polymerase chain reaction reaction.

In Vitro Translation

All subunits were transcribed and translated using the TNT coupled reticulocyte lysate system (Promega). Typically,1 µg of plasmid DNA and 20 µCi of [S]methionine were used in a 50-µl reaction. In all cases transcription was directed by the T7 promoter. (3) cDNA was subcloned into a vector (PAGA-1 vector, kindly provided by Dr. Orly Reiner) containing a poly(A) sequence and the alfalfa mosaic virus leader sequence, which has been previously shown to improve translation efficiency (Jobling and Gehrke, 1987). Synthesis of the desired product was routinely verified by running 5 µl of the translation mixture in a small 11 or 13% polyacrylamide gel (Laemmli, 1970) followed by autoradiography with overnight exposure.

Dimer Formation and Trypsin Digestion

Independently translated beta and subunits were mixed together and incubated at 37 °C for 90 min to allow dimer formation. Because translation was usually more efficient, 10-15 µl of translation mixture were typically added to 50 µl of beta translate. Following incubation, a trypsin digestion assay was performed to verify dimerization. As described previously, 7 µl of beta mixture were treated with 1 µl of 5 µML-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Cooper Biomed) and incubated at 30 °C for 10 min, after which 2 µl of 100 mM benzamidine were added to stop the reaction. The samples were then boiled for 5 min and subjected to SDS-PAGE on 11% polyacrylamide gels. After electrophoresis, gels were stained with Coomassie Blue, destained, soaked in ENHANCE (DuPont NEN) for 1 h, dried, and exposed to film at -70 °C for 1-3 days.

Cross-linking

50 µl of the beta mixture remaining after analysis by tryptic cleavage were passed over an 8-ml AcA 34 column (Sepracor) equilibrated with HMSE (50 mM NaHEPES, pH 7.5, 6 mM MgCl(2), 75 mM sucrose, and 1 mM EDTA) plus 0.1% Lubrol PX at 4 °C, in order to remove DTT and to separate the beta dimers from undimerized beta. 200-µl fractions were collected, and those containing both subunits were pooled and concentrated 5-10-fold using a Centricon-30 concentrator (Amicon). For cross-linking, 30 µl of this sample were mixed with 10 µl of alpha(o) (2-5 µg) purified from bovine brain (Neer et al., 1984) in HMSE or 10 µl of HMSE buffer alone, and the reaction was initiated by the addition of 1.6 µl of freshly prepared 50 mM BMH (Pierce Chemical Co) in Me(2)SO. In control uncross-linked samples, 20 mM DTT was added prior to BMH. After 40 min at 4 °C, DTT (20 mM) and/or Laemmli sample buffer containing 15% beta-mercaptoethanol were added, and the samples were boiled, resolved by SDS-PAGE on 9% polyacrylamide gels, and treated as described previously. The radioactive bands could be visualized after 2-7 days of exposure at -70 °C.

Cross-linking of purified subunits was performed as described previously (Yi et al., 1991); 5-10 µg of bovine brain purified beta or alpha(o)beta (Neer et al., 1984) in HMSE plus 0.4-0.6% Lubrol PX were used for each reaction.


RESULTS

Cross-linking of in Vitro Translated G Protein Subunits

Fig. 1A shows that G protein subunits purified from brain or translated in vitro give a similar pattern of bands cross-linked by BMH (Fig. 1A, indicated by arrows). We previously showed with purified heterotrimers that the two larger molecular mass products (Fig. 1A, marked alphabeta, solid arrows) contain both alpha and beta subunits (2) (Yi et al., 1991). The same two products are obtained with alpha(o) and in vitro translated beta and subunits (see Fig. 1A). Purified beta dimers can also be cross-linked by BMH (Thomas et al., 1993b), giving rise to a product with an apparent molecular mass of 48 kDa (Fig. 1, marked beta, solid arrow). The formation of this band is diminished in the presence of an excess of purified alpha(o) (see the description of cross-linking of alpha(o) to beta(1)(3) dimers below). The identity of the 50kDa band obtained only with in vitro translated subunits (Fig. 1A, open arrow) will be discussed in detail later. None of the three major cross-linked products obtained with in vitro translated subunits are formed when the experiment is carried out with beta alone or alone (Fig. 1B, set A, beta(1) only and set B, (2) only), with or without alpha. Other minor products formed by BMH can be detected after autoradiography. However, it was not our goal to characterize all cross-linked products. We have focused on the three major ones. Note that the bands in set B ((2) only), which was deliberately overexposed, are not cross-linker-dependent. Moreover, we have previously shown that combinations of beta and subtypes that cannot form functional dimers, for example beta(2) and (1), do not generate any of those bands, showing that cross-linking strictly depends on the formation of beta dimers (Spring and Neer, 1994). As expected, the two high molecular mass products (solid arrows, set C in Fig. 1B) are seen whether [S]alpha translated in vitro and purified beta dimers (Thomas et al., 1993a) or [S]beta translated in vitro and purified alpha(o) (as shown in these studies) are used for cross-linking, confirming that both products contain alpha and beta. The faint bands in the middle lane in set C (beta(1)(2) + BMH without alpha(o)) probably result from cross-linking beta to the small amount of alpha subunit present in the reticulocyte lysate (Denker et al., 1992).


Figure 1: Cross-linking of purified and in vitro translated G protein subunits. A, heterotrimers containing alphabeta or beta dimers purified from bovine brain were cross-linked with BMH essentially as described under ``Materials and Methods.'' Similarly, in vitro translated and dimerized beta(1)(2) subunits were mixed with purified alpha(o) (3 µg) and treated with BMH. Cross-linked samples were then resolved on 9% polyacrylamide gels and stained with Coomassie Blue. Lanes containing in vitro translated samples were further processed for autoradiography as detailed under ``Materials and Methods.'' The positions of uncross-linked alpha and beta subunits are indicated by arrowheads. Cross-linked products are shown by arrows. B, in vitro translated beta(1) and (2) were treated separately (sets A and B) or mixed and allowed to dimerize (set C). The cross-linking reaction was carried out either in the absence (- alpha(o)) or presence (+ alpha(o)) of purified alpha(o), and samples were resolved on 9% SDS-PAGE and subjected to autoradiography. The arrowhead shows the position of the radioactive band corresponding to the beta subunit; cross-linked products are indicated by arrows. In the lanes marked - BMH, 20 mM DTT was added prior to BMH. For cross-linker-specific bands compare - BMH and + BMH lanes. Set B was exposed three times longer than sets A and C to make sure that no specific bands were formed. The subunit could not be detected in this gel (9% polyacrylamide), but its presence was confirmed on tricine gels that resolve low molecular mass proteins (Schägger and von Jagow, 1987).



Cross-linking alpha(o)to beta(1)(2)Dimers

The goal of these studies was to identify the cysteine residues in beta and that are involved in cross-linking these subunits to alpha or to each other. The strategy was to mutate the cysteine residues in both subunits and test which mutations interfered with the cross-linking. In order for this strategy to be successful, the mutated beta and subunits must be able to dimerize with each other and to interact with alpha. Otherwise, failure to cross-link could not be attributed to loss of a cysteine involved in cross-linking and would be uninterpretable. We started by individually mutating each one of the 14 cysteine residues in the beta(1) sequence, and we used a well established tryptic protection assay to assess beta dimerization. This assay is based on the observation that when beta is associated with , only one of its 32 potential tryptic cleavage sites is initially accessible, thus generating two fragments: an amino-terminal 14-kDa fragment, which is unstable and therefore difficult to detect on SDS-PAGE, and a stable carboxyl-terminal 24-kDa fragment, which is taken as an indicator of the formation of a properly folded beta dimer (Schmidt and Neer, 1991). As shown in Fig. 2, all of the beta cysteine mutants behaved like the wild type upon trypsin digestion, indicating that they can all dimerize with the (2) subunit and thus can be used for cross-linking.


Figure 2: Trypsin digestion of beta(1)(2) dimers containing beta(1) cysteine mutants. In vitro translated wild type or mutant beta(1) subunits were incubated without (first two lanes) or with (2) and digested with trypsin to verify dimerization. The fragments were resolved on 11% polyacrylamide gels and visualized by autoradiography. The first lane of each pair (- trypsin) corresponds to control undigested sample. The protected carboxyl-terminal fragment is indicated by an arrow. The position of the mutated cysteine in the beta(1) sequence is shown on top of each pair of lanes. The same results were obtained in three independent experiments. The two radioactively labeled bands showing up right below the beta subunit band in control lanes correspond to incomplete beta proteins generated during the in vitro translation as a result of either a premature termination or an internal start from methionine residues located downstream of the initial AUG. These truncated proteins, however, cannot dimerize with the subunit and therefore do not interfere with our experimental procedures.



When dimers containing different beta(1) cysteine mutants and wild type (2) were cross-linked to alpha(o) and the resulting products resolved on SDS-PAGE, we observed that most of the beta(1) mutants were still able to generate the two cross-linked alphabeta bands (Fig. 3, solid arrows). However, two of the mutations affected the cross-linking to alpha(o); mutation of Cys blocked the formation of the upper band, whereas mutation of Cys abolished the lower band. The fact that each of these mutant beta dimers can still form one of the two cross-linked alphabeta products shows that each is still able to interact with alpha. It was previously established in this laboratory that both cross-linked products are generated through a single cysteine residue in the alpha(o) subunit (Cys215), because mutation of that amino acid prevented the formation of both bands (Thomas et al., 1993a). Therefore, each one of the two bands is due to cross-linking Cys in alpha(o) to a different cysteine in beta(1): Cys for the upper band and Cys for the lower one. The possibility existed that might be present in one of the two products, presumably the one with the larger apparent molecular mass. However, because each cross-linked product is affected by mutation of only one cysteine residue in alpha(o) (Cys) and one cysteine residue in beta(1) (Cys or Cys), we conclude that neither product contains an additional cross-linked component, which would have required the involvement of yet another cysteine residue. The distinct mobilities on SDS-PAGE of the two cross-linked products of the same composition are probably due to differences in SDS binding or in the shape of the denatured molecules resulting from the two alternate cross-linking sites in beta.


Figure 3: Cross-linking of beta(1)(2) dimers containing beta cysteine mutants to alpha(o). In vitro translated wild type or mutated beta(1) subunits were dimerized with in vitro translated (2) and treated with BMH in the presence of purified alpha(o) as described. Reactions were stopped by the addition of DTT and/or Laemmli sample buffer, and samples were subjected to SDS-PAGE on 9% polyacrylamide gels followed by autoradiography. Control, uncross-linked samples (- BMH) were incubated with BMH in the presence of excess DTT. The two cross-linked alphabeta products are shown by arrows, and the numbers on top of each pair of lanes indicate which beta(1) cysteine mutant was used. The arrowheads point to the mutants that did not behave like the wild type. Shown here is a representative example of three independent experiments.



Cross-linking beta(1)(2)Dimers

We next analyzed how mutation of the cysteine residues in the beta(1) subunit affected the third cross-linker-specific band obtained with in vitro translated subunits (see Fig. 1, A and B, open arrows). The formation of this cross-linked product was strictly dependent on the presence of dimerized beta subunits, and its mobility on SDS-PAGE was very close to the mobility of the well characterized cross-linked beta band obtained with beta subunits purified from brain (Thomas et al., 1993b). We therefore believed that it corresponded to cross-linked beta (Spring and Neer, 1994). Fig. 4summarizes the results obtained when we treated with BMH beta(1)(2) dimers containing either wild type or mutant beta subunits. All mutants were tested, but for simplicity only some of them are shown; the rest behaved like the wild type. Mutation of Cys, which affected one of the two cross-linked alphabeta bands (see Fig. 3), also inhibits the formation of the 50-kDa cross-linked product. This result was puzzling for us, because previous experimental evidence suggested that was being cross-linked to the 14-kDa amino-terminal tryptic fragment of beta (Thomas et al., 1993b), whereas Cys would be in the carboxyl-terminal 24-kDa fragment. Therefore, to characterize this cross-linked product further and to verify its composition, we individually mutated both cysteines in (2), Cys and Cys, to alanine and serine, respectively. In addition, a double mutant was generated. The mutations did not affect the ability of the subunit to dimerize with beta(1) as deduced from the tryptic proteolysis pattern (Fig. 5A), but to our surprise, neither did they affect the cross-linking (Fig. 5B); even the dimers containing the double mutant lacking both cysteines behaved like the wild type and were able to form the 50-kDa cross-linked product. It was however possible that BMH could be reacting with amino groups with an abnormal pK(a) in (2). If this were the case it should still be possible to label the cross-linked product using [S] and nonradioactive beta. All the appropriate controls were done to make sure that the cross-linked product was being formed, but we still were not able to detect a radioactively labeled cross-linked band under these conditions. Immunoprecipitation experiments using anti- antibodies were also negative. Overall, these negative results indicated that was in fact not present in the 50-kDa cross-linked product (marked by an open arrow in Fig. 1, A and B). Nevertheless, its formation was strictly correlated with the ability of the beta subunit to associate with , and it was a reliable indicator of beta dimerization (Spring and Neer, 1994). We concluded that the beta subunit was being cross-linked through Cys to another small protein (which we will refer to as X) present in the rabbit reticulocyte lysate used for in vitro translation. Preliminary experiments suggest that this unidentified protein might be one of the subunits of hemoglobin (16 kDa), which is by far the most abundant protein in the reticulocyte lysate. Whether this cross-linking to beta reflects any real, functional interaction between beta(1) and hemoglobin or is just a nonspecific reaction with a very concentrated protein remains to be clarified.


Figure 4: Cross-linking of beta(1)(2) dimers containing beta(1) cysteine mutants. Dimers of beta(1)(2) containing wild type or mutated beta(1) subunits were incubated with BMH, and both treated (+ BMH) and untreated (- BMH, 20 mM DTT added before BMH) samples were subjected to SDS-PAGE on 9% polyacrylamide gels followed by autoradiography as described previously. The number of the cysteine residue mutated to alanine in beta(1) is indicated on top of each lane. The closed arrowhead indicates the position of beta(1). The 50-kDa cross-linked product is shown by an arrow. For simplicity only some of the mutants are shown; the rest were indistinguishable from the wild type. The open arrowhead points to the mutant that failed to generate that cross-linked product. This experiment was repeated three times with similar results.




Figure 5: Characterization of (2) cysteine mutants. A, in vitro translated wild type beta(1) subunits were incubated with either wild type (2) or a double mutant (2) with no cysteines in its sequence. Dimerization was subsequently checked by trypsin digestion as described previously. The fragments were resolved on 11% polyacrylamide gels and visualized by autoradiography. The arrow points to the protected carboxyl-terminal beta(1) fragment indicative of dimer formation. Shown here is an example of four independent experiments. B, the same dimers containing wild type or mutated (2) were cross-linked with BMH. Treated (+ BMH) and untreated (- BMH, 20 mM DTT added before BMH) samples were resolved on 9% polyacrylamide gels and processed for autoradiography. The cross-linked product is indicated by an arrow. This experiment was repeated four times with similar results.



Cross-linking beta(1)(3)Dimers

There was a clear discrepancy between the results obtained with in vitro translated subunits, where beta(1) and (2) could not be cross-linked to each other, and the results obtained with beta dimers purified from brain, where beta could be cross-linked to through its 14-kDa amino-terminal region (Thomas et al., 1993b). A major difference between the two samples is the composition of the beta dimers; whereas the in vitro translated samples contain dimers of defined composition (in this case beta(1)(2)), the purified dimers are made up of different beta and isoforms present in brain, including beta(1) and beta(2) (Fong et al., 1987) and (2), (3), (5), and (7) (Cali et al., 1992; Wilcox et al., 1994). The two beta subtypes show a high degree of similarity throughout their sequences. The subunits are much more heterogeneous; in fact (3) contains two additional cysteines (not present in (1), (2), (5), or (7)) flanking the equivalent of Cys in (2) (see diagram in Fig. 6). It was therefore possible that because of those extra cysteines, (3) but not (2) could be cross-linked to beta(1) by BMH. Indeed, when we treated in vitro translated and dimerized beta(1)(3) with BMH, we obtained a cross-linked product with exactly the same apparent molecular mass as the purified cross-linked dimer (Fig. 7, solid arrow). Moreover, the cross-linked product could be labeled with either [S]beta(1) and nonradioactive (3) or nonradioactive beta(1) and [S](3) (Fig. 8, C and D, lower part of gel), indicating that it contains both beta and subunits. Note that the intensity of the beta(1)(3) cross-linked band (closed arrow in Fig. 7) is greater than the beta(1)X band obtained with beta(1)(2) (open arrow in Fig. 7), which is also present in the lanes corresponding to beta(1)(3) (see Fig. 7and Fig. 8, faint band right above beta). As expected, it does not appear when only is radioactively labeled (Fig. 8D)


Figure 6: Comparison of the position of cysteine residues in (1), (2), and (3) sequences. The complete aligned amino acid sequences of bovine (1), (2), and (3) (accession numbers: K03255, M37183, and M58349) are shown. The position of the cysteine residues in each subtype is indicated by arrowheads.




Figure 7: Comparison of cross-linked products obtained with beta dimers purified from brain and beta(1)(2) or beta(1)(3) dimers translated in vitro. Purified beta subunits or in vitro translated dimers of defined composition (beta(1)(2) or beta(1)(3)) were treated in parallel with or without cross-linker, and the resulting products were resolved on 9% polyacrylamide gels and stained with Coomassie Blue. Lanes containing in vitro translated subunits were further processed for autoradiography as described. The arrowhead points to the radioactive band corresponding to beta(1). The position of the cross-linked products is indicated by closed and open arrows. The same experiment was repeated three times with similar results.




Figure 8: Characterization of cross-linked products obtained with beta(1)(3) dimers. beta(1) and (3) subunits were separately translated in vitro in the presence (*) or the absence of [S]methionine, and dimers were formed in which either both subunits (B) or just one of them (C and D) were radioactively labeled. The dimers were then mixed with purified alpha(o) and incubated with cross-linker (+ BMH) or with 20 mM DTT added before the cross-linker (- BMH). The cross-linked products were resolved on 9% polyacrylamide gels and subjected to autoradiography. For comparison a beta(1)(2) sample treated similarly is included (A). Cross-linked products are indicated by arrows. With (3) (but not (2)), we observed an additional cross-linked product at about 60 kDa. It contains both beta and and is not dependent on the presence of alpha. It may represent beta cross-linked to another protein in the reticulocyte lysate or multimers of beta. It was not characterized further. This is a representative example of three independent experiments. Shown in the inset are the bands obtained after cross-linking with BMH purified alpha(o) to purified beta subunits labeled with I-Bolton-Hunter (Thomas et al., 1993b). The samples were run on a 9% polyacrylamide gel, which was subsequently stained with Coomassie Blue, dried, and exposed to autoradiographic film for 2 days. Only the fragment of the gel including those cross-linked products is shown.



In order to determine which cysteine residue in beta(1) is really cross-linked to (3), we tested the beta(1) cysteine mutants again but this time using (3) instead of (2) (Fig. 9, lower part; the effect of mutations on the alphabeta cross-link will be discussed below). The ability of all beta(1) mutants to dimerize with (3) was confirmed with a trypsin digestion assay (data not shown). Fig. 9shows the cross-linking results obtained with the most significant beta(1) mutants, the rest being indistinguishable from the wild type. The C271A mutant, which failed to generate the 50-kDa cross-linked product in experiments with (2) (see Fig. 4), could however be cross-linked to (3). Conversely, mutation of Cys in beta(1), which showed no phenotype when tested with (2) (data not shown), completely abolished the beta(1)(3) cross-linking. Therefore, (3) is cross-linked to beta(1) through Cys, which lies within the 14-kDa amino-terminal beta tryptic fragment as shown in our previous studies with purified protein (Thomas et al., 1993b).


Figure 9: Cross-linking of beta(1)(3) dimers containing beta(1) cysteine mutants. In vitro translated wild type or mutant beta(1) subunits were dimerized with (2) and cross-linked in the presence of alpha(o). Both treated (+ BMH) and untreated (- BMH, 20 mM DTT added before BMH) samples were subjected to SDS-PAGE on 9% polyacrylamide gels followed by autoradiography. Uncross-linked beta(1) is indicated by an arrowhead. Cross-linked products are shown by arrows. The position of the mutated cysteine in the beta(1) sequence is specified on top of each pair of lanes. Shown here is a representative example of three independent experiments.



We next sought to establish which one of the two additional cysteine residues in (3) (Cys or Cys; see diagram in Fig. 6) conferred the ability to be cross-linked to beta by BMH. Following the same strategy, we individually mutated both cysteines to alanines and used tryptic protection assays to confirm that the mutants could still dimerize with beta(1) (Fig. 10A). Subsequently, we cross-linked beta(1)(3) dimers containing either wild type or mutant (3) subunits and showed that mutation of Cys but not Cys prevents the formation of the cross-linked beta band (Fig. 10B, lower part). This cysteine residue in (3) (Cys) is not present in any of the other subtypes and therefore confers a distinctive property to (3).


Figure 10: Characterization of (3) cysteine mutants. A, in vitro translated wild type beta(1) subunits were incubated with either wild type or mutated (3) subunits. Dimerization was subsequently checked on a trypsin digestion assay as described previously. The fragments were resolved on 11% polyacrylamide gels and visualized by autoradiography. The arrow points to the protected carboxyl-terminal beta(1) fragment indicative of dimer formation. B, the same dimers containing wild type or mutated (3) were mixed with alpha(o) and used in a cross-linking assay. Treated (+ BMH) and untreated (- BMH, 20 mM DTT added before BMH) samples were resolved on 9% polyacrylamide gels and processed for autoradiography. Cross-linked products are indicated by arrows. The stronger intensity of the radioactive bands corresponding to the two alphabeta cross-linked products in the lanes containing the Cys mutant was only observed in this particular experiment and does not reflect an increased affinity of the (3) mutant for the beta subunit. The position of the mutated cysteine in the (3) sequence is shown on top of each pair of lanes. The arrowhead signals the mutant that failed to generate a beta cross-link. The same result was obtained in three separate experiments.



Cross-linking alpha(o)to beta(1)(3)Dimers

When the beta(1)(3) dimers were cross-linked to alpha(o), two new cross-linker-specific products became evident (see Fig. 8upper half, sets B and C). They migrate just above the two previously described bands corresponding to cross-linked alphabeta (see diagram in Fig. 11). Moreover, they could be labeled using either [S]beta or [S] (Fig. 8, upper half, sets C and D), showing that besides alpha these two new products contain both beta and subunits. We believe they result from cross-linking beta to both alpha and , which would explain why they could not be detected in experiments carried out with (2) (see Fig. 1and Fig. 3) that cannot be cross-linked to beta. The same products should however be found after cross-linking purified alphabeta subunits. Coomassie staining was not sensitive enough to detect them and gave only faint signals. Whereas all beta dimers can be cross-linked to alpha, only a fraction of them, those containing (3), can produce a beta cross-link and generate the upper alphabeta cross-linked bands. Therefore, in cross-linking experiments with purified subunits, cross-linked alphabeta is less abundant than cross-linked alphabeta. Iodination of purified beta dimers with I-Bolton-Hunter reagent, which reacts strongly with the subunit (Thomas et al., 1993b), allows us to detect all the expected alphabeta and alphabeta products (see Fig. 8, inset). Note that in experiments with in vitro translated subunits, the stronger intensity of the bands corresponding to the two cross-linked alphabeta products as compared with the two alphabeta products (see Fig. 8, set B) reflects the presence of additional radioactively labeled methionines (from the (3) subunit) in those products. When only the beta subunit is radioactive (Fig. 8, set C), the four cross-linked products are approximately equally labeled.


Figure 11: Schematic representation of the major BMH-specific bands obtained with purified or in vitro translated alphabeta subunits after SDS-PAGE. Shown on the left side is the composition of each one of the cross-linked products. The cysteine residues in beta(1) involved in each cross-link are indicated on the right side.



Which are the cysteine residues involved in the formation of those two new cross-linked products? Our hypothesis was that the two new products were the result of attaching to the two previously described forms of cross-linked alphabeta. If this were the case, the same beta cysteine mutations that prevented the formation of the two lower bands (Cys and Cys) should also affect the two additional upper bands. That is exactly what we observed when we tested the beta mutants dimerized with (3) in a cross-linking assay in the presence of alpha(o) (see Fig. 9, upper part). The C204A mutant failed to produce the upper of the two alphabeta bands (as we had previously established) as well as the upper of the two alphabeta bands (for schematic representation of the four products, see Fig. 11). Conversely, C271A mutant failed to produce the lower of the two alphabeta bands and the lower of the two alphabeta bands. As expected, mutation of Cys blocked the formation of the three bands containing leaving the other three (the two cross-linked alphabeta bands and betaX) unaffected. Finally, the results with the (3) cysteine mutants (see Fig. 10B, upper part) confirmed that the same cysteine involved in the beta cross-linking (Cys) is responsible for the formation of the two cross-linked alphabeta bands.

Fig. 11summarizes the results obtained during the characterization of the six major cross-linked products obtained with in vitro translated G protein subunits; starting from below the first band corresponds to beta cross-linked to (3) through Cys, (3) being in turn covalently bound to beta through Cys. Right above is beta cross-linked to a rabbit reticulocyte lysate protein (X) through Cys. The higher molecular mass bands, starting from the bottom, correspond to beta cross-linked to alpha through Cys (lower band) and through Cys (upper band) followed by beta cross-linked to both alpha and again through Cys or Cys (to alpha) and through Cys (to ).


DISCUSSION

beta Cross-linking

We have individually mutated the cysteine residues in beta(1), (2), and (3) to alanine (or to serine in one case) and shown that none of those residues are needed for beta dimerization or for interaction of beta with the alpha subunit. Cys in beta(1) can be cross-linked to Cys in (3) with BMH. Therefore, it is likely that those two cysteines are facing each other. Cys in beta(1) can also form a cupric phenantroline-catalyzed disulfide bond with either Cys or Cys in (1), although it is not known which one of those two cysteines reacts with beta(1) (Bubis and Khorana, 1990). Because a disulfide bond is only about 2 Å long, Cys in beta(1) must be very close to either Cys or Cys in (1). If the conformations of (1) and (3) are similar, then the amino acids in (3) that are the equivalent of Cys and Cys in (1) (Ala and Ala in (3)) should also be close to beta(1). Because we have shown that Cys in (3) can be cross-linked to beta(1), we propose that Ala and Ala lie on the same surface of (3) as Cys and that all face the beta subunit. Defining that surface will help identify what part of (3) is exposed and is therefore likely to be involved in interaction with other proteins such as the alpha subunit or effectors.

The residues in beta(1) and (3) that are cross-linked to each other (Cys in beta(1) and Cys in (3)) lie within regions believed to be important for dimerization. The minimum sequence of necessary to form a native beta dimer is not known. However, removal of 15 amino acids from the amino terminus diminishes but does not entirely block dimerization, whereas removal of 13 amino acids from the carboxyl terminus has little effect (Mende et al., 1995). Therefore, residues important to contact beta probably lie between amino acids 15 and 59. The sequence that defines the selectivity of beta interactions is also located in this central region (Spring and Neer, 1994; Lee et al., 1995). Mutations in the putative amino-terminal alpha-helix in beta(1), where Cys is located, can inhibit beta dimerization (Garritsen et al., 1993). However, selectivity for different subunits is determined by multiple sites within the WD repeat region of the beta subunit (Pronin and Gautam, 1992; Garritsen and Simonds, 1994; Katz and Simon, 1995).

(1) and (2) cannot be cross-linked to beta(1) by BMH. Neither they nor any of the other known subtypes including the newly cloned isoforms (Ong et al., 1995; Ryba and Tirindelli, 1995; Ray et al., 1995) contain a cysteine at a position equivalent to Cys of (3). Therefore, in any preparation, formation of a cross-linked beta product after treatment with BMH suggests the presence of beta(3) dimers. This distinctive property could in turn be used to probe for interaction of beta dimers containing (3) with other proteins.

alphabeta Cross-linking

We have identified two cysteine residues in beta(1) (Cys and Cys) that can be cross-linked to alpha(o) with BMH. Our previous studies show that both cross-linked products involve the same cysteine in alpha(o) (Cys) (Thomas et al., 1993a). The distance from Cys in alpha(o) to either Cys or Cys in beta(1) cannot be longer than 16 Å, the length of the cross-linking reagent, but the flexibility of BMH would allow it to be slightly smaller. The amount of the two alphabeta cross-linked products is usually about equal, suggesting that the probability of the cross-linker reaching each of the two residues in beta is similar. Alternatively, the two cross-linking sites in beta could reflect two different conformations of the heterotrimer, although there is as yet no experimental evidence for differences in beta conformation.

The two cross-linked cysteines are located in the carboxyl-terminal portion of the beta subunit, in agreement with our previous observation with purified subunits that after tryptic digestion of the cross-linked products the alpha subunit remains associated with the 24-kDa carboxyl-terminal beta fragment (Yi et al., 1991). Cys is found in the fourth WD repeat, whereas Cys lies within repeat six. Both are likely to be found in turns. The spatial organization of the seven WD repeats in beta is not known. It is clear however that beta forms a very tightly associated globular structure (Thomas et al. 1993b). Any model of the beta dimer must be compatible with Cys, located in the alpha(2) helix of the alpha subunit, reaching Cys in the fourth repeat and Cys in the sixth repeat through an arm of approximately 16 Å (see Fig. 12). The two cysteines are probably not providing bonds to hold alpha and beta together because they can be mutated without disrupting the alphabeta association. Our findings, however, begin to define an area in beta that faces the alpha subunit, and they pose structural constraints to any model of the configuration of the beta subunit.


Figure 12: Schematic representation of the beta structure: location of the cysteine residues involved in cross-linking to alpha and subunits. The predicted structure of the beta subunit is schematically depicted. Each WD repeat is indicated by a circle. The positions of Cys and Cys are indicated. The helical and GTPase domains of the alpha subunit are indicated by two ovals. Cys is the residue in the GTPase domain of alpha which is cross-linked to beta (Thomas et al., 1993a). The putative alpha-helical amino-terminal segment in beta is shown as a bar. The subunit is shown as a long bar. The site of the beta(1) Cys to (3) Cys cross-link is indicated by a line. The carboxyl-terminal prenyl group is indicated by the zig-zag line.




FOOTNOTES

*
This work was supported by National Institute of Health Grant GM 36259 (to E. J. N.). 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.

§
Recipient of a fellowship from ``Consejo Superior de Investigaciones Cientificas'' (Spain). To whom correspondence should be addressed: Cardiovascular Division, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115. Tel.: 617-732-5874; Fax: 617-732-5132.

Supported by a Samuel A. Levine Fellowship from the American Heart Association, Massachusetts Affiliate.

(^1)
The abbreviations used are: G proteins, guanine nucleotide-binding proteins; BMH, 1,6-bismaleimidohexane; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis.

(^2)
In our previous reports, the two cross-linked alphabeta products were assigned apparent molecular masses of 140 and 122 kDa, respectively. However, we have observed that the mobility of these products on SDS-PAGE, which is anomalously low (122 kDa is more than the sum of the molecular masses of alpha and beta), is affected, in addition, by the percentage of polyacrylamide used to resolve them on SDS-PAGE. Whereas 11% polyacrylamide gels were used in our previous studies, we have now changed to 9% polyacrylamide gels to improve the resolution of the high molecular mass bands. The products now migrate with apparent molecular masses of about 100 and 112 kDa. The mobility of these products relative to markers also varies depending on how long the electrophoresis continues. We frequently run our gels until the 36-kDa beta subunit band is at the bottom of the gel, and we have noted subtle differences in the relative mobilities of the bands; these differences, however, do not cause any ambiguity in identifying them.


ACKNOWLEDGEMENTS

We thank Drs. R. R. Reed and M. I. Simon for providing cDNA clones and Dr. O. Reiner for providing a vector. We are also grateful to all the other members of the Neer laboratory for helpful discussions throughout the course of this work. We thank Paula McColgan for excellent secretarial assistance.


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