INTERSUBUNIT SURFACES IN G-PROTEIN ALPHA-BETA-GAMMA HETEROTRIMERS - ANALYSIS OF CROSS-LINKING AND MUTAGENESIS OF BETA-GAMMA

Heterotrimeric guanine nucleotide binding proteins (G proteins) are ma de up of alpha, beta, and gamma subunits, the last two forming a very tight complex, Stimulation of cell surface receptors promotes dissocia tion of alpha from the beta gamma dimer, which, in turn, allows both c omponents to interact with intracellular enzymes or ion channels and m odulate their activity, At present, little is known about the conforma tion of the beta gamma dimer or about the areas of beta gamma that int eract with alpha, Direct information on the orientation of protein sur faces can be obtained from analysis of chemically cross-linked product s, Previous work in this laboratory showed that 1,6-bismaleimidohexane , which reacts with cysteine residues, specifically cross-links alpha to beta and beta to gamma (Yi, F., Denker, B. M., and Neer, E. J. (199 1) J. Biol. Chem. 266, 3900-3906), To identify the residues in beta an d gamma involved in cross-linking to each other or to alpha, we have m utated the cysteines in beta(1), gamma(2), and gamma(3) and analyzed t he mutated proteins by in vitro translation in a rabbit reticulocyte l ysate, All the mutants were able to form beta gamma dimers that could interact with the alpha subunit, We found that 1,6-bismaleimidohexane can cross-link beta(1) to gamma(3) but not to gamma(2). The cross-link goes from Cys(25) in beta(1) to Cys(30) in gamma(3). This cysteine is absent from any of the other known gamma isoforms and therefore confe rs a distinctive property to gamma(3). The beta subunit in the beta(1) gamma(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) gamma(3). The beta subunit can also be cross-link ed to alpha, giving rise to two products on SDS-polyacrylamide gel ele ctrophoresis, both of which were previously shown to be formed by cros s-linking beta to Cys(215) in alpha(o) (Thomas, T. C., Schmidt, C. J., and Neer, E. J. (1993) Proc, Natl, Acad, Sci, U. S. A. 90, 10295-1029 9). Mutation of Cys(204) in beta(1) abolished one of these two product s, whereas mutation of Cys(271) abolished the other, Because both alph a-beta cross-linked products are formed in approximately equal amounts , Cys(204) and Cys(271) in beta are equally accessible from Cys(215) i n alpha(o). Our findings begin to define intersubunit surfaces, and th ey pose structural constraints upon any model of the beta gamma dimer.