Heterotrimeric guanine nucleotide binding proteins (G proteins) are made up of , , and subunits, the last two forming a very tight complex. Stimulation of cell surface receptors promotes dissociation of from the 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 dimer or about the areas of that interact with . 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 to and to (Yi, F., Denker, B. M., and Neer, E. J.(1991) J. Biol. Chem. 266, 3900-3906). To identify the residues in and involved in cross-linking to each other or to , we have mutated the cysteines in , , and and analyzed the mutated proteins by in vitro translation in a rabbit reticulocyte lysate. All the mutants were able to form dimers that could interact with the subunit. We found that 1,6-bismaleimidohexane can cross-link to but not to . The cross-link goes from Cys in to Cys in . This cysteine is absent from any of the other known isoforms and therefore confers a distinctive property to . The subunit in the dimer can be cross-linked to an unidentified protein in the rabbit reticulocyte lysate, generating a product slightly larger than cross-linked . The subunit can also be cross-linked to , giving rise to two products on SDS-polyacrylamide gel electrophoresis, both of which were previously shown to be formed by cross-linking to Cys in (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 abolished one of these two products, whereas mutation of Cys abolished the other. Because both - cross-linked products are formed in approximately equal amounts, Cys and Cys in are equally accessible from Cys in . Our findings begin to define intersubunit surfaces, and they pose structural constraints upon any model of the dimer.

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