Sites for Gα binding on the G protein β subunit overlap with sites for regulation of phospholipase Cβ and adenylyl cyclase

Heterotrimeric G proteins, composed of alpha and beta gamma subunits, forward signals from transmembrane receptors to intracellular effector enzymes and ion channels. Free beta gamma activates downstream targets, but its action is terminated by association with GDP-liganded alpha subunits. Because alpha can inhibit activation of many effectors by beta gamma, it is likely that the alpha subunit binding surfaces on beta gamma overlap the surfaces necessary for effector activation. To test this hypothesis, we mutated residues on beta shown to contact a in the recently published crystal structures of the alpha beta gamma heterotrimer (Wall, M. A., Coleman, D. E., Lee, E., Iniguez-Lluhi, J. A. Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell 83, 1047-1058; Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319.). The alpha subunit binds to the flat, top surface of the toroidal beta subunit and also extends a helix along the side of the beta subunit at blade I. We mutated four residues on the top surface of beta (H beta(1)[L117A], H beta(1)[D228R], H beta(1)[D246S], and H beta(1)[W332A]) and two residues on the side of beta that contacts alpha (H beta(1)[N88A/K89A]). Each of the mutant proteins was able to form py dimers, but they differed in their ability to bind a and to activate phospholipase C beta(2) (PLC beta(2)), PLC beta(3), and adenylyl cyclase II. Mutation of residues along the side of the torus at blade I diminish affinity for alpha but do not prevent activation of any of the effectors. Mutations on the a binding surface differentially affected PLC beta(2), PLC beta(3), and adenylyl cyclase II. Residues that affect PLC beta and adenylyl cyclase II activity are found on opposite sides of the central tunnel, suggesting that PLC and adenylyl cyclase, like the a subunit, make many contacts on the top surface. None of the mutations affected the ability of beta gamma to inhibit adenylyl cyclase I. We conclude that alpha, PLC beta(2), PLC beta(3), and adenylyl cyclase II share an interaction on the top surface of beta. The importance of individual residues is different for a binding and for effector activation and differs even between closely related isoforms of the same effector.