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

Abstract

Heterotrimeric G proteins, composed of α and βΓ subunits, forward signals from transmembrane receptors to intracellular effector enzymes and ion channels. Free βΓ activates downstream targets, but its action is terminated by association with GDP-liganded α subunits. Because α can inhibit activation of many effectors by βΓ, it is likely that the α subunit binding surfaces on βΓ overlap the surfaces necessary for effector activation. To test this hypothesis, we mutated residues on β shown to contact α in the recently published crystal structures of the αβΓ 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 α subunit binds to the flat, top surface of the toroidal β subunit and also extends a helix along the side of the β subunit at blade 1. We mutated four residues on the top of surface of β (Hβ1[L117A], Hβ1[D228R], Hβ1[D246S], and Hβ1 [W332A]) and two residues on the side of β that contacts α (Hβ1[N88A/K89A]). Each of the mutant proteins was able to form βΓ dimers but they differed in their ability to bind α and to activate phospholipase C β2 (PLCβ2), PLCβ3, and adenylyl cyclase II. Mutation of residues along the side of the torus at blade 1 diminish affinity for α but do not prevent activation of any of the effectors. Mutations on the α binding surface differentially affected PLCβ2, PLCβ3, and adenylyl cyclase II. Residues that affect PLCBβ and adenylyl cyclase II activity are found on opposite sides of the central tunnel, suggesting that PLC and adenylyl cyclase, like the α subunit, make many contacts on the top surface. None of the mutations affected the ability of βΓ to inhibit adenlyl cyclase I. We conclude that α, PLCβ, PLCβ3, and adenylyl cyclase II share an interaction on the top surface of β. The importance of individual residues is different for α binding and for effector activation and differs even between closely related isoforms of the same effector.