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

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.