Loss of association between activated Gαq and Gβγ disrupts receptor-dependent and receptor-independent signaling

The G protein subunit, beta gamma, plays an important role in targeting a subunits to the plasma membrane and is essential for binding and activation of the heterotrimer by heptahelical receptors. Mutation of residues in the N-terminal alpha-helix of as and that contact beta gamma in the crystal structure of alpha(i) reduces binding between alpha and beta gamma, inhibits plasma membrane targeting and palmitoylation of the alpha subunit, and results in G proteins that fail to couple receptor activation to stimulation of effector. Overexpression of beta gamma can recover this loss of signaling through G(s) but not G(q). In fact, a single mutation (I25A) in alpha(q) can block alpha(q)-mediated generation of inositol phosphates. Function is not recovered by beta gamma overexpression nor myristoylation directed plasma membrane localization. Introduction of a Q209L activating mutation with I25A results in a constitutively active aq as expected, but surprisingly a R183C activating mutation does not result in constitutive activity when present with 125A. Examination of binding between alpha and beta gamma via a pull down assay shows that the N-terminal beta gamma-binding mutations inhibit alpha-beta gamma binding significantly more than the R183C or Q209L activating mutations do. Moreover, introduction of the I25A mutation into alpha qRC disrupts co-immunoprecipitation with PLC beta 1. Taken together, results presented here suggest that alpha-beta gamma binding is necessary at a point downstream from receptor activation of the heterotrimeric G protein for signal transduction by a, (c) 2004 Elsevier Inc. All rights reserved.