Functional analysis of a dominant negative mutant of G alpha i2

The key event in receptor-catalyzed activation of heterotrimer G proteins is binding of GTP, which leads to subunit dissociation generating GTP-bound alpha subunits and free beta gamma complexes. We have previously identified a mutation that abolished GTP binding in G alpha o (S47C) and demonstrated that the mutant retained the ability to bind beta gamma and could act in a dominant negative fashion when expressed in Xenopus oocytes (Slepak, V.Z., Quick, M.W., Aragay, A.M., Davidson, N., Lester, H.A., and Simon, M.I. (1993) J. Biol. Chem. 268, 21889-21894). In the current work, we investigated the effects of the homologous mutant of G alpha i2 (S48C) upon signaling pathways reconstituted in transiently transfected COS-7 cells. We found that expression of the G alpha i2 S48C mutant prevented stimulation of phospholipase C (PLC) beta 2 by free beta gamma subunit complexes. This effect of G alpha i S48C was not readily reversible in contrast to the inhibitory effect of wild-type G alpha i2, which could be reversed upon activation of the cotransfected muscarinic M2 receptor, presumably by release of beta gamma from the G protein heterotrimer. Coexpression of G alpha i S48C or the wild-type G alpha i2 also dramatically decreased G16-mediated stimulation of PLC by C5a in the cells transfected with cDNAs encoding C5a receptor and G alpha 16. Activation of PLC via endogenous Gq or G11 in the presence of alpha 1C adrenergic receptors was similarly attenuated by coexpression of G alpha i or G alpha i S48C. Pertussis toxin treatment of the transfected cells enhanced the inhibition of the receptor-stimulated PLC by wild-type G alpha i subunits but did not influence the effects of the dominant negative mutant. The enhancement of the wild-type G alpha i inhibitory effect by pertussis toxin can be explained by stabilization of G alpha i binding to beta gamma as a result of ADP-ribosylation, while G alpha i S48C mutant binds beta gamma irreversibly even without pertussis toxin treatment. Therefore, a feasible mechanism to rationalize the attenuation of the G alpha 16 and Gq/11-mediated activation of PLC by cotransfected G alpha i is the competition between G alpha i and G alpha 16 or Gq/11 for the beta gamma complexes, which are necessary for the G protein coupling with receptors. These experiments provide new evidence for the role of beta gamma in the integration of signals controlling phosphoinositide release through different G alpha families.