Functional Analysis of a Dominant Negative Mutant of Gαi2

Abstract

The key event in receptor-catalyzed activation of heterotrimeric G proteins is binding of GTP, which leads to subunit dissociation generating GTP-bound alpha subunits and free beta complexes. We have previously identified a mutation that abolished GTP binding in Galpha(o) (S47C) and demonstrated that the mutant retained the ability to bind beta 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 Galpha (S48C) upon signaling pathways reconstituted in transiently transfected COS-7 cells. We found that expression of the Galpha S48C mutant prevented stimulation of phospholipase C (PLC) beta2 by free beta subunit complexes. This effect of Galpha(i) S48C was not readily reversible in contrast to the inhibitory effect of wild-type Galpha, which could be reversed upon activation of the cotransfected muscarinic M2 receptor, presumably by release of beta from the G protein heterotrimer. Coexpression of Galpha(i) S48C or the wild-type Galpha also dramatically decreased G-mediated stimulation of PLC by C5a in the cells transfected with cDNAs encoding C5a receptor and Galpha. Activation of PLC via endogenous G(q) or G in the presence of alpha1C adrenergic receptors was similarly attenuated by coexpression of Galpha(i) or Galpha(i) S48C. Pertussis toxin treatment of the transfected cells enhanced the inhibition of the receptor-stimulated PLC by wild-type Galpha(i) subunits but did not influence the effects of the dominant negative mutant. The enhancement of the wild-type Galpha(i) inhibitory effect by pertussis toxin can be explained by stabilization of Galpha(i) binding to beta as a result of ADP-ribosylation, while Galpha(i) S48C mutant binds beta irreversibly even without pertussis toxin treatment. Therefore, a feasible mechanism to rationalize the attenuation of the Galpha and G-mediated activation of PLC by cotransfected Galpha(i) is the competition between Galpha(i) and Galpha or G for the beta complexes, which are necessary for the G protein coupling with receptors. These experiments provide new evidence for the role of beta in the integration of signals controlling phosphoinositide release through different Galpha families.