Functional analysis of a dominant negative mutant of Gαi2

Vladlen Z. Slepak, Arieh Katz, Melvin I. Simon

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45 Scopus citations

Abstract

The key event in receptor-catalyzed activation of heterotrimeric G proteins is binding of GTP, which leads to subunit dissociation generating GTP-bound α subunits and free βγ complexes. We have previously identified a mutation that abolished GTP binding in Gαo (S47C) and demonstrated that the mutant retained the ability to bind βγ 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 Gαi2 (S48C) upon signaling pathways reconstituted in transiently transfected COS-7 cells. We found that expression of the Gαi2 S48C mutant prevented stimulation of phospholipase C (PLC) β2 by free βγ subunit complexes. This effect of Gαi S48C was not readily reversible in contrast to the inhibitory effect of wild-type Gαi2, which could be reversed upon activation of the cotransfected muscarinic M2 receptor, presumably by release of βγ from the G protein heterotrimer. Coexpression of Gαi S48C or the wild-type Gαi2 also dramatically decreased G16-mediated stimulation of PLC by C5a in the cells transfected with cDNAs encoding C5a receptor and Gα16. Activation of PLC via endogenous Gq or G11 in the presence of α1C adrenergic receptors was similarly attenuated by coexpression of Gαi or Gαi S48C. Pertussis toxin treatment of the transfected cells enhanced the inhibition of the receptor-stimulated PLC by wild-type Gαi subunits but did not influence the effects of the dominant negative mutant. The enhancement of the wild-type Gαi inhibitory effect by pertussis toxin can be explained by stabilization of Gαi binding to βγ as a result of ADP-ribosylation, while Gαi S48C mutant binds βγ irreversibly even without pertussis toxin treatment. Therefore, a feasible mechanism to rationalize the attenuation of the Gα16 and Gq/11-mediated activation of PLC by cotransfected Gαi is the competition between Gαi and Gα16 or Gq/11 for the βγ complexes, which are necessary for the G protein coupling with receptors. These experiments provide new evidence for the role of βγ in the integration of signals controlling phosphoinositide release through different Gα families.

Original languageEnglish (US)
Pages (from-to)4037-4041
Number of pages5
JournalJournal of Biological Chemistry
Volume270
Issue number8
DOIs
StatePublished - Feb 24 1995

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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