Rescue of functional interactions between the α(2A)-adrenoreceptor and acylation-resistant forms of G(i1)α by expressing the proteins from chimeric open reading frames

Abstract

Co-expression of the α(2S)-adrenoreceptor with a pertussis toxin- resistant (C351G), but not with an also palmitoylation-resistant (C3S/C351G), form of the α subunit of G(i1) resulted in agonist-induced, pertussis toxin- independent, GTP hydrolysis. Construction and expression of a chimeric fusion protein between the receptor and C351G G(i1)α generated a membrane protein in which the G protein element was activated by receptor agonist. An equivalent fusion protein containing C3S/C351G G(i1)α rescued the ability of receptor agonist to activate this mutant. Fusion proteins of a palmitoylation-resistant (C442A) α(2A)-adrenoreceptor and either C351G or C3S/C351G G(i1)α also responded effectively to agonist. Myristoylation resistant (G2A/C351G) and combined acylation-resistant (G2A/C3S/C351G) mutants of G(i1)α are cytosolic proteins. Expression of these as chimeric α(2A)-adrenoreceptor-G protein fusions restored membrane localization and activation of the G protein by receptor agonist. These studies demonstrate the general utility of generating chimeric fusion proteins to examine receptor regulation of G protein function and that the lack of functional activation of acylation-negative G proteins by a co-expressed receptor is related to deficiencies in cellular targeting and location rather than an inherent incapacity to produce appropriate protein-protein interactions and signal transmission.