Rescue of functional interactions between the alpha2A-adrenoreceptor and acylation-resistant forms of Gi1alpha by expressing the proteins from chimeric open reading frames.
Co-expression of the alpha2A-adrenoreceptor with a pertussis toxin-resistant (C351G), but not with an also palmitoylation-resistant (C3S/C351G), form of the alpha subunit of Gi1 resulted in agonist-induced, pertussis toxin-independent, GTP hydrolysis. Construction and expression of a chimeric fusion protein between the receptor and C351G Gi1alpha generated a membrane protein in which the G protein element was activated by receptor agonist. An equivalent fusion protein containing C3S/C351G Gi1alpha rescued the ability of receptor agonist to activate this mutant. Fusion proteins of a palmitoylation-resistant (C442A) alpha2A-adrenoreceptor and either C351G or C3S/C351G Gi1alpha also responded effectively to agonist. Myristoylation resistant (G2A/C351G) and combined acylation-resistant (G2A/C3S/C351G) mutants of Gi1alpha are cytosolic proteins. Expression of these as chimeric alpha2A-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.
Wise
A
A
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
Milligan
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G
D000215:Acylation; D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000818:Animals; D000068438:Brimonidine Tartrate; D019556:COS Cells; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016366:Open Reading Frames; D011810:Quinoxalines / Q000494:pharmacology; D018341:Receptors, Adrenergic, alpha-2 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D013552:Swine
D000316:Adrenergic alpha-Agonists; D011810:Quinoxalines; D018341:Receptors, Adrenergic, alpha-2; D011993:Recombinant Fusion Proteins; D000068438:Brimonidine Tartrate; D019204:GTP-Binding Proteins