title: A cysteine-3 to serine mutation of the G-protein Gi1 alpha abrogates functional activation by the alpha 2A-adrenoceptor but not interactions with the beta gamma complex.
abstract: Pertussis toxin-resistant (C351G) and also palmitoylation-negative (C3S/C351G), myristoylation-negative (G2A/C351G) and combined acylation-negative (G2A/C3S/C351G) forms of the G-protein Gi1 alpha were expressed in COS-7 cells along with the porcine alpha 2A-adrenoceptor. G2A/C3S/C351G Gi1 alpha and G2A/C351G Gi1 alpha were largely cytosolic and failed to interact with the agonist-occupied alpha 2A-adrenoceptor in membrane preparations. In contrast, C351G Gi1 alpha was almost entirely particulate and the alpha 2-adrenoceptor agonist UK14304 caused a marked stimulation of its GTPase activity and binding of [35S]GTP gamma S which was not prevented by pertussis toxin treatment of the cells. C3S/C351G Gi1 alpha was present in both the particulate and cytosolic fractions but the GTPase activity of the membrane bound fraction was only slightly activated by the alpha 2A-adrenoceptor. Coexpression of C3S/C351G Gi1 alpha and the alpha 2A-adrenoceptor along with beta 1 and gamma 2 subunits increased the P2 membrane complement of the alpha subunit and increased substantially the ratio of membrane bound to cytosolic protein. However, this also failed to allow marked stimulation of high-affinity GTPase activity by the alpha 2A-adrenoceptor despite the increased proportion of G-protein in the P2 membrane fraction. Despite the low fractional activation of C3S/C351G Gi1 alpha by the alpha 2A-adrenoceptor compared to C351G Gi1 alpha, the palmitoylation-resistant G-protein caused a marked reduction in pertussis toxin-resistant, agonist (UK14304)-mediated stimulation of adenylyl cyclase activity. UK14304 caused the same degree of effect on adenylyl cyclase activity in pertussis toxin-treated cells following transfection of the same amounts of C351G Gi1 alpha and C3S/C351G Gi1 alpha, as both appear to act to sequester beta gamma subunits. By contrast, neither G2A/C351G Gi1 alpha nor G2A/C3S/C351G Gi1 alpha resulted in effective regulation of adenylyl cyclase activity.
authors.affiliation: Molecular Pharmacology Group, Institute of Biomedical and Life Sciences, University of Glasgow, Scotland, U.K.
authors.forename: A
authors.initials: A
authors.lastname: Wise
authors.forename: M A
authors.initials: MA
authors.lastname: Grassie
authors.forename: M
authors.initials: M
authors.lastname: Parenti
authors.forename: M
authors.initials: M
authors.lastname: Lee
authors.forename: S
authors.initials: S
authors.lastname: Rees
authors.forename: G
authors.initials: G
authors.lastname: Milligan
mesh_terms: D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000235:genetics*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D019308:Palmitic Acid / Q000378:metabolism; D037342:Pertussis Toxin; D018341:Receptors, Adrenergic, alpha-2 / Q000187:drug effects / Q000502:physiology*; D012694:Serine / Q000235:genetics*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D037361:Adenylate Cyclase Toxin; D018341:Receptors, Adrenergic, alpha-2; D010566:Virulence Factors, Bordetella; D019308:Palmitic Acid; D012694:Serine; D037342:Pertussis Toxin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D000262:Adenylyl Cyclases; D003545:Cysteine