Partial rescue of functional interactions of a nonpalmitoylated mutant of the G-protein G alpha s by fusion to the beta-adrenergic receptor.
Most heterotrimeric G-protein alpha subunits are posttranslationally modified by palmitoylation, a reversible process that is dynamically regulated. We analyzed the effects of Galpha(s) palmitoylation for its intracellular distribution and ability to couple to the beta-adrenergic receptor (betaAR) and stimulate adenylyl cyclase. Subcellular fractionation and immunofluorescence microscopy of stably transfected cyc(-) cells, which lack endogenous Galpha(s), showed that wild-type Galpha(s) was predominantly localized at the plasma membrane, but the mutant C3A-Galpha(s), which does not incorporate [(3)H]palmitate, was mostly associated with intracellular membranes. In agreement with this mislocalization, C3A-Galpha(s) showed neither isoproterenol- or GTPgammaS-stimulated adenylyl cyclase activation nor GTPgammaS-sensitive high-affinity agonist binding, all of which were present in the wild-type Galpha(s) expressing cells. Fusion of C3A-Galpha(s) with the betaAR [betaAR-(C3A)Galpha(s)] partially rescued its ability to induce high-affinity agonist binding and to stimulate adenylyl cyclase activity after isoproterenol or GTPgammaS treatment. In comparison to results with the WT-Galpha(s) and betaAR (betaAR-Galpha(s)) fusion protein, the betaAR-(C3A)Galpha(s) fusion protein was about half as efficient at coupling to the receptor and effector. Chemical depalmitoylation by hydroxylamine of membranes expressing betaAR-Galpha(s) reduced the high-affinity agonist binding and adenylyl cyclase activation to a similar degree as that observed in betaAR-(C3A)Galpha(s) expressing membranes. Altogether, these findings indicate that palmitoylation ensured proper localization of Galpha(s) and facilitated bimolecular interactions of Galpha(s) with the betaAR and adenylyl cyclase.
Ugur
Ozlem
O
Department of Pharmacology and Clinical Pharmacology and Molecular Biology and Technology Research and Development Unit, Ankara University Faculty of Medicine, Sihhiye, 06100 Ankara, Turkey. mugur@dialup.ankara.edu.tr
Onaran
H Ongun
HO
Jones
Teresa L Z
TL
D000262:Adenylyl Cyclases / Q000378:metabolism; D000409:Alanine / Q000235:genetics; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D019811:Hydroxylamine / Q000494:pharmacology; D007424:Intracellular Fluid / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D019308:Palmitic Acid / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics; D051381:Rats; D018343:Receptors, Adrenergic, beta-2 / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D021122:Protein Subunits; D018343:Receptors, Adrenergic, beta-2; D011993:Recombinant Fusion Proteins; D019811:Hydroxylamine; D019308:Palmitic Acid; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases; D003545:Cysteine; D000409:Alanine