Partial Rescue of Functional Interactions of a Nonpalmitoylated Mutant of the G-Protein Gαs by Fusion to the β-Adrenergic Receptor

• Published in: Biochemistry (Easton) 2003-03, Vol.42 (9), p.2607-2615
• Main Authors: Ugur, Ozlem, Onaran, H. Ongun, Jones, Teresa L. Z
• Format: Article
• Language: English
• Subjects: Adenylyl Cyclases - metabolism; Alanine - genetics; Animals; Cell Membrane - drug effects; Cell Membrane - genetics; Cell Membrane - metabolism; Cysteine - genetics; Gene Expression Regulation - genetics; GTP-Binding Protein alpha Subunits, Gs - chemistry; GTP-Binding Protein alpha Subunits, Gs - deficiency; GTP-Binding Protein alpha Subunits, Gs - genetics; GTP-Binding Protein alpha Subunits, Gs - metabolism; Humans; Hydroxylamine - pharmacology; Intracellular Fluid - metabolism; Mice; Mutagenesis, Site-Directed; Palmitic Acid - metabolism; Protein Binding - drug effects; Protein Binding - genetics; Protein Subunits - chemistry; Protein Subunits - deficiency; Protein Subunits - genetics; Protein Subunits - metabolism; Protein Transport - drug effects; Protein Transport - genetics; Rats; Receptors, Adrenergic, beta-2 - biosynthesis; Receptors, Adrenergic, beta-2 - chemistry; Receptors, Adrenergic, beta-2 - genetics; Receptors, Adrenergic, beta-2 - metabolism; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Transfection; Tumor Cells, Cultured
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi026470i

Most heterotrimeric G-protein α subunits are posttranslationally modified by palmitoylation, a reversible process that is dynamically regulated. We analyzed the effects of Gαs palmitoylation for its intracellular distribution and ability to couple to the β-adrenergic receptor (βAR) and stimulate adenylyl cyclase. Subcellular fractionation and immunofluorescence microscopy of stably transfected cyc- cells, which lack endogenous Gαs, showed that wild-type Gαs was predominantly localized at the plasma membrane, but the mutant C3A-Gαs, which does not incorporate [3H]palmitate, was mostly associated with intracellular membranes. In agreement with this mislocalization, C3A-Gαs showed neither isoproterenol- or GTPγS-stimulated adenylyl cyclase activation nor GTPγS-sensitive high-affinity agonist binding, all of which were present in the wild-type Gαs expressing cells. Fusion of C3A-Gαs with the βAR [βAR-(C3A)Gαs] partially rescued its ability to induce high-affinity agonist binding and to stimulate adenylyl cyclase activity after isoproterenol or GTPγS treatment. In comparison to results with the WT-Gαs and βAR (βAR-Gαs) fusion protein, the βAR-(C3A)Gαs fusion protein was about half as efficient at coupling to the receptor and effector. Chemical depalmitoylation by hydroxylamine of membranes expressing βAR-Gαs reduced the high-affinity agonist binding and adenylyl cyclase activation to a similar degree as that observed in βAR-(C3A)Gαs expressing membranes. Altogether, these findings indicate that palmitoylation ensured proper localization of Gαs and facilitated bimolecular interactions of Gαs with the βAR and adenylyl cyclase.