title: Extra-long Gαs variant XLαs protein escapes activation-induced subcellular redistribution and is able to provide sustained signaling.
abstract: Murine models indicate that Gαs and its extra-long variant XLαs, both of which are derived from GNAS, markedly differ regarding their cellular actions, but these differences are unknown. Here we investigated activation-induced trafficking of Gαs and XLαs, using immunofluorescence microscopy, cell fractionation, and total internal reflection fluorescence microscopy. In transfected cells, XLαs remained localized to the plasma membrane, whereas Gαs redistributed to the cytosol after activation by GTPase-inhibiting mutations, cholera toxin treatment, or G protein-coupled receptor agonists (isoproterenol or parathyroid hormone (PTH)(1-34)). Cholera toxin treatment or agonist (isoproterenol or pituitary adenylate cyclase activating peptide-27) stimulation of PC12 cells expressing Gαs and XLαs endogenously led to an increased abundance of Gαs, but not XLαs, in the soluble fraction. Mutational analyses revealed two conserved cysteines and the highly charged domain as being critically involved in the plasma membrane anchoring of XLαs. The cAMP response induced by M-PTH(1-14), a parathyroid hormone analog, terminated quickly in HEK293 cells stably expressing the type 1 PTH/PTH-related peptide receptor, whereas the response remained maximal for at least 6 min in cells that co-expressed the PTH receptor and XLαs. Although isoproterenol-induced cAMP response was not prolonged by XLαs expression, a GTPase-deficient XLαs mutant found in certain tumors and patients with fibrous dysplasia of bone and McCune-Albright syndrome generated more basal cAMP accumulation in HEK293 cells and caused more severe impairment of osteoblastic differentiation of MC3T3-E1 cells than the cognate Gαs mutant (gsp oncogene). Thus, activated XLαs and Gαs traffic differently, and this may form the basis for the differences in their cellular actions.
authors: Endocrine Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114.
authors: Zun
authors: Z
authors: Liu
authors: Endocrine Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114; Pediatric Endocrinology, Marmara University School of Medicine Hospital, 34662 Istanbul, Turkey.
authors: Serap
authors: S
authors: Turan
authors: Laboratory for G Protein-coupled Receptor Biology, Department of Pharmacology and Chemical Biology, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15213.
authors: Vanessa L
authors: VL
authors: Wehbi
authors: Endocrine Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114; Laboratory for G Protein-coupled Receptor Biology, Department of Pharmacology and Chemical Biology, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15213.
authors: Jean-Pierre
authors: JP
authors: Vilardaga
authors: Endocrine Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114. Electronic address: bastepe@helix.mgh.harvard.edu.
authors: Murat
authors: M
authors: Bastepe
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002454:Cell Differentiation; D002462:Cell Membrane / Q000378:metabolism; D002772:Cholera Toxin / Q000737:chemistry; D002864:Chromogranins; D000242:Cyclic AMP / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D010006:Osteoblasts / Q000166:cytology; D016716:PC12 Cells; D051381:Rats; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D002864:Chromogranins; D044168:Receptor, Parathyroid Hormone, Type 1; D002772:Cholera Toxin; D000242:Cyclic AMP; C498642:Gnas protein, mouse; D019205:GTP-Binding Protein alpha Subunits, Gs