title: The effects of agonist stimulation and beta(2)-adrenergic receptor level on cellular distribution of gs(alpha) protein.
abstract: This study examines the effects of adrenergic ligands, cholera toxin, forskolin, and varying levels of beta(2) adrenergic receptors (beta(2)AR) on the cellular distribution of Gs(alpha) subunits in CHO cells. Localization of Gs(alpha) was evaluated by confocal microscopy and beta(2)AR-mediated signalling was assessed by adenylyl cyclase (AC) activity. In cells expressing 0.2 pmol/mg protein beta(2)ARs (WT18), the localization of Gs(alpha) subunit was restricted to the plasma membrane region. Isoproterenol (ISO), cholera toxin or forskolin elicited redistribution of cellular Gs(alpha) so that Gs(alpha) appeared as intense spots throughout the plasma membrane as well as the cytoplasm. Exposure to a neutral beta(2)AR antagonist, alprenolol, prevented the ISO-stimulated Gs(alpha) translocation from peripheral to inner cytoplasm. In cells expressing high level of beta(2)ARs (8.2 pmol/mg) (WT4), basal and ISO-stimulated AC activities were significantly elevated when compared to the values detected in WT18 clone, suggesting a positive correlation between receptor expression and receptor-mediated signalling. Basal Gs(alpha) distribution in this group was similar to that observed in ISO-, cholera toxin-, or forskolin-stimulated WT18 clone. ISO, cholera toxin, or forskolin did not change the distribution of Gs(alpha) significantly when tested in WT4 clone. No difference in the cellular level of Gs(alpha) protein between WT18 and WT4 clones was detected. Alprenolol did not affect the distribution of Gs(alpha) in WT4 clone. ICI 118,551, a negative beta(2)AR antagonist, altered Gs(alpha) distribution from a dispersed basal pattern to a membrane-confined pattern. The latter appearance was similar to that observed in unstimulated WT18 clone. Taken together, these data suggest that: (1) enhanced beta(2)AR-Gs(alpha) coupling induced by agonist stimulation or by increased expression of beta(2)ARs remodel the cellular distribution of Gs(alpha); (2) the alteration in Gs(alpha) distribution induced by beta(2)AR overexpression provides evidence for agonist-independent interaction of beta(2)AR and Gs(alpha), that can be inhibited by a negative antagonist but not by a neutral antagonist; and (3) forskolin influences the activity state of Gs(alpha) that displays a Gs(alpha) distribution pattern comparable to that observed when Gs(alpha) is activated via beta(2)AR stimulation or directly by cholera toxin.
authors.affiliation: Ankara University Faculty of Medicine, Department of Histology-Embryology, Sihhiye 06100, Ankara, Turkey.
authors.forename: A
authors.initials: A
authors.lastname: Can
authors.forename: K
authors.initials: K
authors.lastname: Sayar
authors.forename: E
authors.initials: E
authors.lastname: Friedman
authors.forename: C
authors.initials: C
authors.lastname: Ambrosio
authors.forename: E
authors.initials: E
authors.lastname: Erdemli
authors.forename: H
authors.initials: H
authors.lastname: Gurdal
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D058666:Adrenergic beta-2 Receptor Agonists; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000319:Adrenergic beta-Antagonists / Q000494:pharmacology; D000526:Alprenolol / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells / Q000737:chemistry / Q000201:enzymology; D002772:Cholera Toxin / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D018613:Microscopy, Confocal; D018343:Receptors, Adrenergic, beta-2 / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection
chemical_list: D058666:Adrenergic beta-2 Receptor Agonists; D000318:Adrenergic beta-Agonists; D000319:Adrenergic beta-Antagonists; D018343:Receptors, Adrenergic, beta-2; D005576:Colforsin; D000526:Alprenolol; D002772:Cholera Toxin; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases; D007545:Isoproterenol