title: Coupling of the alpha 2-adrenergic receptor to the inhibitory G-protein Gi and adenylate cyclase in HT29 cells.
abstract: Previous studies have established that the human colon carcinoma cell line HT29 expresses an alpha 2-adrenergic receptor of the alpha 2A subtype, which is negatively coupled to adenylate cyclase. The purpose of the present study was to examine the mechanisms of alpha 2-adrenergic signal transduction in these cells. [32P]ADP-ribosylation with pertussis toxin and immunoblots using antibodies specific for the Gi alpha-subunits indicated that two distinct Gi-proteins (Gi2 and Gi3) were present in HT29-cell membranes. Treatment of intact cells with pertussis toxin resulted in a time-dependent decrease in the amount of [32P]ADP-ribosylatable Gi2 and Gi3, which coincided with a diminution in the number of alpha 2-adrenergic receptors in high-affinity state for agonists and with a progressive loss of ability of UK14304 to inhibit forskolin-stimulated accumulation of cyclic AMP. When membranes were [32P]ADP-ribosylated with cholera toxin in the absence of exogenous added guanine nucleotides, radioactivity was incorporated into a 45 kDa polypeptide representing Gs, as well as into 40-41 kDa polypeptides corresponding to Gi3 and Gi2. The amount of radioactivity incorporated into the two GiS under basal conditions was decreased by addition of the alpha 2-antagonist RX821002. It was not significantly affected by addition of clonidine (partial alpha 2-agonist), but was doubled by the addition of UK14304 (full alpha 2-agonist). This effect was blocked by RX821002. Study of adenylate cyclase activity indicated that preincubation of HT29 membranes with the antibody AS/7 (anti-alpha i1/alpha i2), but not with the antibody EC/2 (anti-alpha i3), attenuated the inhibitory effect of UK14304 on forskolin-stimulated adenylate cyclase. These data demonstrate that the alpha 2A-adrenergic receptor is coupled to both Gi2 and Gi3, and identify Gi2 as the major mediator of inhibition of adenylate cyclase in HT29 cells.
authors.affiliation: INSERM U317, Institut Louis Bugnard, CHU Rangueil, Toulouse, France.
authors.forename: A
authors.initials: A
authors.lastname: Remaury
authors.forename: D
authors.initials: D
authors.lastname: Larrouy
authors.forename: D
authors.initials: D
authors.lastname: Daviaud
authors.forename: B
authors.initials: B
authors.lastname: Rouot
authors.forename: H
authors.initials: H
authors.lastname: Paris
mesh_terms: D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000316:Adrenergic alpha-Agonists / Q000378:metabolism / Q000494:pharmacology; D000317:Adrenergic alpha-Antagonists / Q000378:metabolism / Q000494:pharmacology; D000068438:Brimonidine Tartrate; D004146:Dioxanes / Q000378:metabolism / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D019329:Idazoxan / Q000031:analogs & derivatives; D037342:Pertussis Toxin; D011810:Quinoxalines / Q000378:metabolism / Q000494:pharmacology; D011942:Receptors, Adrenergic, alpha / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D000316:Adrenergic alpha-Agonists; D000317:Adrenergic alpha-Antagonists; D004146:Dioxanes; D011810:Quinoxalines; D011942:Receptors, Adrenergic, alpha; D010566:Virulence Factors, Bordetella; D000068438:Brimonidine Tartrate; C058525:2-methoxyidazoxan; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D019329:Idazoxan