title: Characterization of isoprenaline- and salmeterol-stimulated interactions between beta2-adrenoceptors and beta-arrestin 2 using beta-galactosidase complementation in C2C12 cells.
abstract: beta-Arrestin is an adaptor protein that has been shown to couple G protein-coupled receptors (GPCRs) to clathrin-coated pits and target them for subsequent internalization. More recently, beta-arrestin 2 has also been shown to be involved in the activation of mitogen-activated protein kinase cascades by G protein-coupled receptors independently of G protein activation. Direct interactions between proteins can be monitored using enzyme complementation between two inactive deletion mutants of beta-galactosidase (beta-gal; Deltaalpha and Deltaomega). In the present study, we have used fusion proteins of the human beta(2)-adrenoceptor (C-terminal beta-gal Deltaalpha) and beta-arrestin 2 (beta-gal Deltaomega) to study directly the pharmacology of this interaction in C2C12 cells expressing the beta(2)-adrenoceptor-beta-gal Deltaalpha fusion protein at low physiological levels (38.2 +/- 2.7 fmol . mg protein(-1)). Isoprenaline, noradrenaline, and adrenaline (-log EC(50) = 5.9, 5.5, and 5.7, respectively) stimulated an association between the beta(2)-adrenoceptor and beta-arrestin 2 at much higher concentrations than required for activation of cAMP accumulation (-log EC(50) = 7.6, 6.3, and 7.7, respectively). This was sensitive to inhibition by the beta(2)-adrenoceptor antagonists propranolol, timolol, and ICI 118551. Both salbutamol and terbutaline behaved as partial agonists of beta-gal complementation. Furthermore, the long-acting beta(2)-agonist salmeterol (-log K(D) for inhibition of [(3)H]CGP12177 binding = 8.7) behaved as an antagonist of isoprenaline-stimulated beta(2)-adrenoceptor-arrestin 2 interactions (-log K(D) = 8.0), whereas acting as a full agonist of cAMP accumulation in the same cells (-log EC(50) = 9.2). These data suggest that salmeterol can discriminate between receptor-G(S) protein and receptor-arrestin 2 complexes (in terms of efficacy and affinity) in a way that is favorable for its long duration of action.
authors.affiliation: Institute of Cell Signaling, Medical School, Queen's Medical Centre, Nottingham, UK.
authors.forename: Alison A
authors.initials: AA
authors.lastname: Carter
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Hill
mesh_terms: D000318:Adrenergic beta-Agonists / Q000378:metabolism / Q000494:pharmacology*; D000319:Adrenergic beta-Antagonists / Q000378:metabolism; D000420:Albuterol / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D019390:Arrestins / Q000187:drug effects*; D016466:CHO Cells; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D005816:Genetic Complementation Test; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology*; D009154:Mutation / Q000502:physiology; D011412:Propanolamines / Q000378:metabolism; D011433:Propranolol / Q000378:metabolism; D018343:Receptors, Adrenergic, beta-2 / Q000187:drug effects*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000068299:Salmeterol Xinafoate; D014162:Transfection; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
chemical_list: C000605254:ARRB2 protein, human; D000318:Adrenergic beta-Agonists; D000319:Adrenergic beta-Antagonists; D019390:Arrestins; D011412:Propanolamines; D018343:Receptors, Adrenergic, beta-2; D011993:Recombinant Fusion Proteins; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D005576:Colforsin; C026777:ICI 118551; D000068299:Salmeterol Xinafoate; D011433:Propranolol; D000242:Cyclic AMP; D001616:beta-Galactosidase; D007545:Isoproterenol; D000420:Albuterol; C033394:CGP 12177