title: Human alpha1D-adrenoceptor phosphorylation and desensitization.
abstract: Rat-1 fibroblast were transfected with a plasmid containing the cDNA of the human alpha(1D)-adrenoceptor. A cell line was isolated that stably expressed the receptor as evidenced by BMY 7378-sensitive noradrenaline-induced increases in intracellular calcium concentration. The effect of noradrenaline was blocked by active phorbol esters; such blockade was mediated by protein kinase C (PKC) as evidenced by its inhibition by staurosporine or the downregulation of this protein kinase. Radioligand binding experiments showed expression of receptors with high affinity for [3H]tamsulosin (K(D) 0.30 +/- 0.05 nM) but low density (B(max) 35 +/- 4 fmol/mg protein). The receptors had the expected orders of potency for agonists (adrenaline = noradrenaline > oxymetazoline) and antagonists (BMY 7378 > 5-methyl-urapidil = phentolamine). Photoaffinity labeling identified the receptor as a band of M(r) 70-80kDa, which could be immunoprecipitated with a selective anti-alpha(1D)-adrenoceptor antiserum. In cells metabolically labeled with radioactive phosphate the adrenoceptor was identified as a phosphoprotein whose phosphorylation state was increased by the agonist, noradrenaline, and by phorbol myristate acetate. The data indicate that the human alpha(1D)-adrenoceptor function was regulated through phosphorylation by PKC.
authors.affiliation: Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Apartado Postal 70-248, Mexico D.F. 04510, Mexico. agarcia@ifc.unam.mx
authors.forename: J Adolfo
authors.initials: JA
authors.lastname: García-Sáinz
authors.forename: C Ekaterina
authors.initials: CE
authors.lastname: Rodríguez-Pérez
authors.forename: M Teresa
authors.initials: MT
authors.lastname: Romero-Avila
mesh_terms: D058668:Adrenergic alpha-1 Receptor Antagonists; D000317:Adrenergic alpha-Antagonists / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D006801:Humans; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D018340:Receptors, Adrenergic, alpha-1 / Q000235:genetics / Q000378:metabolism*
chemical_list: C547719:ADRA1D protein, human; C547721:Adra1d protein, rat; D058668:Adrenergic alpha-1 Receptor Antagonists; D000317:Adrenergic alpha-Antagonists; D010879:Piperazines; D018340:Receptors, Adrenergic, alpha-1; D011493:Protein Kinase C; C053428:BMY 7378