Noradrenaline, oxymetazoline and phorbol myristate acetate induce distinct functional actions and phosphorylation patterns of α 1A -adrenergic receptors

• Published in: Biochimica et biophysica acta. Molecular cell research 2017-12, Vol.1864 (12), p.2378
• Main Authors: Alcántara-Hernández, Rocío, Hernández-Méndez, Aurelio, Romero-Ávila, M Teresa, Alfonzo-Méndez, Marco A, Pupo, André S, García-Sáinz, J Adolfo
• Format: Article
• Language: English
• Subjects: Calcium Signaling - drug effects; Cell Line, Tumor; Gene Expression Regulation, Neoplastic - drug effects; Humans; MAP Kinase Signaling System - drug effects; Mass Spectrometry; Norepinephrine - pharmacology; Oxymetazoline - pharmacology; Phosphorylation - genetics; Protein Kinase C - genetics; Proteolysis; Receptors, Adrenergic, alpha-1 - genetics; Receptors, Adrenergic, alpha-1 - metabolism; Tetradecanoylphorbol Acetate - pharmacology
• ISSN: 0167-4889
• DOI: 10.1016/j.bbamcr.2017.09.002

In LNCaP cells that stably express α -adrenergic receptors, oxymetazoline increased intracellular calcium and receptor phosphorylation, however, this agonist was a weak partial agonist, as compared to noradrenaline, for calcium signaling. Interestingly, oxymetazoline-induced receptor internalization and desensitization displayed greater effects than those induced by noradrenaline. Phorbol myristate acetate induced modest receptor internalization and minimal desensitization. α -Adrenergic receptor interaction with β-arrestins (colocalization/coimmunoprecipitation) was induced by noradrenaline and oxymetazoline and, to a lesser extent, by phorbol myristate acetate. Oxymetazoline was more potent and effective than noradrenaline in inducing ERK 1/2 phosphorylation. Mass spectrometric analysis of immunopurified α -adrenergic receptors from cells treated with adrenergic agonists and the phorbol ester clearly showed that phosphorylated residues were present both at the third intracellular loop and at the carboxyl tail. Distinct phosphorylation patterns were observed under the different conditions. The phosphorylated residues were: a) Baseline and all treatments: T233; b) noradrenaline: S220, S227, S229, S246, S250, S389; c) oxymetazoline: S227, S246, S381, T384, S389; and d) phorbol myristate acetate: S246, S250, S258, S351, S352, S401, S402, S407, T411, S413, T451. Our novel data, describing the α -AR phosphorylation sites, suggest that the observed different phosphorylation patterns may participate in defining adrenoceptor localization and action, under the different conditions examined.