title: Heterologous, PKC-Mediated Desensitization of Human Histamine H3 Receptors Expressed in CHO-K1 Cells.
abstract: Desensitization is a major mechanism to regulate the functional response of G protein-coupled receptors. In this work we studied whether the human histamine H3 receptor of 445 amino acids (hH3R445) experiences heterologous desensitization mediated by PKC activation. Bioinformatic analysis indicated the presence of Serine and Threonine residues susceptible of PKC-mediated phosphorylation on the third intracellular loop and the carboxyl terminus of the hH3R445. In CHO-K1 cells stably transfected with the hH3R445 direct PKC activation by phorbol 12-myristate 13-acetate (TPA, 200 nM) abolished H3R-mediated inhibition of forskolin-stimulated cAMP accumulation. Activation of endogenous purinergic receptors by ATP (adenosine 5'-triphosphate, 10 μM) increased the free calcium intracellular concentration ([Ca(2+)]i) confirming their coupling to phospholipase C stimulation. Incubation with ATP also abolished H3R-mediated inhibition of forskolin-induced cAMP accumulation, and this effect was prevented by the PKC inhibitors Ro-31-8220 and Gö-6976. Pre-incubation with TPA or ATP reduced H3R-mediated stimulation of [(35)S]-GTPγS binding to membranes from CHO-K1-hH3R445 cells by 39.7 and 54.2 %, respectively, with no change in the agonist potency, and the effect was prevented by either Ro-31-8220 or Gö-6976. Exposure to ATP or TPA also resulted in the loss of cell surface H3Rs (-30.4 and -45.1 %) as evaluated by [(3)H]-NMHA binding to intact cells. These results indicate that the hH3R445 undergoes heterologous desensitization upon activation of receptors coupled to PKC stimulation.
authors.affiliation: Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados del IPN, Av. IPN 2508, Zacatenco, 07360, México, D.F., Mexico.
authors.forename: Wilber
authors.initials: W
authors.lastname: Montejo-López
authors.affiliation: Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados del IPN, Av. IPN 2508, Zacatenco, 07360, México, D.F., Mexico.
authors.forename: Nayeli
authors.initials: N
authors.lastname: Rivera-Ramírez
authors.affiliation: Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados del IPN, Av. IPN 2508, Zacatenco, 07360, México, D.F., Mexico.
authors.forename: Juan
authors.initials: J
authors.lastname: Escamilla-Sánchez
authors.affiliation: Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados del IPN, Av. IPN 2508, Zacatenco, 07360, México, D.F., Mexico.
authors.forename: Ubaldo
authors.initials: U
authors.lastname: García-Hernández
authors.affiliation: Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados del IPN, Av. IPN 2508, Zacatenco, 07360, México, D.F., Mexico. jaarias@fisio.cinvestav.mx.
authors.forename: José-Antonio
authors.initials: JA
authors.lastname: Arias-Montaño
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002227:Carbazoles / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D003412:Cricetulus / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D018100:Receptors, Histamine H3 / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D002227:Carbazoles; D007211:Indoles; D018100:Receptors, Histamine H3; C081021:Go 6976; D005576:Colforsin; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; C064758:Ro 31-8220
keywords: CHO-K1 cells; Desensitization; H3 receptor; Histamine; PKC