Effect of inhibitors of mitogen-activated protein kinase kinase on a 1B -adrenoceptor phosphorylation R. Alca ´ntara-Herna ´ndez & J. Adolfo Garcı ´a-Sa ´inz Instituto de Fisiologı ´a Celular, Universidad Nacional Auto ´noma de Me ´xico, Ap. Postal 70-248, Mexico D.F. 04510 Summary 1 Mitogen-activated protein kinases mediate hormone ⁄ neurotransmitter action on prolifera- tion and differentiation and participate in receptor regulation. The effect of inhibitors of mitogen-activated kinase kinase (MEK) on a 1B -adrenoceptor phosphorylation state and function was studied using different cell lines. It was observed that at nanomolar concentrations the MEK inhibitors, PD98059 (2¢-amino-3¢-methoxyflavone) and UO126 [1,4- (diamino-2,3-dicyano ⁄ 1,4-bis-(2-aminophenylthio)-butadiene], increased a 1B -adrenoceptor phosphorylation and diminished the functional response of this receptor to noradrenaline. These agents did not alter the action of lysophosphatidic acid. 2 Staurosporine (IC 50  0.8 nM) (a general protein kinase inhibitor) and bis-indolyl-maleimide I (IC 50  200 nM) (a selective protein kinase C inhibitor) inhibited PD98059-induced a 1B - adrenoceptor phosphorylation. In contrast, neither wortmannin (phosphoinositide 3-kinase inhibitor) nor genistein (protein tyrosine kinase inhibitor) had any effect. The data suggest the possibility that MEK might exert control on the activity of the enzymes that regulate receptor phosphorylation, such as G-protein-coupled receptor kinases, protein kinase C or ser- ine ⁄ threonine protein phosphatases. 3 Coimmunoprecipitation studies showed a constant association of total extracellular signal- regulated kinase 2 (ERK2) with a 1B -adrenoceptors. Association of phospho-ERK 1 ⁄ 2 to a 1B - adrenoceptors increased not only in response to agonist but also in response to agents that increase a 1B -adrenoceptor and ERK1 ⁄ 2 phosphorylation [such as endothelin-1, phorbol 12- myristate-13-acetate (PMA) and epidermal growth factor (EGF)]; not surprisingly, PD98059 decreased this effect. 4 Our data show that blockade of MEK activity results in increased a 1B -adrenoceptor phosphorylation, diminished adrenoceptor function and perturbation of receptor–ERK1 ⁄ 2 interaction. Keywords: a 1B -adrenoceptors, receptor desensitization, receptor phosphorylation, MAPK, MEK Introduction The mitogen-activated protein kinase (MAPK) cascade is involved in physiological processes of cardinal importance for the survival of cells and organisms, including proliferation, growth, vascu- lar remodelling, apoptosis, survival and differenti- ation, among many others (Robinson & Cobb, 1997; Kolch, 2000; Nishimoto & Nishida, 2006; Meloche & Pouyssegur, 2007). These actions seem to involve one or more of the four known MAPK branches, i.e. p38, JNK (c-Jun N-terminal kinase), extracellular signal-regulated kinases 1 and 2 (ERK1 ⁄ 2) and ERK5. For many of these pathways, activation of the MAPK cascade alters gene expres- sion. A vast amount of literature has documented the ability of hormones, neurotransmitters and growth factors (acting through receptor tyrosine kinases and G-protein-coupled receptors) to in- crease the activity of the MAPK cascade and the different pathways involved (see reviews in Gut- kind, 1998; Luttrell, 2002; Marinissen & Gutkind, 2001; Meloche & Pouyssegur, 2007; Robinson & Cobb, 1997; Schlessinger, 2000; Schlessinger & 13 Autonomic & Autacoid Pharmacology 2009, 29, 13–23 Ó 2009 Blackwell Publishing Ltd Correspondence: J. Adolfo Garcı ´a-Sa ´inz doi: 10.1111/j.1474-8673.2009.00427.x 13 Autonomic฀&฀Autacoid Pharmacology