José Vázquez-Prado

α1-Adrenoceptors : function and phosphorylation

Abstract This review focuses on α 1 -adrenoceptor phosphorylation and function. Most of what is currently known is based on studies on the hamster α 1B -adrenoceptor. It is known that agonist stimulation leads to homologous desensitization of these receptors and current evidence indicates that such decrease in receptor activity is associated with receptor phosphorylation. Such receptor phosphorylation seems to involve G protein-receptor kinases and the receptor phosphorylation sites have been located in the carboxyl tail (Ser 404 , Ser 408 , and Ser 410 ). There is also evidence showing that in addition to desensitization, receptor phosphorylation is associated with internalization and roles of β-arrestins have been observed. Direct activation of protein kinase C leads to receptor desensitization/internalization associated with phosphorylation; the protein-kinase-C-catalyzed receptor phosphorylation sites have been also located in the carboxyl tail (Ser 394 and Ser 400 ). Activation of G q -coupled receptors, such as the endothelin ET A receptor induces α 1B -adrenoceptor phosphorylation and desensitization. Such effect involves protein kinase C and a yet unidentified tyrosine kinase. Activation of G i -coupled receptors, such as the lysophosphatidic acid receptor, also induces α 1B -adrenoceptor phosphorylation and desensitization. These effects involve protein kinase C and phosphatidyl inositol 3-kinase. Interestingly, activation of epidermal growth factor receptors also induces α 1B -adrenoceptor phosphorylation and desensitization involving protein kinase C and phosphatidyl inositol 3-kinase. A pivotal role of these kinases in heterologous desensitization is evidenced.

α1-Adrenoceptors: Subtypes, Signaling, and Roles in Health and Disease

Alpha 1-adrenoceptors mediate some of the main actions of the natural catecholamines, adrenaline, and noradrenaline. They participate in many essential physiological processes, such as sympathetic neurotransmission, modulation of hepatic metabolism, control of vascular tone, cardiac contraction, and the regulation of smooth muscle activity in the genitourinary system. It is now clear that alpha 1-adrenoceptors mediate, in addition to immediate effects, longer term actions of catecholamines such as cell growth and proliferation. In fact, adrenoceptor genes can be considered as protooncogenes. Over the past years, considerable progress has been achieved in the molecular characterization of different alpha 1-adrenoceptor subtypes. Three main subtypes have been characterized pharmacologically and in molecular terms. Splice variants, truncated isoforms, and polymorphisms have also been detected. Similarly, it is now clear that these receptors are coupled to several classes of G proteins that, therefore, are capable of modulating different signaling pathways. In the present article, some of these aspects are reviewed, together with the distribution of the subtypes in different tissues and some of the known roles of these receptors in health and disease.

Activation of Endothelin ETA Receptors Induces Phosphorylation of α1b-Adrenoreceptors in Rat-1 Fibroblasts

The effect of endothelin-1 on the phosphorylation of α1b-adrenoreceptors, transfected into rat-1 fibroblasts, was studied. Basal α1b-adrenoreceptor phosphorylation was markedly increased by endothelin-1, norepinephrine, and phorbol esters. The effect of endothelin-1 was dose dependent (EC50 ≈ 1 nm), reached its maximum 5 min after stimulation, and was inhibited by BQ-123, an antagonist selective for ETA receptors. Endothelin-1-induced α1b-adrenoreceptor phosphorylation was attenuated by staurosporine or genistein and essentially abolished when both inhibitors were used together. The effect of norepinephrine was not modified by either staurosporine or genistein alone, and it was only partially inhibited when both were used together. These data suggest the participation of protein kinase C and tyrosine kinase(s) in endothelin-1-induced receptor phosphorylation. However, phosphoaminoacid analysis revealed the presence of phosphoserine and traces of phosphothreonine, but not of phosphotyrosine, suggesting that the putative tyrosine kinase(s), activated by endothelin, could act in a step previous to receptor phosphorylation. The effect of endothelin-1 on α1b-adrenoreceptor phosphorylation was not mediated through pertussis toxin-sensitive G proteins. Calcium mobilization induced by norepinephrine was diminished by endothelin-1. Norepinephrine and endothelin-1 increased [35S]GTPγS binding to control membranes. The effect of norepinephrine was abolished in membranes obtained from cells pretreated with endothelin-1. Interestingly, genistein plus staurosporine inhibited this effect of the endothelial peptide. Endothelin-1 did not induce α1b-adrenoreceptor internalization. Our data indicate that activation of ETA receptors by endothelin-1 induces α1b-adrenoreceptor phosphorylation and alters G protein coupling.

Crosstalk: phosphorylation of α1b-adrenoceptors induced through activation of bradykinin B2 receptors

The possibility that bradykinin could modulate α1b‐adrenoceptors was studied. It was observed that bradykinin and kallidin increased α1b‐adrenoceptor phosphorylation and that such effect was also blocked by Hoe 140. Interestingly, the ability of norepinephrine to increase intracellular calcium concentration was not altered by pretreatment of the cells with bradykinin, i.e. bradykinin induced α1b‐adrenoceptor phosphorylation but this did not lead to receptor desensitization.

α1-Adrenoceptor subtype activation increases proto-oncogene mRNA levels. Role of protein kinase C

Abstract Noradrenaline increased the mRNA levels of c-fos and c-jun in rat-1 fibroblast lines stably expressing the cloned α1-adrenoceptor subtypes. The efficacy to induce the expression of c-fos mRNA was similar for the three cell lines (α1d=α1b=α1a) but different for c-jun (α1a≥α1b>α1d). The EC50 values were also different: ≈5 nM (c-fos) and ≈300 nM (c-jun) for cells transfected with the α1a subtype, ≈30 nM (c-fos) and ≈300 nM (c-jun) for cells transfected with the α1b subtype and ≈300 nM (c-fos and c-jun) for those transfected with the α1d subtype. Staurosporine and protein kinase C down-regulation blocked such effects, indicating a role of this protein kinase. Endothelin-1 (10 nM) also increased the levels of c-fos and c-jun mRNAs. These actions of endothelin-1 were unaffected by staurosporine and protein kinase C down-regulation. It is concluded that activation of any of the three cloned subtypes can increase the levels of c-fos and c-jun mRNAs and that protein kinase C plays a major role in mediating such effects.

Regulation of the human bradykinin B2 receptor expressed in sf21 insect cells: A possible role for tyrosine kinases

The functional regulation of the human bradykinin B2 receptor expressed in sf21 cells was studied. Human bradykinin B2 receptors were immunodetected as a band of 75–80 kDa in membranes from recombinant baculovirus‐infected cells and visualized at the plasma membrane, by confocal microscopy, using an antibody against an epitope from its second extracellular loop. B2 receptors, detected in membranes by [3H‐bradykinin] binding, showed a Kd of 0.66 nmol/L and an expression level of 2.57 pmol/mg of protein at 54 h postinfection. In these cells, bradykinin induced a transient increase of intracellular calcium ([Ca2+]i) in fura 2‐AM loaded sf21 cells, and promoted [35S]‐GTPγS binding to membranes. The effects of bradykinin were dose dependent (with an EC50 of 50 nmol/L for calcium mobilization) and were inhibited by N‐α‐adamantaneacetyl‐D‐Arg‐[Hyp3,Thi5,8,D‐phe7]‐Bk, a specific B2 receptor antagonist. When the B2 antagonist was applied at the top of the calcium transient, it accelerated the decline of the peak, suggesting that calcium mobilization at this point was still influenced by receptor occupation. No calcium mobilization was elicited by 1 μmol/L (Des‐Arg9)‐Bk, a B1 receptor agonist that did not inhibit the subsequent action of 100 nmol/L bradykinin. No effect of bradykinin was detected in uninfected cells or cells infected with the wild‐type baculovirus. Bradykinin‐induced [Ca2+]i mobilization was increased by genistein and tyrphostin A51. These tyrosine kinase inhibitors did not modify basal levels of [Ca2+]i. Homologous desensitization of the B2 receptor was observed after repeated applications of bradykinin, which resulted in attenuated changes in intracellular calcium. In addition, genistein promoted an increased response to a third exposure to the agonist when applied after washing the cells that had been previously challenged with two increasing doses of bradykinin. Genistein did not affect the calcium mobilization induced by activation of the endogenous octopamine G protein‐coupled receptor or by thapsigargin. The B2 receptor, detected by confocal microscopy in unpermeabilized cells, remained constant at the surface of cells stimulated with bradykinin for 10 min, in the presence or absence of genistein. Agonist‐promoted phosphorylation of the B2 receptor was markedly accentuated by genistein treatment. Phosphoaminoacid analysis revealed the presence of phosphoserine and traces of phosphothreonine, but not phosphotyrosine, suggesting that the putative tyrosine kinase(s), activated by bradykinin, could act in a step previous to receptor phosphorylation. Interestingly, genistein prevented agonist‐induced G protein uncoupling from B2 receptors, determined by in vitro bradykinin‐stimulated [35S]‐GTPγS binding, in membranes from bradykinin pretreated cells. Our results suggest that tyrosine kinase(s) regulate the activity of the human B2 receptor in sf21 cells by affecting its coupling to G proteins and its phosphorylation. J. Cell. Biochem. 76:658–673, 2000.

Protein kinase C-mediated phosphorylation and desensitization of human α1b-adrenoceptors

Human alpha(1b)-adrenoceptors stably expressed (B(max) approximately 800 fmol/mg membrane protein) in mouse fibroblasts were able to increase intracellular Ca(2+) and inositol phosphate production in response to noradrenaline. Activation of protein kinase C desensitized the alpha(1b)-adrenergic-mediated actions but did not block the ability of the cells to respond to lysophosphatidic acid. Inhibition or downregulation of protein kinase C also blocked the action of the tumor promoter on the adrenergic effects. Photolabeling experiments indicated that the receptor has an apparent molecular weight of approximately 80 kDa. The receptors were phosphorylated in the basal state and such phosphorylation was increased when the cells were incubated with phorbol myristate acetate or noradrenaline. Incubation of the cells with phorbol myristate acetate or noradrenaline blocked noradrenaline-promoted [35S]GTP-gamma-S binding to membranes, suggesting receptor-G protein uncoupling. The results indicate that activation of protein kinase C blocked/desensitized human alpha(1b)-adrenoceptors and that such effect was associated to receptor phosphorylation.

Intracellular Calcium and α1b-Adrenoceptor Phosphorylation

Abstract Background Desensitization of G protein-coupled receptors is associated with receptor phosphorylation. Two groups of kinases seem to participate in such receptor phosphorylation, i. e., second messenger-activated protein kinases and G protein-coupled receptor kinases. Calcium seems to play a role in the phosphorylation of some G protein-coupled receptors. The role of calcium in α 1b -adrenoceptor phosphorylation has not been critically assessed. Methods Rat-1 fibroblasts stably expressing the hamster α 1b -adrenergic receptor were used. To study receptor phosphorylation cells metabolically labeled with [ 32 P]Pi were lysed and the receptor immunoprecipitated using a polyclonal antibody generated against the receptor carboxyl terminal decapeptide. Intracellular calcium was determined by using Fura-2 fluorescence. Results Norepinephrine, endothelin-1, and lysophosphatidic acid increased intracellular calcium concentration. All these agents and phorbol myristate acetate (PMA) induce α 1b -adrenoceptor phosphorylation. The intracellular chelator, BAPTA, abolished the increase in intracellular calcium induced by the previously mentioned agents but did not affect the receptor phosphorylation induced by norepinephrine, PMA, or lysophosphatidic acid. Under these conditions, receptor phosphorylation induced by endothelin was slightly but consistently decreased. Thapsigargin increased intracellular calcium concentration but was unable to induce α 1b -adrenoceptor phosphorylation and decreased PMA-induced receptor phosphorylation. No increase in receptor phosphorylation was observed when calcium ionophores were used. Conclusions Our data indicate that an increase in [Ca 2 +]i is not sufficient to induce α 1b -adrenoceptor phosphorylation and that buffering of [Ca 2 +]i does not alter the receptor phosphorylation induced by norepinephrine, lysophosphatidic acid, and PMA. A marginal role of calcium in the α 1b -adrenoceptor phosphorylation induced by endothelin-1 cannot be discarded.