S Salim1, A N Desai, M Taneja, D C Eikenburg. 1. Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX 77204-5037, USA.
Abstract
BACKGROUND AND PURPOSE: A polymorphism of the human alpha(2B)-adrenoceptor (Del(301-303)-alpha(2B)-adrenoceptor) has been described, and this receptor exhibits reduced G-protein-coupled receptor kinase (GRK) phosphorylation and impaired short-term desensitization. Expression of the Del(301-303)-alpha(2B)-adrenoceptor also is associated with an increased risk for myocardial infarction in humans. Recent evidence from our laboratory suggests a quantitative relationship between cellular GRK3 expression levels and the sensitivity of the alpha(2B)-adrenoceptor to agonist-induced down-regulation. Therefore, the present study was undertaken to study agonist-induced down-regulation of the wild-type (WT)- and Del(301-303)-alpha(2B)-adrenoceptor in a neuronal cell model. EXPERIMENTAL APPROACH: Haemagglutinin (HA) epitope-tagged WT- and Del(301-303)-alpha(2B)-adrenoceptor containing plasmids were constructed and the receptors were stably or transiently transfected in neuroblastoma/glioma hybrid NG108 cells. The expression levels in stable transfects were approximately 50 fmol x mg(-1). These cells were used to examine agonist-induced down-regulation and phosphorylation of the WT- and Del(301-303)-alpha(2B)-adrenoceptor. KEY RESULTS: The Del(301-303)-alpha(2B)-adrenoceptor, compared with the WT-alpha(2B-)adrenoceptor, displayed reduced adrenaline-stimulated (20 microM) phosphorylation and did not down-regulate in response to adrenaline (20-1000 microM). Using immunofluorescence labelling, we observed that transiently transfected WT-alpha(2B)-adrenoceptors internalized upon adrenaline treatment whereas the Del(301-303)-alpha(2B)-adrenoceptor did not. Finally, we determined the effect of adrenaline on the Del(301-303)-alpha(2B)-adrenoceptor in cells stably over-expressing GRK3 3-fold. In spite of the GRK3 over-expression, 20-1000 microM ADR failed to down-regulate or to increase phosphorylation of the Del(301-303)-alpha(2B)-adrenoceptor in these cells. CONCLUSIONS AND IMPLICATIONS: The results suggest that the 301-303 deletion mutation of the alpha(2B)-adrenoceptor eliminates agonist-induced down-regulation, an effect that cannot be overcome by increasing agonist concentration or by modest GRK3 over-expression.
BACKGROUND AND PURPOSE: A polymorphism of the human alpha(2B)-adrenoceptor (Del(301-303)-alpha(2B)-adrenoceptor) has been described, and this receptor exhibits reduced G-protein-coupled receptor kinase (GRK) phosphorylation and impaired short-term desensitization. Expression of the Del(301-303)-alpha(2B)-adrenoceptor also is associated with an increased risk for myocardial infarction in humans. Recent evidence from our laboratory suggests a quantitative relationship between cellular GRK3 expression levels and the sensitivity of the alpha(2B)-adrenoceptor to agonist-induced down-regulation. Therefore, the present study was undertaken to study agonist-induced down-regulation of the wild-type (WT)- and Del(301-303)-alpha(2B)-adrenoceptor in a neuronal cell model. EXPERIMENTAL APPROACH: Haemagglutinin (HA) epitope-tagged WT- and Del(301-303)-alpha(2B)-adrenoceptor containing plasmids were constructed and the receptors were stably or transiently transfected in neuroblastoma/glioma hybrid NG108 cells. The expression levels in stable transfects were approximately 50 fmol x mg(-1). These cells were used to examine agonist-induced down-regulation and phosphorylation of the WT- and Del(301-303)-alpha(2B)-adrenoceptor. KEY RESULTS: The Del(301-303)-alpha(2B)-adrenoceptor, compared with the WT-alpha(2B-)adrenoceptor, displayed reduced adrenaline-stimulated (20 microM) phosphorylation and did not down-regulate in response to adrenaline (20-1000 microM). Using immunofluorescence labelling, we observed that transiently transfected WT-alpha(2B)-adrenoceptors internalized upon adrenaline treatment whereas the Del(301-303)-alpha(2B)-adrenoceptor did not. Finally, we determined the effect of adrenaline on the Del(301-303)-alpha(2B)-adrenoceptor in cells stably over-expressing GRK3 3-fold. In spite of the GRK3 over-expression, 20-1000 microM ADR failed to down-regulate or to increase phosphorylation of the Del(301-303)-alpha(2B)-adrenoceptor in these cells. CONCLUSIONS AND IMPLICATIONS: The results suggest that the 301-303 deletion mutation of the alpha(2B)-adrenoceptor eliminates agonist-induced down-regulation, an effect that cannot be overcome by increasing agonist concentration or by modest GRK3 over-expression.