Chronic adrenaline treatment fails to down-regulate the Del301–303-α2B-adrenoceptor in neuronal cells

Background and purpose: A polymorphism of the human α 2B -adrenoceptor (Del 301 - 303 -α 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 -α 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 α 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 -α 2B -adrenoceptor in a neuronal cell model. Experimental approach: Haemagglutinin (HA) epitope-tagged WT- and Del 301 - 303 -α 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 ~50 fmol·mg -1 . These cells were used to examine agonist-induced down-regulation and phosphorylation of the WT- and Del 301 - 303 -α 2B -adrenoceptor. Key results: The Del 301 - 303 -α 2B -adrenoceptor, compared with the WT-α 2B -adrenoceptor, displayed reduced adrenaline-stimulated (20 μM) phosphorylation and did not down-regulate in response to adrenaline (20-1000 μM). Using immunofluorescence labelling, we observed that transiently transfected WT-α 2B -adrenoceptors internalized upon adrenaline treatment whereas the Del 301 - 303 -α 2B -adrenoceptor did not. Finally, we determined the effect of adrenaline on the Del 301 - 303 -α 2B -adrenoceptor in cells stably over-expressing GRK3 3-fold. In spite of the GRK3 over-expression, 20-1000 μM ADR failed to down-regulate or to increase phosphorylation of the Del 301 - 303 -α 2B -adrenoceptor in these cells. Conclusions and implications: The results suggest that the 301-303 deletion mutation of the α 2B -adrenoceptor eliminates agonist-induced down-regulation, an effect that cannot be overcome by increasing agonist concentration or by modest GRK3 over-expression.