title: Chronic adrenaline treatment fails to down-regulate the Del301-303-alpha2B-adrenoceptor in neuronal cells.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  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
  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.
authors.affiliation: Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX 77204-5037, USA.
authors.forename: S
authors.initials: S
authors.lastname: Salim
authors.forename: A N
authors.initials: AN
authors.lastname: Desai
authors.forename: M
authors.initials: M
authors.lastname: Taneja
authors.forename: D C
authors.initials: DC
authors.lastname: Eikenburg
mesh_terms: D045744:Cell Line, Tumor; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics*; D004334:Drug Administration Schedule; D004837:Epinephrine / Q000008:administration & dosage*; D006801:Humans; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D011110:Polymorphism, Genetic / Q000187:drug effects / Q000502:physiology*; D018341:Receptors, Adrenergic, alpha-2 / Q000235:genetics* / Q000378:metabolism*; D017384:Sequence Deletion
chemical_list: C547771:ADRA2B protein, human; D018341:Receptors, Adrenergic, alpha-2; D004837:Epinephrine