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pubmed:abstractText |
We have investigated whether Ezrin-radixin-moesin (ERM)-binding phosphoprotein-50/Na(+)/H(+) exchanger regulatory factor (EBP50/NHERF), a PDZ domain-containing phosphoprotein, is associated with the human kappa opioid receptor (hkor) and whether it regulates the trafficking and signaling of the hkor. When expressed in CHO cells stably transfected with the FLAG-tagged hkor (FLAG-hkor), EBP50/NHERF co-immunoprecipitated with FLAG-hkor, and the PDZ domain I, but not the PDZ domain II, of EBP50/NHERF was involved in the interaction. Treatment with the agonist (-)-(trans)-3,4- dichloro-N-methyl-N-[2-(1-pyrrolidiny)cyclohexyl]benzeneacetamide (U50,488H) enhanced the association of EBP50/NHERF with FLAG-hkor. Expression of EBP50/NHERF, but not a truncated form lacking the ERM-binding domain, abolished U50,488H-induced down-regulation of FLAG-hkor, which was apparently due to an increase in the recycling rate of internalized receptors. However, expression of EBP50/NHERF did not affect U50,488H binding affinity and U50,488H-stimulated [(35)S]guanosine 5'-3-O-(thio)triphosphate binding and p42/p44 MAP kinase activation, nor did it affect U50,488H-induced desensitization and internalization of FLAG-hkor. To determine the motif of FLAG-hkor involved in EBP50/NHERF binding, we generated two mutants, FLAG-hkor-A and FLAG-hkor-EE, in which one Ala or two Glu residues were added to the C terminus, respectively. Neither FLAG-hkor-A nor FLAG-hkor-EE co-immunoprecipitated with EBP50/NHERF, and U50,488H-induced down-regulation of FLAG-hkor-A and FLAG-hkor-EE were not affected by expression of EBP50/NHERF. Thus, EBP50/NHERF binds to the C terminus of FLAG-hkor and blocks the down-regulation of FLAG-hkor. The C-terminal sequence of the hkor, NKPV, is distinctly different from the sequence D(S/T)XL, the optimal C-terminal motif in the beta(2)-adrenergic receptor for EBP50/NHERF binding. EBP50/NHERF may have a broader binding specificity and may interact with a subset of G protein-coupled receptors to serve as a recycling signal for these receptors.
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pubmed:affiliation |
Department of Pharmacology, Center for Substance Abuse Research, Temple University School of Medicine, 3420 N. Broad Street, Philadelphia, PA 19140, USA.
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