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Papers In Press, published online ahead of print May 9, 2002
Department of Pharmacology, Temple University School of Medicine, Philadelphia, PA 19140
Corresponding Author: lliuche{at}astro.temple.edu
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 {kapp} 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 the 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 U50,488H enhanced the association of EBP50/NHERF with the Flag-hkor. Expression of EBP50/NHERF, but not a truncated form lacking the ERM-binding domain, abolished U50,488H-induced down-regulation of the 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 [35S]GTP
J. Biol. Chem, 10.1074/jbc.M200058200
Submitted on January 3, 2002
Revised on May 8, 2002
Accepted on May 9, 2002
EBP50/NHERF blocks U50,488H-induced down-regulation of the human
opioid receptor by enhancing its recycling rate
S binding and p42/p44 MAP kinase activation, nor did it affect U50,488H-induced desensitization and internalization of the Flag-hkor. To determine the motif of the 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 the Flag-hkor and blocks the down-regulation of the Flag-hkor. The C-terminal sequence of the hkor, NKPV, is distinctly different from the sequence D-S/T-X-L, the optimal C-terminal motif in the 
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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