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J. Biol. Chem., Vol. 279, Issue 24, 25002-25009, June 11, 2004

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Opioid Receptor Interacts with Na⁺/H⁺-exchanger Regulatory Factor-1/Ezrin-Radixin-Moesin-binding Phosphoprotein-50 (NHERF-1/EBP50) to Stimulate Na⁺/H⁺ Exchange Independent of G_i/G_o Proteins^*

Peng Huang, Deborah Steplock, Edward J. Weinman, Randy A. Hall¶, Zhe Ding, Jianguo Li, Yulin Wang, and Lee-Yuan Liu-Chen||

From the Department of Pharmacology and Center for Substance Abuse Research, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, the Department of Medicine and Department of Physiology, University of Maryland School of Medicine, and Medical Service, Department of Veterans Affairs Medical Center, Baltimore, Maryland 21201, and the ^¶Department of Pharmacology, Rollins Research Center, Emory University School of Medicine, Atlanta, Georgia 30322

We previously showed that Na⁺/H⁺-exchanger regulatory factor-1/Ezrin-radixin-moesin-binding phosphoprotein-50 (NHERF-1/EBP50) co-immunoprecipitated with the human opioid receptor (hKOR) and that its overexpression blocked the agonist U50,488H-induced hKOR down-regulation by enhancing recycling. Here, we show that glutathione S-transferase (GST)-hKOR C-tail interacted with purified NHERF-1/EBP50, whereas GST or GST-C-tails of µ or opioid receptors did not. GST-hKOR C-tail, but not GST, bound HA-NHERF-1/EBP50 transfected into Chinese hamster ovary cells and endogenous NHERF-1/EBP50 in opossum kidney proximal tubule epithelial cells (OK cells). The PDZ domain I, but not II, of NHERF-1/EBP50 was involved in the interaction. Association of NHERF-1/EBP50 with hKOR C-tail enhanced oligomerization of NHERF-1/EBP50. NHERF-1/EBP50 was previously shown to regulate Na⁺/H⁺-exchanger 3 (NHE3) activities in OK cells. We found stimulation of OK cells with U50,488H significantly enhanced Na⁺/H⁺ exchange, which was blocked by naloxone but not by pertussis toxin pretreatment, indicating it is mediated by KORs but independent of G_i/G_o proteins. In OKH cells, a subclone of OK cells expressing a much lower level of NHERF-1/EBP50, U50,488H had no effect on Na⁺/H⁺ exchange, although it enhanced p44/42 mitogen-activated protein kinase phosphorylation via G_i/G_o proteins similar to that in OK cells. Stable transfection of NHERF-1/EBP50 into OKH cells restored the stimulatory effect of U50,488H upon Na⁺/H⁺ exchange. Thus, NHERF-1/EBP50 binds directly to KOR, and this association plays an important role in accelerating Na⁺/H⁺ exchange. We hypothesize that binding of the KOR to NHERF-1/EBP50 facilitates oligomerization of NHERF-1/EBP50, leading to stimulation of NHE3. This study provides the first direct evidence that a G protein-coupled receptor through association with NHERF-1/EBP-50 stimulates NHE3.

Received for publication, December 8, 2003 , and in revised form, April 6, 2004.

^* This work was supported by National Institutes of Health (NIH) Grants DA04745, DA11263, DA17302, and P30 DA13429 (to L.-Y. L.-C.), NIH Grant DK55881 and Research Service, Department of Veterans Affairs grant (to E. J. W.), and grants from the NIH and W. M. Keck Foundation (to R. A. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: Dept. of Pharmacology, Temple University School of Medicine, 3420 N. Broad St., Philadelphia, PA 19140. Tel.: 215-707-4188; Fax: 215-707-7068; E-mail: lliuche{at}astro.temple.edu.

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