The opioid ligand binding of human l-opioid receptor is modulated by novel splice variants of the receptor Hack Sun Choi * , Chun Sung Kim, Cheol Kyu Hwang, Kyu Young Song, Wei Wang, Yu Qiu, Ping-Yee Law, Li-Na Wei, Horace H. Loh Department of Pharmacology, Medical School, University of Minnesota, Minneapolis, MN 55455, USA Received 3 March 2006 Available online 23 March 2006 Abstract The pharmacological actions of morphine and morphine-like drugs, such as heroin, mediate primarily through the l-opioid receptor (MOR). It has been proposed that the functional diversity of MOR may be related to alternative splicing of the MOR gene. Although a number of MOR mRNA splice variants have been reported, their biological function has been controversial. In this study, two novel splice variants of the human MOR gene were discovered. Splice variants 1 and 2 (here called the SV1 and SV2) retain different portions of intron I. In vitro translation of SV1 and SV2 produced proteins with the predicted molecular weights. The splice variant proteins were identical to the wild-type MOR-1 up to the first transmembrane domains, but were different after the first intracellular loop domains. SV1 and SV2 of hMOR were present in human neuroblastoma NMB cells and human whole brain confirmed by RT-PCR. In a receptor binding assay, cells expressing the SV1 and SV2 do not exhibit binding to [ 3 H]diprenorphine. The formations of MOR Æ SV1 and MOR Æ SV2 heterodimers were demonstrated by co-immunoprecipitation and bioluminescence resonance energy transfer between MOR and splice variants. Co-transfection of MOR-GFP and SV-DsRed gene showed that MOR and SV protein co-localized at the cytoplasmic membrane. In NMB cells expressing human MOR gene, transfection of SV1 or SV2 reduced binding activity of the endogenous MOR. These data support a potential role of SV1 and SV2 proteins as possible biological modulator of human l-opioid receptor. Ó 2006 Elsevier Inc. All rights reserved. Keywords: MOR, l-Opioid receptor; BRET, Bioluminescence resonance energy transfer; Rluc, Renilla luciferase; DsRed, Destabilized red fluorescence protein; SV, Splice variant; TM, Transmembrane domain Morphine has been used as a potent clinical analgesic for pain killing, but has serious limitations such as toler- ance and dependence. The opioid receptors are classified into three major types (l, d, and j) that were determined by numerous pharmacological reports and molecular clon- ing [1]. All three types of opioid receptors belong to the superfamily of G-protein-coupled receptors (GPCRs). Pre- vious studies suggested that the l-opioid receptor (MOR) plays important roles in morphine-induced analgesia, toler- ance, and dependence as indicated from pharmacological studies and analysis of MOR knockout mice [2,3]. Upon binding with opioids, MOR is able to couple to G-proteins and to regulate adenylyl cyclase, intracellular calcium, inwardly rectifying potassium channels, MAP kinase, and other messengers, which further trigger a cascade of intra- cellular events [4]. MOR is expressed mainly in the central nervous system (CNS), but densities of MOR vary in different parts of the brain that may be related to differing roles of MOR in spe- cific brain regions [5]. Furthermore, MOR is also expressed in other tissues, such as immune cells [6]. The presence of MOR in immune cells can elucidate the reason why drug abusers (opioid and heroin) become more susceptible to external pathogens after weakening the body’s immune sys- tem by chronic drug use, especially well described in HIV- infected opiate users [7–9]. The l-opioid receptor, MOR-1, was the first cloned l-opioid receptor cDNA and consisted of 4 exons [10–12]. Alternative splicing variants of MOR have been 0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.03.084 * Corresponding author. Fax: +1 612 625 8408. E-mail address: choix074@umn.edu (H.S. Choi). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 343 (2006) 1132–1140 BBRC