Elsevier

Biochemical Pharmacology

Volume 62, Issue 3, 1 August 2001, Pages 349-355
Biochemical Pharmacology

Molecular cloning and pharmacological characterization of the rat sigma1 receptor1

https://doi.org/10.1016/S0006-2952(01)00666-9Get rights and content

Abstract

In an effort to further understand the pharmacology of sigma receptors, we have cloned the rat homolog of the sigma1 receptor. We isolated a cDNA clone (rs2-2) from rat brain tissue using reverse transcriptase-polymerase chain reaction (RT-PCR) and 5′ and 3′ rapid amplification of cDNA ends (RACE) that encoded a full-length sequence of 223 amino acids. The predicted protein sequence of the clone has high homology with that of the murine (93.3%), guinea pig (93.7%), and human (96%) sigma1 receptors. Northern analysis showed a major mRNA band of approximately 1.8 kb. RT-PCR revealed the presence of the mRNA in all the tissues tested, with high levels in the brain, spinal cord, liver, thymus, adrenal glands, and kidneys. When expressed in Chinese hamster ovary (CHO) cells, the level of sigma1 binding increased markedly, and the binding profile was consistent with sigma1 sites. However, measurable levels of sigma1 binding present in the cell lines before transfection made the interpretation of these results difficult. To ensure that the binding reflected the transfected protein, we tagged the receptors with a hemagglutinin (HA) epitope at the amino terminus and examined binding in immunoprecipitated receptors. Western analysis using an antisera against the HA epitope revealed a molecular weight of ∼28 kDa, close to the predicted value. The receptor binding profile of the immunopurified receptor was consistent with that seen with traditional sigma1 binding sites. Thus, rs2-2.HA encodes a high-affinity [3H](+)-pentazocine binding site with characteristics of a rat sigma1 receptor.

Introduction

Sigma receptors were originally proposed based upon the actions of the benzomorphan opiate (±)-SKF-10047 [1]. However, the pharmacology of (±)-SKF-10047 is complex and involves more than one receptor class. The sites defined as sigma today are not opioid and represent a unique type of receptor. Two subtypes of sigma receptors have been proposed based upon binding profiles, with the sigma1 receptor displaying high affinity for (+)-pentazocine and haloperidol [2]. Sigma receptors have been observed across many species and are present at high levels in the CNS, the immune system, and the liver [3], [4], [5], [6], [7]. Within the CNS, the distribution of sigma receptors is heterogeneous [6]. Pharmacologically, sigma1 receptors comprise a potent anti-opioid analgesic system [8], [9], [10], [11], [12], [13], [14]. Administered with opioid analgesics, however, sigma1 receptor agonists, such as (+)-pentazocine, have markedly diminished analgesic potency. These systems also appear to be tonically active, since sigma1 antagonists enhance opioid analgesia. Although sigma1 receptors modulate analgesia mediated through all the opioid receptors, kappa analgesics are most affected by tonic sigma1 activity. Furthermore, the level of this activity varies among strains of mice.

The sigma1 receptor was first cloned from guinea pig liver [15]. Based on the homology with the guinea pig sigma1 cDNA, human [16] and mouse [17] sigma1 receptors have also been cloned. The structure of the sigma1 receptor shows no homology with opioid receptors or other known mammalian proteins. Its predicted amino acid sequence suggests two transmembrane domains, demonstrating that it does not correspond to a traditional G-protein-coupled receptor. Understanding the molecular actions of this receptor is a major goal toward establishing its function(s). However, differences at the level of the cDNA encoding the receptor among species can complicate molecular studies on the receptor. The rat has been used extensively to evaluate the neuropharmacology of many varieties of drugs. To explore the sigma1 receptor function at a molecular level in rats, we have now cloned the rat homolog of the sigma1 receptor.

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Materials and methods

[3H](+)-Pentazocine was purchased from the New England Nuclear Corp. (+)-Pentazocine and (−)-pentazocine were gifts from the Research Technology Branch of NIDA. DTG and CHAPS were purchased from Research Biochemicals International. Anti-HA monoclonal antibody was purchased from BabCO. Fetal bovine serum was purchased from BioWhittaker. Antibiotics and other media were purchased from GIBCO. All other chemicals were purchased from the Sigma Chemical Co.

Glass fiber filters (No. 32) for receptor

Cloning of the rat sigma1 receptor

The clone isolated using RT-PCR and 5′ and 3′ RACE, rs2-2, had a predicted protein sequence of 223 amino acids and was highly homologous to that of the mouse (93.3% identity) [17], guinea pig (93.7% identity) [15], and human (96.0% identity) [16] sigma1 receptors (Fig. 1). The predicted amino acid sequence using Kyte-Doolittle analysis suggested two potential transmembrane domains, similar to the other clones and consistent with a membrane protein.

The rat sigma1 receptor is widely distributed

Discussion

Sigma1 receptors are part of a potent anti-opioid system. Sigma1 agonists greatly reduce opioid analgesia, while antagonists potentiate opioid actions by eliminating the tonic sigma receptor activity. Yet our understanding of sigma receptors remains limited. Rats are well suited to explore the pharmacological role of sigma1 receptors. To examine the role of sigma1 receptors at the molecular level, we have isolated and cloned the rat homologue of the sigma1 receptor. Its structure is closely

Acknowledgments

This work was supported, in part, from a Senior Scientist Award (DA00220), a grant from the National Institute on Drug Abuse to G.W.P. (DA06241), and a core grant from the National Cancer Institute to the Memorial Sloan-Kettering Cancer Center (CA08748).

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Abbreviations: DTG, N,N′-di(o-tolyl-guanidine); HA, hemagglutinin; RT-PCR, reverse transcriptase-polymerase chain reaction; RACE, rapid amplification of cDNA ends; and CHO, Chinese hamster ovary.

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