| 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. © 2001 Elsevier Science Inc. |
