Abstract
The sigma1 receptor acts as a chaperone at the endoplasmic reticulum, associates with multiple proteins in various cellular systems, and involves in a number of diseases, such as addiction, pain, cancer and psychiatric disorders. The sigma1 receptor is encoded by the single copy SIGMAR1 gene. The current study identifies five alternatively spliced variants of the mouse sigma1 receptor gene using a polymerase chain reaction cloning approach. All the splice variants are generated by exon skipping or alternative 3’ or 5’ splicing, producing the truncated sigma1 receptor. Similar alternative splicing has been observed in the human SIGMAR1 gene based on the molecular cloning or genome sequence prediction, suggesting conservation of alternative splicing of SIGMAR1 gene. Using quantitative polymerase chain reactions, we demonstrate differential expression of several splice variants in mouse tissues and brain regions. When expressed in HEK293 cells, all the splice variants fail to bind sigma ligands, implicating that each truncated region in these splice variants is important for ligand binding. However, co-immunoprecipitation (Co-IP) study in HEK293 cells co-transfected with tagged constructs reveals that all the splice variants maintain their ability to physically associate with a mu opioid receptor (mMOR-1), providing useful information to correlate the motifs/sequences necessary for their physical association. Furthermore, a competition Co-IP study showed that all the variants can disrupt in a dose-dependent manner the dimerization of the original sigma1 receptor with mMOR-1, suggesting a potential dominant negative function and providing significant insights into their function.
Highlights
Sigma receptors were initially proposed following the pharmacological studies with the benzomorphan opiate (±)SKF-10047 [1]
To isolate the mouse Sigmar1 splice variants, we used a PCR approach with an upstream sense primer in the first exon and a downstream antisense primer in the fourth exon. This led to the identification of five splice variants, mouse sigma1 receptor 1a (mSIG-1A), mSIG-1B, mSIG-1C, mSIG-1D and mSIG-1E (Figs 1 & 2). mSIG-1A and mSIG-1E are exon skipping variants that skip exon 3 and exon 2, respectively
Splicing from exon 3 to exon 4b in mSIG-1C leads to truncation of 38 amino acids encoded by exon 4a. mSIG-1D is a splice variant derived from combination of alternative 5’ splicing in exon 1, exon 2 skipping and alternative 3’ splicing in exon 3
Summary
Sigma receptors were initially proposed following the pharmacological studies with the benzomorphan opiate (±)SKF-10047 [1]. Initially thought to be related to opioid receptors, the sigma receptor is recognized as a distinct protein unrelated to any traditional classes of receptor, including opioid receptors [2, 3]. Alternative pre-mRNA splicing of sigma receptor gene, Sigmar and The Experimental Therapeutics Center of Memorial Sloan Kettering Cancer Center. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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