Generation of the mu opioid receptor (MOR-1) protein by three new splice variants of the Oprm gene.

Using 5' RACE, we have isolated four additional exons of the mu opioid receptor gene (Oprm), resulting in a gene spanning over 250 kb. The four new exons are contained within eight additional splice variants containing exon 11 at the 5' terminus. Exon 11, which is under the control of a previously unknown upstream promoter, and exon 12 are located approximately 10 kb and approximately 8 kb upstream from exon 1, respectively. Exon 13 and 14 are located between exons 1 and 2. The regional distributions of the variants, as determined by reverse transcription-PCR, varied among themselves and were distinct from that of MOR-1, implying region-specific RNA processing. Three variants (MOR-1H, MOR-1I, and MOR-1J) contained two potential translational start points, with the translational start point in exon 1 producing proteins identical to the original MOR-1 protein. When expressed, the receptor binding of these three variants was indistinguishable from that of MOR-1. The remaining eight proteins using the translation start point in exon 11 were all truncated, with three (MOR-1G, MOR-1M, and MOR-1N) predicting proteins of only six transmembrane domains and the rest giving proteins under 10 kDa. Western blots with an exon 11-specific antiserum revealed bands consistent with the six transmembrane domain proteins within the brain, but the shorter proteins were not detected. Thus, the MOR-1 protein can be generated by four different splice variants of the Oprm gene under the control of two physically distinct promoters. Although the truncated proteins are expressed in brain with a unique regional distribution, their functional significance remains unknown.

Isolation and expression of a novel alternatively spliced mu opioid receptor isoform, MOR-1F.

The MOR-1 gene is large, with a recent study reporting nine exons spanning 250 kb which combine to yield six different mu opioid receptor splice variants. We now report the isolation of exon 10, which is contained within yet another splice variant, MOR-1F, which is composed of exons 1, 2, 3, 10, 6, 7, 8 and 9. Exon 10 comprises 186 bp which predict a unique 58 amino acid sequence extending beyond exon 3. It has been mapped between exons 4 and 6 and has flanking consensus splice sequences. On Northern blot analysis, the MOR-1F mRNA is smaller than the other MOR-1 mRNAs. When expressed in CHO cells, MOR-1F binds the mu opioid radioligand [3H]DAMGO with high affinity (K(D) = 1.04+/-0.03 nM). Competition studies demonstrated the selectivity of the variant for mu opioid ligands, supporting its classification within the mu opioid receptor family.