| Abstract: |
Our previous studies identified at least fifteen exons in the mouse Oprm gene, which spans 250 kb and whose combinations generate sixteen alternatively spliced variants. In the present studies, we report the isolation and characterization of four additional variants from the mouse Oprm gene. To obtain the mouse homolog of rat MOR-1B previously reported, we first isolated the mouse exon 5 from a BAC genomic clone containing the Oprm gene with the rat exon 5 as a probe. Using a RT-PCR approach with the primers from the mouse exon 5, we isolated mouse MOR-1B (mMOR-1B1) and other four new variants, mMOR-1B2, mMOR-1B3, mMOR-1B4 and mMOR-1B5. All the variants contained the same exons 1, 2 and 3 as the original mMOR-1, but with a different fourth exons that derived from alternative splicing within exon 5. Similar to the other C-terminal variants, these new variants contained a distinct carboxyl tail ranging from two to thirty-nine amino acids. Thus now there were total twelve functional variants in the mouse Oprm gene which were generated from alternative splicing downstream of exon 3. Expression of the variant mRNAs in mouse brain was examined by Northern blot with the exon 5 probes. We also explored the pharmacology of the variants by using opioid binding in CHO cells stably transfected with the variant cDNAs. All the variants except mMOR-1B4 revealed binding profiles similar to those of the original mMOR-1. Although mMOR-1B4 bound antagonists such as CTAP, diprenorphine and naloxone with high affinity, mu agonists, such as DAMGO and morphine, displayed markedly lower affinities for the expressed receptor. Immunohistochemical distribution of mMOR-1B4 was also examined using an antibody raised against an specific sequence from MOR-1B4. |
