Molecular identification and functional expression of mu 3, a novel alternatively spliced variant of the human mu opiate receptor gene.
Studies from our laboratory have revealed a novel mu opiate receptor, mu 3, which is expressed in both vascular tissues and leukocytes. The mu 3 receptor is selective for opiate alkaloids and is insensitive to opioid peptides. We now identify the mu 3 receptor at the molecular level using a 441-bp conserved region of the mu 1 receptor. Sequence analysis of the isolated cDNA suggests that it is a novel, alternatively spliced variant of the mu opiate receptor gene. To determine whether protein expressed from this cDNA exhibits the biochemical characteristics expected of the mu 3 receptor, the cDNA clone was expressed in a heterologous system. At the functional level, COS-1 cells transfected with the mu 3 receptor cDNA exhibited dose-dependent release of NO following treatment with morphine, but not opioid peptides (i.e., Met-enkephalin). Naloxone was able to block the effect of morphine on COS-1 transfected cells. Nontransfected COS-1 cells did not produce NO in the presence of morphine or the opioid peptides at similar concentrations. Receptor binding analysis with [(3)H]dihydromorphine further supports the opiate alkaloid selectivity and opioid peptide insensitivity of this receptor. These data suggest that this new mu opiate receptor cDNA encodes the mu 3 opiate receptor, since it exhibits biochemical characteristics known to be unique to this receptor (opiate alkaloid selective and opioid peptide insensitive). Furthermore, using Northern blot, RT-PCR, and sequence analysis, we have demonstrated the expression of this new mu variant in human vascular tissue, mononuclear cells, polymorphonuclear cells, and human neuroblastoma cells.
Neuroscience Research Institute, State University of New York, Old Westbury, NY 11568, USA.
Patrick
P
Cadet
Kirk J
KJ
Mantione
George B
GB
Stefano
D017398:Alternative Splicing / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive / Q000235:genetics; D015152:Blotting, Northern; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D015870:Gene Expression / Q000502:physiology; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D009292:Narcotic Antagonists / Q000378:metabolism / Q000494:pharmacology; D009928:Organ Specificity / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D017450:Receptors, Opioid, mu / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D009292:Narcotic Antagonists; C485764:OPRM1 protein, human; D020033:Protein Isoforms; D017450:Receptors, Opioid, mu