title: Association of a lower molecular weight protein to the mu-opioid receptor demonstrated by (125)I-beta-endorphin cross-linking studies.
abstract: Cross-linking experiments using the (125)I-beta-endorphin revealed the presence of several receptor-related species in cell lines expressing endogenous opioid receptors, including a small molecular mass protein (approximately 22 kDa). Previous reports have suggested that this 22-kDa (125)I-beta-endorphin cross-linked protein could be the degradative product from a higher molecular mass species, i.e., a fragment of the receptor. To determine if this protein is indeed a degraded receptor fragment, (125)I-beta-endorphin was cross-linked to the (His)(6) epitope-tagged mu-opioid receptor (His-mu) stably expressed in the murine neuroblastoma Neuro(2A) cells. Similar to earlier reports with cell lines expressing endogenous receptors, two major bands of 72- and 25-kDa proteins were specifically cross-linked. Initial cross-linking experiments indicated the absolute requirement of the high-affinity (125)I-beta-endorphin binding to the mu-opioid receptor prior to the appearance of the low molecular weight species, suggesting that the 22-kDa protein could be a degraded fragment of the receptor. However, variations in the ratios of these protein bands being cross-linked by several homo- or heterobifunctional cross-linking agents were observed. Although neither the carboxyl terminus mu-opioid receptor-specific antibodies nor the antibodies against the epitope at the amino terminus of the receptor could recognize the 22-kDa protein, this (125)I-beta-endorphin cross-linked species could be coimmunoprecipitated with the receptor antibodies or could be isolated with a nickel resin affinity chromatography. The direct physical association of the 22-kDa protein with the receptor was demonstrated also by the observation that the 22-kDa protein could not bind to the nickel resin alone, but that its binding to the nickel resin was restored in the presence of the His-mu. Taken together, these results suggest that the 22-kDa protein cross-linked by (125)I-beta-endorphin is not a degradative product, but a protein located within the proximity of the mu-opioid receptor, and that it is tightly associated with the receptor.
authors.affiliation: Department of Pharmacology, Medical School, University of Minnesota, Minneapolis, Minnesota, USA. ping@mail.ahc.umn.edu
authors.forename: P Y
authors.initials: PY
authors.lastname: Law
authors.forename: S J
authors.initials: SJ
authors.lastname: Tine
authors.forename: L A
authors.initials: LA
authors.lastname: McLeod
authors.forename: H H
authors.initials: HH
authors.lastname: Loh
mesh_terms: D000818:Animals; D003432:Cross-Linking Reagents; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000378:metabolism; D006639:Histidine; D007457:Iodine Radioisotopes; D051379:Mice; D008970:Molecular Weight; D009020:Morphine / Q000378:metabolism; D009447:Neuroblastoma / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D017450:Receptors, Opioid, mu / Q000378:metabolism*; D013004:Somatostatin / Q000031:analogs & derivatives / Q000378:metabolism; D014407:Tumor Cells, Cultured; D001615:beta-Endorphin / Q000378:metabolism*
chemical_list: D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D011506:Proteins; D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; C049352:phenylalanyl-cyclo(cysteinyltyrosyl-tryptophyl-ornithyl-threonyl-penicillamine)threoninamide; D006639:Histidine; D013004:Somatostatin; D001615:beta-Endorphin; D009020:Morphine