Association of a lower molecular weight protein to the mu-opioid receptor demonstrated by I-125-beta-endorphin cross-linking studies

Cross-linking experiments using the I-125-beta-endorphin revealed the prese nce of several receptor-related species in cell lines expressing endogenous opioid receptors, including a small molecular mass protein (similar to 22 kDa). Previous reports have suggested that this 22-kDa I-125-beta-endorphin cross-linked protein could be the degradative product from a higher molecu lar mass species, i.e., a fragment of the receptor. To determine if this pr otein is indeed a degraded receptor fragment, I-125-beta-endorphin was cros s-linked to the (His), epitope-tagged Ir-opioid receptor (His-mu) stably ex pressed in the murine neuroblastoma Neuro, cells. Similar to earlier report s with cell lines expressing endogenous receptors, two major bands of 72- a nd 25-kDa proteins were specifically cross-linked. Initial cross-linking ex periments indicated the absolute requirement of the high-affinity I-125-bet a-endorphin binding to the mu-opioid receptor prior to the appearance of th e 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 heterobifunction al cross-linking agents were observed. Although neither the carboxyl termin us mu-opioid receptor-specific antibodies nor the antibodies against the ep itope at the amino terminus of the receptor could recognize the 22-kDa prot ein, this I-125-beta-endorphin cross-linked species could be coimmunoprecip itated with the receptor antibodies or could be isolated with a nickel resi n affinity chromatography. The direct physical association of the 22-kDa pr otein with the receptor was demonstrated also by the observation that the 2 2-kDa protein could not bind to the nickel resin alone, but that its bindin g to the nickel resin was restored in the presence of the His-mu. Taken tog ether, these results suggest that the 22-kDa protein cross-linked by I-125- beta-endorphin is not a degradative product, but a protein located within t he proximity of the mu-opioid receptor, and that it is tightly associated w ith the receptor.