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

Cross-linking experiments using the ¹²⁵I-β-endorphin revealed the presence of several receptor-related species in cell lines expressing endogenous opioid receptors, including a small molecular mass protein (~22 kDa). Previous reports have suggested that this 22-kDa ¹²⁵I-β-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, ¹²⁵I-β-endorphin was cross-linked to the (His)₆ epitope-tagged μ-opioid receptor (His-μ) 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 ¹²⁵I-β-endorphin binding to the μ-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 μ-opioid receptor-specific antibodies nor the antibodies against the epitope at the amino terminus of the receptor could recognize the 22-kDa protein, this ¹²⁵I-β-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-υ. Taken together, these results suggest that the 22-kDa protein cross-linked by ¹²⁵I-β-endorphin is not a degradative product, but a protein located within the proximity of the μ-opioid receptor and that it is tightly associated with the receptor.