Association of a Lower Molecular Weight Protein to the mu -Opioid Receptor Demonstrated by super(125)I- beta -Endorphin Cross-Linking Studies
Cross-linking experiments using the super(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 ( similar to 22 kDa). Previous reports have suggested that this 22-kDa super(125)I...
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Veröffentlicht in: | Journal of neurochemistry 2000-07, Vol.75 (1), p.164-173 |
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Zusammenfassung: | Cross-linking experiments using the super(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 ( similar to 22 kDa). Previous reports have suggested that this 22-kDa super(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, super(125)I- beta -endorphin was cross-linked to the (His) sub(6) epitope-tagged mu -opioid receptor (His- mu ) stably expressed in the murine neuroblastoma Neuro sub(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 super(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 super(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 super(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. |
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ISSN: | 0022-3042 |
DOI: | 10.1046/j.1471-4159.2000.0750164.x |