Law P Y, Tine S J, McLeod L A, Loh H H
Department of Pharmacology, Medical School, University of Minnesota, Minneapolis, Minnesota, USA.
J Neurochem. 2000 Jul;75(1):164-73. doi: 10.1046/j.1471-4159.2000.0750164.x.
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.
使用¹²⁵I-β-内啡肽进行的交联实验表明，在表达内源性阿片受体的细胞系中存在几种与受体相关的物质，包括一种小分子质量蛋白（约22 kDa）。先前的报道表明，这种22 kDa的¹²⁵I-β-内啡肽交联蛋白可能是高分子质量物质的降解产物，即受体的一个片段。为了确定该蛋白是否确实是降解的受体片段，将¹²⁵I-β-内啡肽与在小鼠神经母细胞瘤Neuro(2A)细胞中稳定表达的(His)₆表位标记的μ-阿片受体（His-μ）进行交联。与早期关于表达内源性受体的细胞系的报道相似，72 kDa和25 kDa蛋白的两条主要条带被特异性交联。最初的交联实验表明，在低分子量物质出现之前，¹²⁵I-β-内啡肽与μ-阿片受体的高亲和力结合是绝对必需的，这表明22 kDa蛋白可能是受体的降解片段。然而，观察到几种同双功能或异双功能交联剂交联这些蛋白条带的比例存在差异。尽管羧基末端μ-阿片受体特异性抗体和针对受体氨基末端表位的抗体都不能识别22 kDa蛋白，但这种¹²⁵I-β-内啡肽交联物质可以与受体抗体共同免疫沉淀，或者可以通过镍树脂亲和色谱法分离。22 kDa蛋白与受体的直接物理关联也通过以下观察得到证明：22 kDa蛋白不能单独与镍树脂结合，但在His-μ存在的情况下其与镍树脂的结合得以恢复。综上所述，这些结果表明，¹²⁵I-β-内啡肽交联的22 kDa蛋白不是降解产物，而是位于μ-阿片受体附近的一种蛋白，并且它与受体紧密相关。
