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Association of a Lower Molecular Weight Protein to the μ‐Opioid Receptor Demonstrated by 125 I‐β‐Endorphin Cross‐Linking Studies
Author(s) -
Law P. Y.,
Tine S. J.,
McLeod L. A.,
Loh H. H.
Publication year - 2000
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.2000.0750164.x
Subject(s) - receptor , μ opioid receptor , opioid receptor , epitope , microbiology and biotechnology , antibody , molecular mass , opioid peptide , 5 ht5a receptor , biochemistry , biology , chemistry , opioid , genetics , enzyme
: Cross‐linking experiments using the 125 I‐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 125 I‐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, 125 I‐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 125 I‐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 125 I‐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‐μ. Taken together, these results suggest that the 22‐kDa protein cross‐linked by 125 I‐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.