AGONIST-INDUCED DESENSITIZATION AND DOWN-REGULATION OF DELTA-OPIOID RECEPTORS ALTER THE LEVELS OF THEIR I-125 BETA-ENDORPHIN CROSS-LINKED PRODUCTS IN SUBCELLULAR-FRACTIONS FROM NG108-15 CELLS

The delta opioid binding sites in subcellular fractions from NG108-15 cells were characterized with respect to their relative molecular size and levels under conditions of receptor adaptation. Endorphin was cro ss-linked to preparations enriched in plasma membranes (P-20), nuclear membranes or nuclear matrices. Five cross-linked bands appear in all subcellular fractions. The largest molecular size reaction product in nuclear matrix preparations (similar to 72 kDa) differed from that in the other two fractions (similar to 83 kDa). Immunoblot analyses with an antibody to the delta, opioid receptor gave a P-20 band pattern sim ilar to that for the corresponding cross-linked products. To determine which cross-linked products in P-20 are glycoproteins, labeled membra nes were solubilized and purified by wheat germ agglutinin chromatogra phy. The absence of a similar to 36 kDa band after purification sugges ts that this product is not a glycoprotein. The remaining four bands w ere present in N-acetyl-D-glucosamine eluates, although their % distri bution changes in favor of the largest molecular size band (similar to 83 kDa). Immunoblotting of the eluate gave a single diffuse band at s imilar to 73 kDa, suggesting the native glycoprotein has a molecular s ize in the 70-80 kDa range. Etorphine-induced desensitization of cell surface receptors increased the amount of some cross-linked products a ssociated with nuclear membranes. The same treatment did not affect th e relative density of the four larger molecular size bands in P-20, bu t increased the density of the similar to 26 kDa product two fold. Eto rphine-induced down-regulation evoked an elevation of cross-linked pro ducts in nuclear matrix preparations, while all band densities of P-20 were diminished. These results suggest that nuclear matrix associated opioid binding sites represent internalized, truncated forms of the g lycosylated delta opioid receptor found in P-20.