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 cross-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 similar to that for the corresponding cross-linked products. To determine which cross-linked products in P-20 are glycoproteins, labeled membranes were solubilized and purified by wheat germ agglutinin chromatography. The absence of a similar to 36 kDa band after purification suggests that this product is not a glycoprotein. The remaining four bands were present in N-acetyl-D-glucosamine eluates, although their % distribution changes in favor of the largest molecular size band (similar to 83 kDa). Immunoblotting of the eluate gave a single diffuse band at similar to 73 kDa, suggesting the native glycoprotein has a molecular size in the 70-80 kDa range. Etorphine-induced desensitization of cell surface receptors increased the amount of some cross-linked products associated with nuclear membranes. The same treatment did not affect the relative density of the four larger molecular size bands in P-20, but increased the density of the similar to 26 kDa product two fold. Etorphine-induced down-regulation evoked an elevation of cross-linked products 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 glycosylated delta opioid receptor found in P-20.