Requirement of receptor internalization for opioid stimulation of mitogen-activated protein kinase: biochemical and immunofluorescence confocal microscopic evidence.

Previously, we implicated the opioid receptor (OR), Gbetagamma subunits, and Ras in the opioid activation of extracellular signal-regulated protein kinase (ERK), a member of the mitogen-activated protein (MAP) kinase family involved in mitogenic signaling. We now report that OR endocytosis also plays a role in the opioid stimulation of ERK activity. COS-7 and HEK-293 cells were cotransfected with the cDNA of delta-, mu;-, or kappa-OR, dynamin wild-type (DWT), or the dominant suppressor mutant dynamin K44A, which blocks receptor endocytosis. The activation of ERK by opioid agonists in the presence of DWT was detected. In contrast, parallel ectopic coexpression of the K44A mutant with OR, followed by agonist treatment, resulted in a time-dependent attenuation of ERK activation. Immunofluorescence confocal microscopy of delta-OR and DWT-cotransfected COS-7 cells revealed that agonist exposure for 10 min resulted in an ablation of cell surface delta-OR immunoreactivity (IR) and an intensification of cytoplasmic (presumably endosomal) staining as seen in the absence of overexpressed DWT. After 1 hr of delta-agonist exposure the cells displayed substantial internalization of delta-OR IR. If the cells were cotransfected with delta-OR and dynamin mutant K44A, OR IR was retained on the cell surface even after 1 hr of delta-agonist treatment. Parallel immunofluorescence confocal microscopy, using an anti-ERK antibody, showed that agonist-induced time-dependent ERK IR trafficking into perinuclear and nuclear loci was impaired in the internalization-defective cells. Thus, both biochemical and immunofluorescence confocal microscopic evidence supports the hypothesis that the opioid activation of ERK requires receptor internalization in transfected mammalian cells.

Agonist-induced desensitization and down-regulation of delta opioid receptors alter the levels of their 125I-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. 125I-beta-Endorphin was cross-linked to preparations enriched in plasma membranes (P20), nuclear membranes or nuclear matrices. Five cross-linked bands appear in all subcellular fractions. The largest molecular size reaction product in nuclear matrix preparations (approximately 72 kDa) differed from that in the other two fractions-(approximately 83 kDa). Immunoblot analyses with an antibody to the delta opioid receptor gave a P20 band pattern similar to that for the corresponding cross-linked products. To determine which cross-linked products in P20 are glycoproteins, labeled membranes were solubilized and purified by wheat germ agglutinin chromatography. The absence of a approximately 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 (approximately 83 kDa). Immunoblotting of the eluate gave a single diffuse band at approximately 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 P20, but increased the density of the approximately 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 P20 were diminished. These results suggest that nuclear matrix associated opioid binding sites represent internalized, truncated forms of the glycosylated delta opioid receptor found in P20.

Buprenorphine differentially alters opioid receptor adaptation in rat brain regions.

Previous in vivo studies revealed that the mixed agonist-antagonist buprenorphine can down-regulate mu and up-regulate delta 2 and kappa 1 opioid receptors in rat brain. In this report brain regional differences in opioid receptor adaptation were addressed. Rats received i.p. injections with buprenorphine (0.5-2.5 mg/kg) and were killed 20 h later. Membranes from 7 brain regions were analyzed for mu (3H-[D-Ala2,N-mephe4,Gly-ol5] enkephalin), kappa 1 (3H-U-69593), delta 1 (3H-[D-Pen2, D-Pen5] enkephalin) and delta 2 (3H-deltorphin II) receptor binding parameters. Buprenorphine induced down-regulation of mu receptors in frontal cortex, occipital cortex, thalamus, hippocampus, striatum and brain stem. Kd values for 3H-[D-Ala2,N-mephe4,Gly-ol5] enkephalin were unchanged from controls. Up-regulation of kappa 1 receptors was observed in frontal, parietal, occipital cortexes and striatum. Binding to delta 2 sites was elevated in frontal and parietal cortexes. Buprenorphine did not alter delta 1 binding in any of the regions examined. Changes in opioid receptor adaptation induced by buprenorphine were further supported by data from cross-linking of 125I-beta-endorphin to cortical membrane preparations. A reduction in a 60- to 65-kDa band was detected in frontal and occipital cortices in which binding assays revealed down-regulation of mu receptors. In parietal cortex neither the 60- to 65-kDa product nor Bmax changes were observed. These results indicate that buprenorphine is a useful tool to study brain opioid receptor adaptation in vivo and the information accrued may be relevant to the mode of action of this drug in the treatment of heroin and cocaine abuse.