Stereospecific opiate-binding sites occur in coated vesicles

Abstract

We prepared clathrin-coated vesicles from bovine forebrain utilizing sucrose or deuterium oxide-Ficoll density gradient centrifugation followed by permeation chromatography. Homogeneity was monitored by electron microscopy (EM) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). EM revealed that the predominant (up to 98% of the total) organelles were coated vesicles and empty hexagonal baskets. Diameters of the coated vesicles ranged from 37 to 120 nm with a mean of 65.2 +/- 2.2. Upon SDS-PAGE of the coated vesicle fraction, the most prominent band appeared at 180,000 daltons. There were also three additional bands at 100,000, 50,000 and 35,000 daltons, giving the overall pattern characteristic of coated vesicles. Both 0.5 nM tritiated naltrexone and etorphine displayed specific binding to coated vesicles. Naltrexone binding in coated vesicles from gradient fractions was increased 2.5-fold over the original 100,000 X g pellet. An additional 4-fold enrichment in specific binding was observed after permeation chromatography which was concomitant with an increase in the volume density of coated vesicles in electron micrographs. Naltrexone binding was stereospecific and etorphine binding was inhibited by 100 mM NaCl (40%). Both naltrexone and etorphine binding were inhibited by 50 microM guanyl-5'-yl imidodiphosphate (40 to 50%). In summary, purified bovine brain-coated vesicles contained high affinity stereospecific opiate alkaloid-binding sites with characteristic opioid binding properties.

The Interaction of a Constitutively Active Arrestin with the Arrestin-Insensitive 5-HT2A Receptor Induces Agonist-Independent Internalization

“…Arrestin binding also promotes the targeting of desensitized receptors to clathrin-coated pits for their internalization (Ferguson, 2001). This well-documented targeting of GPCRs to the clathrin-coated pit/endosome pathway (Roth et al, 1981;Bennett et al, 1985;Chuang et al, 1986;von Zastrow and Kobilka, 1992) is critical because it allows for the resensitization and recycling of receptors (Yu et al, 1993;Pippig et al, 1995) and may be involved in down-regulation (Gray and Roth, 2002).…”

Detection and characterization of 3H-clonidine binding sites in coated vesicles isolated from bovine brain.

“…9-Adrenergic receptors have been reported to be present in coated vesicles and supposed to be inter nalized by the vesicles, resulting in desensi tization of this receptor on the plasma mem brane (4). In addition, muscarinic acetyl choline (5) and opioid receptors (6) were shown to be present in coated vesicles. a2 Adrenergic receptors (a2-receptors) are in volved in the control of a number of important physiological processes and suggested to relate to an etiology of depression (7,8).…”

Opioid Receptors of Neuroblastoma Cells Are in Two Domains of the Plasma Membrane that Differ in Content of G Proteins

“…Because a redistribution of these receptors from the heavy to the light membrane fraction has been observed during desensitization, it was suggested that receptors in the light membrane fraction represent a population of desensitized receptors that are seques-tered away from the cell surface, probably by internalization Waldo et al, 1983;Sibley and Lefkowitz, 1985). For opioid receptors, Law et al ( 1984) have demonstrated internalization into endosomes after exposure of NG 108-15 cells to an opioid agonist, and Bennett et al (1985) found opioid receptor binding in clathrin-coated vesicles isolated from bovine brain. This can be taken as an indication that opioid receptors in the light membrane fraction are internalized receptors.…”