To assess whether internalization of beta-adrenergic receptor occurs in the CNS, we have isolated clathrin-coated vesicles from bovine forebrain and examined them for the presence of beta-adrenergic receptor binding and adenylate cyclase activities. A coated vesicle enriched preparation isolated by successive D2O-Ficoll density gradient centrifugations was applied to a glass bead permeation column to achieve further purification. Two major peaks of protein were eluted from the column and monitored by electron microscopy and SDS-PAGE. Peak II contained almost exclusively coated vesicles (98%), whereas peak I, which appeared in the void volume, contained larger smooth vesicles and few coated vesicles. beta-Adrenergic receptor binding to peaks I and II was measured with 125I-cyanopindolol (CYP) as ligand in Sepharose 4B column assays. 125I-CYP was found to bind specifically and saturably to both peaks I and II with a Bmax of 28 +/- 4 and 32 +/- 3 fmol/mg protein, respectively. 3H-CGP 12177, a hydrophilic beta-adrenergic receptor ligand, did not label receptors present in peak II, but it specifically bound to synaptic plasma membranes (SPM) prepared from bovine hippocampus and, to a lesser extent, to peak I. These results suggest that receptors present in coated vesicles are cryptic in nature. In the displacement of 125I-CYP binding by (-)-isoproterenol, addition of 50 microM GppNHp caused a significant "right shift" with SPM and peak I but not the peak II preparation. Adenylate cyclase activities could also be detected in both peaks I and II (specific activities, 21 +/- 0.6 and 24 +/- 0.5 pmol cAMP/mg protein/min, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

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.