Detection and characterization of beta-adrenergic receptors and adenylate cyclase in coated vesicles isolated from bovine brain

Abstract

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)

Detection of G Proteins in Bovine Brain Clathrin Coated Vesicles with Common Alpha and Beta Subunits Antibodies.

“…The similarity in the expression pattern of the common alpha and beta subunits of G-proteins in CV to the one typical of bovine brain demonstrates that CV play a role in the subcellular transport of these guanine nucleotide regulatory proteins. The results are also consistent with studies that showed the presence of adenylate cyclase activity in brain CV (3,5,6). These findings suggest that receptors and their transduction machinery are co-transported in CV during their export or internalization in neurons.…”

“…Clathrin coated vesicles (CV) participate in the transport and recycling of hormones, cellu lar membranes and their constituent mole cules, i.e., receptors (1)(2)(3)(4)(5)(6)(7). In neuronal tis sue, CV have been shown to transport opiate, muscarinic, beta-adrenergic, alpha-adrenergic, and adenosine (Al) receptors (1,3,(5)(6)(7).…”

“…In neuronal tis sue, CV have been shown to transport opiate, muscarinic, beta-adrenergic, alpha-adrenergic, and adenosine (Al) receptors (1,3,(5)(6)(7). Some of these studies demonstrated the pres ence of receptors functionally coupled to adenylate cyclase, thus providing indirect evi dence for the presence of the transductory guanine nucleotide regulatory proteins (G-pro teins) in CV.…”

Presence of phospholipase C in coated vesicles from bovine brain Dual regulatory effects of GTP‐analogs

“…Recently, evidence for the presence of the components of the adenylate cyclase system such as receptors that mediate stimulation [16], receptors that mediate inhibition [ 17,181, the catalytic activity and Gproteins [l&19] have been described in coated vesicles. These are subcellular organelles involved in endocytotic and exocytotic protein trafficking within the cell [20].…”

Bovine brain coated vesicles contain guanine nucleotide regulatory proteins.

“…,8-Adrenergic receptors have been reported to be present in coated vesicles and supposed to be internalized by the vesicles, leading to desensitization of this receptor on the plasma membrane (2). In addition, muscarinic acetyl choline (3), opioid (4), a2 -adrenergic (5) and adenosine receptors (6) have been demon strated to be present in the coated vesicles.…”