Bovine Brain Coated Vesicles Contain Adenosine A₁ Receptors. Presence of Adenylate Cyclase Coupled to the Receptor

Abstract

Clathrin-coated vesicles purified from bovine brain express adenosine A1 receptor binding activity. N6-Cyclohexyl[3H]adenosine [( 3H]CHA), an agonist for the A1 receptor, binds specifically to coated vesicles. High and low agonist affinity states of the receptor for the radioligand [3H]CHA with KD values of 0.18 and 4.4 nM, respectively, were detected. The high purity of coated vesicles was established by assays for biochemical markers and by electron microscopy. Binding competition experiments using agonists (N6CHA, N-cyclopentyladenosine, 5'-(N-ethylcarboxamido)adenosine, and N6-[(R)- and N6-[(S)-phenylisopropyl]adenosine) and antagonists (theophylline, 3-isobutyl-1-methylxanthine, and caffeine) confirmed the typical adenosine A1 nature of the binding site. This binding site presents stereospecificity for N6-phenylisopropyladenosine, showing 33 times more affinity for N6-[(R)- than for N6-[(S)-phenylisopropyl]adenosine. The specific binding of [3H]CHA in coated vesicles is regulated by guanine nucleotides. [3H]CHA specific binding was decreased by 70% in the presence of the hydrolysis-resistant GTP analogue guanyl-5-yl-imidodiphosphate. Bovine brain coated vesicles present adenylate cyclase activity. This activity was modulated by forskolin and CHA. The results of this study support the evidence that adenosine A1 receptors present in coated vesicles are coupled to adenylate cyclase activity through a Gi protein.

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.…”

“…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. The recent development and availability of antisera of designed specificity for the alpha and beta subunits of G-proteins permits their detection and characterization (5,(8)(9)(10)(11)(12)(13). Taking advantage of the analytical power of Western blots, we have aimed in this study to provide direct immunochemical evi dence for the presence of two of the main sub units of the heterotrimeric G protein com plexes in CV from bovine brain.…”

“…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.…”

“…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).…”

Bovine brain coated vesicles contain guanine nucleotide regulatory proteins.

“…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. Some of the receptors in the coated vesicles are suggested to be associated with adenylate cyclase via guanine nucleotide regulatory pro teins (G-proteins) (3,6). No direct evidence, however, has been obtained for the presence of G-proteins in the vesicles.…”