G-protein-coupled A1 adenosine receptors in coated vesicles of mammalian
brain: characterization by radioligand binding and photoaffinity
labelling
Cell Signal, 4 (6):
737-45 (November 1992)Gonzalez-Calero, G Cubero, A Klotz, K N In Vitro Research Support,
Non-U.S. Gov't England Cellular signalling Cell Signal. 1992 Nov;4(6):737-45..Zusammenfassung
A1 adenosine receptors in coated vesicles have been characterized
by radioligand binding and photoaffinity labelling. Saturation experiments
with the antagonist 8-cyclopentyl-1,3-3Hdipropyl-xanthine (3HDPCPX)
gave a Kd value of 0.7 nM and a Bmax value of 82 +/- 13 fmol/mg protein.
For the highly A1-selective agonist 2-chloro-N6-3Hcyclopentyladenosine
(3HCCPA) a Kd value of 1.7 nM and a Bmax value of 72 +/- 29 fmol/mg
protein was estimated. Competition of agonists for 3HDPCPX binding
gave a pharmacological profile with R-N6-phenylisopropyladenosine
(R-PIA) > CCPA > S-PIA > 5'-N-ethylcarboxamidoadenosine (NECA), which
is identical to brain membranes. The competition curves were best
fitted according to a two-site model, suggesting the existence of
two affinity states. GTP shifted the competition curve for CCPA to
the right and only one affinity state similar to the low affinity
state in the absence of GTP was detected. The photoreactive agonist
2-azido-N6-125I-p-hydroxyphenylisopropyladenosine (125IAHPIA) specifically
labelled a single protein with an apparent molecular weight of 35,000
in coated vesicles, which is identical to A1 receptors labelled in
brain membranes. Therefore, coated vesicles contain A1 adenosine
receptors with similar binding characteristics as membrane-bound
receptors, including GTP-sensitive high-affinity agonist binding.
Photoaffinity labelling data suggest that A1 receptors in these vesicles
are not a processed receptor form. These results confirm that A1
receptors in coated vesicles are coupled to a G-protein, and it appears
that the A1 receptor systems in coated vesicles and in plasma membranes
are identical.