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Agonist‐Induced Internalization and Recycling of the Human A 3 Adenosine Receptors
Author(s) -
Trincavelli Maria Letizia,
Tuscano Daniela,
Cecchetti Paolo,
Falleni Alessandra,
Benzi Luca,
Klotz KarlNorbert,
Gremigni Vittorio,
Cattabeni Flaminio,
Lucacchini Antonio,
Martini Claudia
Publication year - 2000
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.2000.0751493.x
Subject(s) - internalization , receptor , agonist , adenosine receptor , adenosine , homologous desensitization , chinese hamster ovary cell , adenylyl cyclase , adenosine a3 receptor , adenosine a1 receptor , biology , g protein coupled receptor , microbiology and biotechnology , adenosine a2b receptor , endocrinology , chemistry , medicine , biochemistry
A 3 adenosine receptors have been proposed to play an important role in the pathophysiology of cerebral ischemia with a regimen‐dependent nature of the therapeutic effects probably related to receptor desensitization and down‐regulation. Here we studied the agonist‐induced internalization of human A 3 adenosine receptors in transfected Chinese hamster ovary cells, and then we evaluated the relationship between internalization and signal desensitization and resensitization. Binding of N 6 ‐(4‐amino‐3‐[ 125 I]iodobenzyl)adenosine‐5′‐ N ‐methyluronamide to membranes from Chinese hamster ovary cells stably transfected with the human A 3 adenosine receptor showed a profile typical of these receptors in other cell lines ( K D = 1.3 ± 0.08 n M ; B max = 400 ± 28 fmol/mg of proteins). The iodinated agonist, bound at 4°C to whole transfected cells, was internalized by increasing the temperature to 37°C with a rate constant of 0.04 ± 0.034 min ‐1 . Agonist‐induced internalization of A 3 adenosine receptors was directly demonstrated by immunogold electron microscopy, which revealed the localization of these receptors in plasma membranes and intracellular vesicles. Moreover, short‐term exposure of these cells to the agonist caused rapid desensitization as tested in adenylyl cyclase assays. Subsequent removal of the agonist led to restoration of the receptor function and recycling of the receptors to the cell surface. The rate constant of receptor recycling was 0.02 ± 0.0017 min ‐1 . Blockade of internalization and recycling demonstrated that internalization did not affect signal desensitization, whereas recycling of internalized receptors was implicated in the signal resensitization.