Agonist-induced internalization and recycling of the human A(3) adenosine receptors: Role in receptor desensitization and resensitization

A(3) adenosine receptors have been proposed to play an important role in th e pathophysiology of cerebral ischemia with a regimen-dependent nature of t he therapeutic effects probably related to receptor desensitization and dow n-regulation. Here we studied the agonist-induced internalization of human A(3) adenosine receptors in transfected Chinese hamster ovary cells, and th en we evaluated the relationship between internalization and signal desensi tization and resensitization, Binding of N-6-(4-amino-3-[I-125]iodobenzyl)a denosine-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 nM; B-m ax = 400 +/- 28 fmol/mg of proteins). The iodinated agonist, bound at 4 deg rees C to whole transfected cells, was internalized by increasing the tempe rature to 37 degrees C with a rate constant of 0.04 +/- 0.034 min(-1). Agon ist-induced internalization of A(3) adenosine receptors was directly demons trated by immunogold electron microscopy, which revealed the localization o f these receptors in plasma membranes and intracellular vesicles. Moreover, short-term exposure of these cells to the agonist caused rapid desensitiza tion as tested in adenylyl cyclase assays, Subsequent removal of the agonis t led to restoration of the receptor function and recycling of the receptor s to the cell surface. The rate constant of receptor recycling was 0.02 +/- 0.0017 min(-1). Blockade of internalization and recycling demonstrated tha t internalization did not affect signal desensitization, whereas recycling of internalized receptors was implicated in the signal resensitization.