Agonist‐Induced Internalization and Recycling of the Human A₃Adenosine Receptors

Abstract

Abstract: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-5Ј-Nmethyluronamide 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 ϭ 1.3 Ϯ 0.08 nM; B max ϭ 400 Ϯ 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 Ϯ 0.034 min Ϫ1 . 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 Ϯ 0.0017 min Ϫ1 .

Mechanisms of desensitization of the adrenocorticotropin response to arginine vasopressin in ovine anterior pituitary cells

“…It is of interest that, at the concentration used, ConA only partially inhibited the ability of AVP pretreatment to induce desensitization. This concentration (0·25 mg/ml) has been shown, in some instances, to inhibit completely internalization of a variety of GPCRs (Pippig et al 1995), while in others it only partially inhibited internalization (Trincavelli et al 2000). If receptor internalization was completely inhibited by ConA in the perifused anterior pituitary cell system used in these experiments, the remaining desensitization must have been the result of another desensitization process or processes.…”

“…The involvement of receptor internalization in the regulation of the ACTH response to AVP was investigated with the lectin concanavalin A (ConA) to block receptor internalization (Pippig et al 1995, Trincavelli et al 2000. The experimental protocol employed was based on that which we have previously used to investigate resensitization of the ACTH response to AVP (Hassan et al 2003).…”

Role of adenosine A3 receptors on CA1 hippocampal neurotransmission during oxygen–glucose deprivation episodes of different duration

“…The adenosine A 3 receptor is particularly susceptible to phosphorylation by G protein-coupled receptor kinases [17,50]. Studies using CHO cells stably expressing the A 3 receptors have demonstrated that, following a few minutes of exposure to the agonist, there is rapid phosphorylation of the A 3 receptor, a reduction in number of A 3 high affinity agonist binding sites [16], and receptor internalization [19]. In human astrocytoma cells, brief exposure (1-60 min) to nanomolar concentrations of Cl-IB-MECA results in a rapid uncoupling of A 3 receptors and receptor internalization while a long-lasting (1-24 h) Cl-IB-MECA treatment leads to receptor down-regulation which recovers after several hours [20].…”

“…This effect may be attributable to desensitization of A 3 receptors. In fact, both human and rat A 3 receptors are desensitized within a few minutes after agonist exposure [16][17][18][19][20][21].…”

Translocation of arrestin induced by human A3 adenosine receptor ligands in an engineered cell line: Comparison with G protein-dependent pathways

“…The OD values were converted to actual pERK concentrations (pg/ml) based on the standard curve of the recombinant pERK standards provided in the kit. For the desensitization studies [20,21], cells were incubated with agonists at 37°C for 24 h. The medium was then replaced with agonist-free medium and cells were treated with an A 3 AR agonist MRS3558 at various time points.…”