Agonist-Induced Internalization and Recycling of the Human A 3 Adenosine Receptors: Role in Receptor Desensitization and Resensitization Maria Letizia Trincavelli, Daniela Tuscano, *Paolo Cecchetti, ²Alessandra Falleni, *Luca Benzi, ‡Karl-Norbert Klotz, ²Vittorio Gremigni, §Flaminio Cattabeni, Antonio Lucacchini, and Claudia Martini Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, *Endocrinologia e Metabolismo, Ortopedia e Traumatologia, Medicina del Lavoro, and ² Morfologia Umana e Biologia Applicata, Universita ` di Pisa, Pisa; §Istituto di Scienze Farmacologiche, Universita ` di Milano, Milano, Italia; and ‡Institut fu ¨r Pharmakologie und Toxikologie, Universita ¨t Wu ¨rzburg, Wu ¨rzburg, Germany Abstract: A 3 adenosine receptors have been proposed to play an important role in the pathophysiology of cere- bral ischemia with a regimen-dependent nature of the therapeutic effects probably related to receptor desensi- tization and down-regulation. Here we studied the ago- nist-induced internalization of human A 3 adenosine re- ceptors in transfected Chinese hamster ovary cells, and then we evaluated the relationship between internaliza- tion and signal desensitization and resensitization. Bind- ing 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 adeno- sine receptor showed a profile typical of these receptors in other cell lines (K D = 1.3  0.08 nM; 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 im- munogold electron microscopy, which revealed the local- ization 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 impli- cated in the signal resensitization. Key Words: Human A 3 adenosine receptors—Internalization—Desensitization— Resensitization. J. Neurochem. 75, 1493–1501 (2000). Extracellular purine nucleosides have well-docu- mented functions as neurotransmitters and neuromodu- lators (Phillis and Wu, 1981; Brundege and Dunwiddie, 1997). These purines, interacting with specific adenosine receptor (AR) subtypes (A 1 ,A 2A ,A 2B , and A 3 ) can act to regulate neural development, the proliferation and apo- ptosis of glial and brain capillary endothelial cells, neural plasticity, and the response to disease processes (Neary and Norenberg, 1992; Rathbone et al., 1992). Recent data suggest a trophic role for adenosine in brain repair mechanisms following trauma and brain ischemia, when it is released in large amounts by dam- aged or dying cells. In particular, the chronic activation of A 3 ARs by agonists was found to be cerebroprotective against N-methyl-D-aspartate-induced seizures in mice (von Lubitz et al., 1995) and against ischemia-associated neurodegeneration in gerbils (von Lubitz et al., 1994a, 1999). Opposite effects, i.e., enhanced mortality and extensive neuronal destruction in the hippocampus of ischemic animals, were induced by acute agonist admin- istration (von Lubitz et al., 1994a). Such regimen-depen- dent inversion of agonist-induced effects might be re- lated to adaptive changes of brain A 3 ARs (agonist- induced desensitization) in a way similar to that hypothesized for other AR subtypes (Barraco, 1991; Abbracchio et al., 1992; von Lubitz et al., 1993, 1994b). Received January 17, 2000; revised manuscript received May 17, 2000; accepted May 19, 2000. Address correspondence and reprint requests to Dr. C. Martini at Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotec- nologie, Universita ` di Pisa, Via Bonanno 6, 56126 Pisa, Italy. E-mail: cmartini@farm.unipi.it Abbreviations used: [ 125 I]AB-MECA, N 6 -(4-amino-3-[ 125 I]iodoben- zyl)adenosine-5'-N-methyluronamide; AR, adenosine receptor; CHO, Chinese hamster ovary; GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; IB-MECA, N 6 -(3-iodobenzyl)- adenosine-5'-N-methyluronamide; NECA, 5'-N-ethylcarboxamido- adenosine; PBS, phosphate-buffered saline; R-PIA, (-)-N 6 -(2-phenyl- isopropyl)adenosine. 1493 Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 2000 International Society for Neurochemistry