75 Molecular and Cellular Biochemistry 206: 75–89, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands. Differential agonist-induced desensitization of P2Y 2 nucleotide receptors by ATP and UTP Betty Velázquez, 1 Richard C. Garrad, 2 Gary A. Weisman 2 and Fernando A. González 1 1 Department of Chemistry, University of Puerto Rico, Rio Piedras Campus, San Juan, Puerto Rico; 2 Department of Biochemistry, University of Missouri-Columbia, Columbia, MO, USA Received 28 June 1999; accepted 4 November 1999 Abstract The equal potency and efficacy of the agonists, ATP and UTP, pharmacologically distinguish the P2Y 2 receptor from other nucleotide receptors. Investigation of the desensitization of the P2Y 2 receptors is complicated by the simultaneous expression of different P2 nucleotide receptor subtypes. The co-expression of multiple P2 receptor subtypes in mammalian cells may have led to contradictory reports on the efficacy of the natural agonists of the P2Y 2 receptor to induce desensitization. We decided to investigate the desensitization of human and murine isoforms of the P2Y 2 receptor, and to rigorously examine their signaling and desensitization properties. For these purposes, we used 1321N1 astrocytoma cells stably transfected with the human or murine P2Y 2 receptor cDNA, as well as human A431 cells that endogenously express the receptor. The mobilization of intracellular calcium by extracellular nucleotides was used as a functional assay for the P2Y 2 receptors. While ATP and UTP activated the murine and human P2Y 2 receptors with similar potencies (EC 50 values were 1.5–5.8 μM), ATP was ~ 10-fold less potent (IC 50 = 9.1–21.2 μM) than UTP (IC 50 = 0.7–2.9 μM) inducing homologous receptor desensitization in the cell systems examined. Individual cell analyses of the rate and dose dependency of agonist-induced desensitization demonstrated that the murine receptor was slightly more resistant to desensitization than its human counterpart. To our knowledge, this is the first individual cell study that has compared the cellular heterogeneity of the desensitized states of recombinant and endogenously expressed receptors. This comparison demonstrated that the recombinant system conserved the cellular regulatory elements needed to attenuate receptor signaling by desensitization. (Mol Cell Biochem 206: 75–89, 2000) Key words: P2 nucleotide receptors, intracellular calcium mobilization, receptor desensitization, individual cell measurements Introduction P2Y nucleotide receptors are a family of G protein-coupled receptors that participate in a variety of biological processes such as vasodilatation, aggregation of platelets, and cell growth among others [1]. To date, six subtypes of P2Y receptors have been cloned and functionally expressed [2]. The P2Y 2 receptor, formerly termed P 2U purinoceptor, is the sole subtype of the nucleotide receptor family that can be activated with equal potency and efficacy by ATP and UTP [3, 4]. Upon stimulation by agonist, P2Y 2 receptors activate phospholipase C and therefore increase the production of inositol phosphates and cause an increase in the con- centration of intracellular free calcium ([Ca 2+ ] i ) [3]. Desensitization is a physiological process by which receptor systems become unresponsive to agonists following prior exposure. Little is known about the mechanisms for P2Y 2 receptor desensitization, although various reports have described the phenomenon [5–11]. Nucleotides (acting through the P2Y 2 nucleotide receptor) have been shown to induce Cl – secretion in airway epithelial cells that is non-CFTR (cystic fibrosis transmembrane-conductance regulator)- dependent [12–14]. This observation raised the possibility that P2Y 2 receptor agonists could be used in the treatment of Address for offprints: F.A. Gonzalez, Department of Chemistry, University of Puerto Rico, P.O. Box 23346, San Juan, PR 00931-3346