Gary A. Weisman

The P2 Nucleotide Receptors

Part I. Historical Perspectives of P2 Nucleotide Receptors. History of Extracellular Nucleotides and Their Receptors, G. Burnstock. Part II. Pharmacological and Molecular Characterization of P2 Nucleotide Receptors. P2x Receptors: Cloned and Expressed, R. J. Evans, A. Surprenant, and R. A. North. The Cloning and Expression of G Protein-Coupled P2Y Nucleotide Receptors, G. A. Weisman, L. Erb, R. C. Garrad, J. T. Turner, and F. A. Gonzalez. Structure Activity Relationships of P2 Receptor Agonists and Antagonists, K. A. Jacobson, Y.-C. Kim, E. Camaioni, and A. M. van Rhee. Pharmacological Selectivities of Molecularly Defined Subtypes of P2Y Receptors, T. K. Harden, R. A. Nicholas, J. R. Schachter, E. R. Lazarowski, and J. L. Boyer. P2 Receptor Modeling and Identification of Ligand Binding Sites, A. M. van Rhee. Part III. Mechanisms of P2 Nucleotide Receptor Action. Nucleotide Receptors Coupling to the Phospholipase C Signaling Pathway, J.-M. Boeynaems, D. Communi, R. Janssens, S. Motte, B. Robaye, and S. Pirotton. Cyclic AMP and Tyrosine Kinase Cascades in the Regulation of Cellular Function by P2Y Nucleotide Receptors, M. R. Boarder. Characteristics of Ligand-Gated Ion Channel P2 Nucleotide Receptors, C. Kennedy, S. E. Hickman, and S. C. Silverstein. Cell Responses Initiated by Ecto-kinases, J. S. Fedan. Part IV. Physiological Roles for P2 Nucleotide Receptors. Functions of Extracellular Nucleotides in Peripheral and Central Neuronal Tissues, E. M. Silinsky, I. von Kugelgen, A. D. Smith and D. P. Westfall. The Role of Purinoceptors in the Regulation of Lung Surfactant Secretion, S. A. Rooney. The Role of Nucleotide Receptors in the Cardiovascular System, S. Pirotton, C. Desgranges, G. Vassort, and J.-M. Boeynaems. Physiological Roles for P2 Receptors in Platelets, Visceral Smooth Muscle, and the Immune and Endocrine Systems, S. Hourani, F. DiVirgilio, and M.-M. Loubatieres-Mariani. Therapeutic Applications for Nucleotides in Lung Disease, S. H. Donaldson and R. C.Boucher. Part V. Future Vistas. P2 Nucleotide Receptors: Future Vistas, J. S. Fedan, J. T. Turner, and G. A. Weisman. Index.

The Cloning and Expression of G Protein-Coupled P2Y Nucleotide Receptors

The existence of cell surface receptors for extracellular adenine nucleotides in mammalian cells has been postulated for decades (Burn-stock, 1972), based on scores of reports on diverse responses to these molecules in a wide variety of tissues and cell types (Dubyak and El-Moatassim, 1993). Pharmacological studies have provided evidence for the expression of two major types of nucleotide receptors: those belonging to the G protein-coupled receptor family and those belonging to the ligand-gated ion channel family. Nucleotide receptors (P2 purinergic receptors) are distinct from P1 receptors for adenosine, and, from results of earlier studies, were thought to be selective for purine nucleotides, in particular adenosine 5′-triphosphate (ATP), adenosine 5′-diphosphate (ADP) and various analogs, including 2MeSATP, α,β-MeATP, and β,γ-MeATP. Recent studies have demonstrated that uridine nucleotides are equally or more effective than adenine nucleotides in activating several G protein-coupled P2 receptor subtypes, raising a question as to the appropriateness of referring to this family of receptors as “purinergic.” Nonetheless, nucleotide receptors have retained the designation “P2,” which now refers to both their purine- and/or pyrimidine-based nucleotide agonists.

Erratum: Cloning and expression of a human P(2U) nucleotide receptor, a target for cystic fibrosis pharmacotherapy (Proceedings of the National Academy of Sciences of the United States of America (April 12, 1994) 91:8 (3275-3279))

The Cl- secretory pathway that is defective in cystic fibrosis (CF) can be bypassed by an alternative pathway for Cl- transport that is activated by extracellular nucleotides. Accordingly, the P2 receptor that mediates this effect is a therapeutic target for improving Cl- secretion in CF patients. In this paper, we report the sequence and functional expression of a cDNA cloned from human airway epithelial (CF/T43) cells that encodes a protein with properties of a P2U nucleotide receptor. With a retrovirus system, the human airway clone was stably expressed in 1321N1 astrocytoma cells, a human cell line unresponsive to extracellular nucleotides. Studies of inositol phosphate accumulation and intracellular Ca2+ mobilization induced by extracellular nucleotides in 1321N1 cells expressing the receptor identified this clone as the target receptor in human airway epithelia. In addition, we independently isolated an identical cDNA from human colonic epithelial (HT-29) cells, indicating that this is the same P2U receptor that has been functionally identified in other human tissues. Expression of the human P2U receptor (HP2U) in 1321N1 cells revealed evidence for autocrine ATP release and stimulation of transduced receptors. Thus, HP2U expression in the 1321N1 cell line will be useful for studying autocrine regulatory mechanisms and in screening of potential therapeutic drugs.

Future Directions in P2 Receptor Research

The identification and characterization of receptors for extracellular nucleotides has benefited substantially from modern molecular biological techniques, as this volume has attempted to chronicle. The rapid advancement provided by these molecular approaches has also highlighted perhaps the most important challenge facing researchers in the area—the identification of the cloned P2 receptor subtype(s) involved in each of the wide array of physiological and pathological processes affected by extracellular nucleotides. Information in the preceding chapters inspires confidence that this essential task will be facilitated in the near future by the availability of additional P2 receptor subtype-selective ligands, hopefully both agonists and antagonists. Although the number of cloned P2 receptors is already impressive, evidence indicates the existence of additional subtypes and it seems reasonable to expect a few surprises as the full range of signaling molecules for extracellular nucleotides becomes known. It is essential to establish whether or not other nucleotides, in addition to adenosine triphosphate (ATP) and adenosine diphosphate (ADP), function as endogenous extracellular signaling molecules through P2 receptors. If uridine triphosphate (UTP) or other nucleotides are shown to serve such a role, a re-evaluation of the current nucleotide receptor classification scheme may be required.