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J Biol Chem, Vol. 273, Issue 45, 29437-29444, November 6, 1998

Structural Basis of Agonist-induced Desensitization and Sequestration of the P2Y₂ Nucleotide Receptor
CONSEQUENCES OF TRUNCATION OF THE C TERMINUS

Richard C. Garrad, Miguel A. Otero^¶, Laurie Erb, Patty M. Theiss, Lane L. Clarke^**, Fernando A. Gonzalez^¶, John T. Turner^§§, and Gary A. Weisman

From the Departments of  Biochemistry,  Veterinary Biomedical Sciences, and ^§§ Pharmacology and the ^** Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri 65212 and the ^¶ Department of Chemistry, University of Puerto Rico, Rio Piedras Campus, San Juan, Puerto Rico, 00931

Molecular determinants of P2Y₂ receptor desensitization and sequestration have been investigated. Wild-type P2Y₂ receptors and a series of five C-terminal truncation mutants of the receptor were epitope-tagged and stably expressed in 1321N1 cells. These constructs were used to assess the importance of the intracellular C terminus on 1) UTP-stimulated increases in intracellular calcium concentration, 2) homologous desensitization of the receptor, and 3) agonist-induced decreases in cell-surface density (receptor sequestration) of epitope-tagged receptors using fluorescence-activated cell sorting. The potency and efficacy of UTP were similar for the wild-type and all mutant P2Y₂ receptors. Truncation of 18 or more amino acids from the C terminus increased by ~30-fold the concentration of UTP necessary to desensitize the receptor. Both the rate and magnitude of UTP-induced receptor sequestration were decreased with progressively larger truncations of the C terminus. Furthermore, the recovery from sequestration was slower for the most extensively truncated receptor. Complete desensitization was obtained with >50% of the original receptor complement remaining on the cell surface. Protein kinase C activation, which desensitizes the P2Y₂ receptor, had no effect on sequestration, consistent with the ideas that desensitization and sequestration are discrete events and that agonist occupancy is required for receptor sequestration.

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Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.

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