Molecular determinants of P2Y receptor desensitization and sequestration have been investigated. Wild-type P2Y 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 P2Y 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 P2Y 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.
This research was originally published in the Journal of Biological Chemistry 273, no. 45 29437-29444. © the American Society for Biochemistry and Molecular Biology and authors.
Garrad, Richard C., Miguel A. Otero, Laurie Erb, Patty M. Theiss, Lane L. Clarke, Fernando A. Gonzalez, John T. Turner, and Gary A. Weisman. "Structural basis of agonist-induced desensitization and sequestration of the P2Y2 nucleotide receptor: consequences of truncation of the C terminus." Journal of Biological Chemistry 273, no. 45 (1998): 29437-29444.
Journal of Biological Chemistry