Structural basis of agonist-induced desensitization and sequestration of the P2Y2 nucleotide receptor: Consequences of truncation of the C terminus
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. 2 2 2 2
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