STRUCTURAL BASIS OF AGONIST-INDUCED DESENSITIZATION AND SEQUESTRATIONOF THE P2Y(2) NUCLEOTIDE RECEPTOR - CONSEQUENCES OF TRUNCATION OF THEC-TERMINUS

Molecular determinants of P2Y(2) receptor desensitization and sequestr ation have been investigated. Wildtype P2Y(2) receptors and a series o f five C-terminal truncation cation mutants of the receptor were epito pe-tagged and stably expressed in 1321N1 cells. These constructs were used to assess the importance of the intracellular C terminus on 1) UT P stimulated increases in intracellular calcium concentration, 2) homo logous desensitization of the receptor, and 3) agonist-induced decreas es in cell-surface density (receptor sequestration) of epitope-tagged receptors using fluorescence-activated cell sorting. The potency and e fficacy of UTP were similar for the wild-type and all mutant P2Y(2) re ceptors, Truncation of 18 or more amino acids from the C terminus incr eased by similar to 30-fold the concentration of UTP necessary to dese nsitize the receptor. Both the rate and magnitude of UTP-induced recep tor sequestration were decreased with progressively larger truncations of the C terminus, Furthermore, the recovery from sequestration was s lower for the most extensively truncated receptor. Complete desensitiz ation was obtained with >50% of the original receptor complement remai ning on the cell surface. Protein kinase C activation, which desensiti zes the P2Y(2) receptor, had no effect on sequestration, consistent wi th the ideas that desensitization and sequestration are discrete event s and that agonist occupancy is required for receptor sequestration.