Abstract
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 approximately 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.
Highlights
Molecular determinants of P2Y2 receptor desensitization and sequestration have been investigated
Responses to extracellular nucleotides are mediated by P2 receptors that belong to two receptor superfamilies: P2Y G protein-coupled receptors (GPCRs)1 and P2X ligand-gated ion channels
Five cDNAs encoding P2Y2 receptors with different length C-terminal truncations (Fig. 1) were constructed by polymerase chain reaction (PCR) and expressed in 1321N1 cells to determine the role of the C terminus in agonist-induced desensitization of receptor-mediated calcium mobilization and receptor sequestration
Summary
Mutagenesis, Epitope Tagging, and Subcloning—Wild-type murine P2Y2 receptor cDNA was subcloned into the retroviral vector pLXSN at the EcoRI/BamHI sites of the multiple cloning site. The P2Y2-1321N1 cells were washed with 2 ml of ice-cold Hepesbuffered saline (pH 7.4) and incubated at 4 °C for 1 h with gentle rocking in 1 ml of Hepes-buffered saline containing 10 g of anti-HA antibody 12CA5 (Boehringer Mannheim) that was affinity-purified as follows. 8.5), dialyzed against phosphate-buffered saline (PBS), and stored for use at a protein concentration of 1.5 mg/ml as estimated by a modification (18) of the method of Lowry et al (19). Calculation of Relative Receptor Densities—Cells expressing P2Y2 receptors were diluted from 1:2 to 1:8 with nontransfected 1321N1 cells, and the amount of fluorescein isothiocyanate fluorescence was measured in a Spex dual-excitation/emission spectrofluorometer using 488 and 525 nm for the excitation and emission wavelengths, respectively. A standard curve of fluorescence was constructed with HEK-293 cells heterologously expressing N-terminally HA-tagged 2-adrenergic receptors (Bmax ϭ 500 fmol/mg of total cell protein (20)) diluted with nontransfected HEK-293 cells
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