title: ATP binding to a unique site in the type-1 S2- inositol 1,4,5-trisphosphate receptor defines susceptibility to phosphorylation by protein kinase A.
abstract: The subtype- and splice variant-specific modulation of inositol 1,4,5-trisphosphate receptors (InsP3R) by interaction with cellular factors plays a fundamental role in defining the characteristics of Ca2+ release in individual cell types. In this study, we investigate the binding properties and functional consequences of the expression of a putative nucleotide binding fold (referred to as the ATPC site) unique to the S2- splice variant of the type-1 InsP3R (InsP3R-1), the predominant splice variant in peripheral tissue. A glutathione S-transferase fusion protein encompassing amino acids 1574-1765 of the S2- InsP3R-1 and including the glycine-rich motif Gly-Tyr-Gly-Glu-Lys-Gly bound ATP specifically as measured by fluorescent trinitrophenyl-ATP binding. This binding was completely abrogated by a point mutation (G1690A) in the nucleotide binding fold. The functional sensitivity of S2- InsP3R-1 constructs was evaluated in DT40-3KO-M3 cells, a null background for InsP3R, engineered to express muscarinic M3 receptors. The S2- InsP3R-1 containing the G1690A mutation was markedly less sensitive to agonist stimulation than wild type S2- InsP3R-1 or receptors containing a similar (Gly --> Ala) mutation in the established nucleotide binding sites in InsP3R-1 (the ATPA and ATPB sites). The ATP sensitivity of InsP3-induced Ca2+ release, however, was not altered by the G1690A mutation when measured in permeabilized DT40-3KO cells, suggesting a unique role for the ATPC site. Ca2+ release was dramatically potentiated following activation of cAMP-dependent protein kinase in DT40-3KO cells transiently expressing wild type S2- InsP3R or Gly --> Ala mutations in the ATPA and ATPB sites, but phosphorylation of the receptor and the potentiation of Ca2+ release were absent in cells expressing the G1690A mutation in S2- InsP3R. These data indicate that ATP binding specifically to the ATPC site in S2- InsP3R-1 controls the susceptibility of the receptor to protein kinase A-mediated phosphorylation, contributes to the functional sensitivity of the S2- InsP3R-1 and ultimately the sensitivity of cells to agonist stimulation.
authors.affiliation: Department of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester Medical Center, Rochester, New York 14642.
authors.forename: Larry E
authors.initials: LE
authors.lastname: Wagner
authors.affiliation: Department of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester Medical Center, Rochester, New York 14642.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Betzenhauser
authors.affiliation: Department of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester Medical Center, Rochester, New York 14642. Electronic address: David_Yule@urmc.rochester.edu.
authors.forename: David I
authors.initials: DI
authors.lastname: Yule
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015220:Calcium Channels / Q000737:chemistry*; D002460:Cell Line; D002645:Chickens; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D043587:Receptor, Muscarinic M3 / Q000737:chemistry; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry*
chemical_list: D015220:Calcium Channels; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D043587:Receptor, Muscarinic M3; D018160:Receptors, Cytoplasmic and Nuclear; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases