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J. Biol. Chem., Vol. 281, Issue 25, 17410-17419, June 23, 2006

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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^*

From the Department of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester Medical Center, Rochester, New York 14642

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.

Received for publication, February 10, 2006 , and in revised form, March 29, 2006.

^* This work was supported by National Institutes of Health (NIH) Grants RO1-DK54568, R01-DE14756, and R01-DE16999 (to D. I. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by NIDCR, NIH, Grant T32-DE07202.

² To whom correspondence should be addressed. Tel.: 585-273-2154; E-mail: David_Yule{at}urmc.rochester.edu.

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