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J. Biol. Chem., Vol. 279, Issue 44, 46242-46252, October 29, 2004

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Functional Consequences of Phosphomimetic Mutations at Key cAMP-dependent Protein Kinase Phosphorylation Sites in the Type 1 Inositol 1,4,5-Trisphosphate Receptor^*

Larry E. Wagner, II, Wen-Hong Li¶, Suresh K. Joseph||, and David I. Yule**

From the Department of Pharmacology and Physiology, University of Rochester, Rochester, New York 14642, the Departments of Cell Biology and Biochemistry, University of Texas, Southwestern Medical Center, Dallas, Texas 75390-9039, and the ^||Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Regulation of Ca²⁺ release through inositol 1,4,5-trisphosphate receptors (InsP₃R) has important consequences for defining the particular spatio-temporal properties of intracellular Ca²⁺ signals. In this study, regulation of Ca²⁺ release by phosphorylation of type 1 InsP₃R (InsP₃R-1) was investigated by constructing "phosphomimetic" charge mutations in the functionally important phosphorylation sites of both the S2+ and S2- InsP₃R-1 splice variants. Ca²⁺ release was investigated following expression in Dt-40 3ko cells devoid of endogenous InsP₃R. In cells expressing either the S1755E S2+ or S1589E/S1755E S2- InsP₃R-1, InsP₃-induced Ca²⁺ release was markedly enhanced compared with nonphosphorylatable S2+ S1755A and S2- S1589A/S1755A mutants. Ca²⁺ release through the S2- S1589E/S1755E InsP₃R-1 was enhanced 8-fold over wild type and 50-fold when compared with the nonphosphorylatable S2- S1589A/S1755A mutant. In cells expressing S2- InsP₃R-1 with single mutations in either S1589E or S1755E, the sensitivity of Ca²⁺ release was enhanced 3-fold; sensitivity was midway between the wild type and the double glutamate mutation. Paradoxically, forskolin treatment of cells expressing either single Ser/Glu mutation failed to further enhance Ca²⁺ release. The sensitivity of Ca²⁺ release in cells expressing S2+ S1755E InsP₃R-1 was comparable with the sensitivity of S2- S1589E/S1755E InsP₃R-1. In contrast, mutation of S2+ S1589E InsP₃R-1 resulted in a receptor with comparable sensitivity to wild type cells. Expression of S2- S1589E/S1755E InsP₃R-1 resulted in robust Ca²⁺ oscillations when cells were stimulated with concentrations of -IgM antibody that were threshold for stimulation in S2- wild type InsP₃R-1-expressing cells. However, at higher concentrations of -IgM antibody, Ca²⁺ oscillations of a similar period and magnitude were initiated in cells expressing either wild type or S2- phosphomimetic mutations. Thus, regulation by phosphorylation of the functional sensitivity of InsP₃R-1 appears to define the threshold at which oscillations are initiated but not the frequency or amplitude of the signal when established.

Received for publication, May 26, 2004 , and in revised form, August 5, 2004.

^* This work was supported in part by National Institutes of Health Grants RO1-DK54568, R01-DE14756, PO1 DE13539 (to D. I. Y.), and RO1-DK34804 (to S. K. J.). 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.

^¶ Supported by Research Grant I-1510 from the Robert A. Welch Foundation and a Career Development award from the American Diabetes Association.

^** To whom correspondence should be addressed. Tel.: 585-273-2154; Fax: 585-670-0394; E-mail: david_yule{at}urmc.rochester.edu.

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