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J. Biol. Chem., Vol. 280, Issue 35, 31011-31018, September 2, 2005

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Role of Phospholipase C- Domains in Ca²⁺-dependent Phosphatidylinositol 4,5-Bisphosphate Hydrolysis and Cytoplasmic Ca²⁺ Oscillations^*

Michail Nomikos, Lynda M. Blayney, Mark G. Larman, Karen Campbell¶, Andreas Rossbach, Christopher M. Saunders, Karl Swann¶, and F. Anthony Lai||

From the Cell Signalling Laboratory, Wales Heart Research Institute, School of Medicine, Cardiff University, Cardiff CF14 4XN, the Department of Anatomy and Developmental Biology, University College London, London WC1E 6BT, and the ^¶Department of Obstetrics and Gynaecology, School of Medicine, Cardiff University, Cardiff CF14 4XN, United Kingdom

The sperm-specific phospholipase C- (PLC) elicits fertilization-like Ca²⁺ oscillations and activation of embryo development when microinjected into mammalian eggs (Saunders, C. M., Larman, M. G., Parrington, J., Cox, L. J., Royse, J., Blayney, L. M., Swann, K., and Lai, F. A. (2002) Development (Camb.) 129, 3533-3544; Cox, L. J., Larman, M. G., Saunders, C. M., Hashimoto, K., Swann, K., and Lai, F. A. (2002) Reproduction 124, 611-623). PLC may represent the physiological stimulus for egg activation and development at mammalian fertilization. PLC is the smallest known mammalian PLC isozyme, comprising two EF hand domains, a C2 domain, and the catalytic X and Y core domains. To gain insight into PLC structure-function, we assessed the ability of PLC and a series of domain-deletion constructs to cause phosphatidylinositol 4,5-bisphosphate hydrolysis in vitro and also to generate cytoplasmic Ca²⁺ changes in intact mouse eggs. PLC and the closely related PLC1 had similar K_m values for phosphatidylinositol 4,5-bisphosphate, but PLC was around 100 times more sensitive to Ca²⁺ than was PLC1. Notably, specific phosphatidylinositol 4,5-bisphosphate hydrolysis activity was retained in PLC constructs that had either EF hand domains or the C2 domain removed, or both. In contrast, Ca²⁺ sensitivity was greatly reduced when either one, or both, of the EF hand domains were absent, and the Hill coefficient was reduced upon deletion of the C2 domain. Microinjection into intact mouse eggs revealed that all domain-deletion constructs were ineffective at initiating Ca²⁺ oscillations. These data suggest that the exquisite Ca²⁺-dependent features of PLC regulation are essential for it to generate inositol 1,4,5-trisphosphate and Ca²⁺ oscillations in intact mouse eggs.

Received for publication, January 18, 2005 , and in revised form, June 30, 2005.

^* This work was supported by a School of Medicine Ph.D. studentship (to M. N.), an Education and Learning Wales Knowledge Exploitation Fund grant and Cardiff Partnership Fund grant (to F. A. L.), and by a Wellcome Trust grant (to K. S.). 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.

^|| To whom correspondence should be addressed. Tel.: 44-29-2074-2338; Fax: 44-29-2074-3500; E-mail: lait{at}cf.ac.uk.

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