Carroll D J, Ramarao C S, Mehlmann L M, Roche S, Terasaki M, Jaffe L A
Department of Physiology, University of Connecticut Health Center, Farmington, Connecticut 06032, USA.
J Cell Biol. 1997 Sep 22;138(6):1303-11. doi: 10.1083/jcb.138.6.1303.
Although inositol trisphosphate (IP3) functions in releasing Ca2+ in eggs at fertilization, it is not known how fertilization activates the phospholipase C that produces IP3. To distinguish between a role for PLCgamma, which is activated when its two src homology-2 (SH2) domains bind to an activated tyrosine kinase, and PLCbeta, which is activated by a G protein, we injected starfish eggs with a PLCgamma SH2 domain fusion protein that inhibits activation of PLCgamma. In these eggs, Ca2+ release at fertilization was delayed, or with a high concentration of protein and a low concentration of sperm, completely inhibited. The PLCgammaSH2 protein is a specific inhibitor of PLCgamma in the egg, since it did not inhibit PLCbeta activation of Ca2+ release initiated by the serotonin 2c receptor, or activation of Ca2+ release by IP3 injection. Furthermore, injection of a PLCgamma SH2 domain protein mutated at its phosphotyrosine binding site, or the SH2 domains of another protein (the phosphatase SHP2), did not inhibit Ca2+ release at fertilization. These results indicate that during fertilization of starfish eggs, activation of phospholipase Cgamma by an SH2 domain-mediated process stimulates the production of IP3 that causes intracellular Ca2+ release.
尽管肌醇三磷酸（IP3）在受精时可促使卵子释放Ca2+，但尚不清楚受精过程是如何激活产生IP3的磷脂酶C的。为了区分PLCγ（其两个src同源2结构域与激活的酪氨酸激酶结合时被激活）和PLCβ（由G蛋白激活）的作用，我们向海星卵中注射了一种抑制PLCγ激活的PLCγ SH2结构域融合蛋白。在这些卵子中，受精时Ca2+的释放被延迟，或者在高浓度蛋白和低浓度精子的情况下被完全抑制。PLCγ SH2蛋白是卵子中PLCγ的特异性抑制剂，因为它不抑制由5-羟色胺2c受体引发的Ca2+释放的PLCβ激活，也不抑制通过注射IP3引起的Ca2+释放激活。此外，注射在其磷酸酪氨酸结合位点发生突变的PLCγ SH2结构域蛋白，或另一种蛋白（磷酸酶SHP2）的SH2结构域，均不抑制受精时的Ca2+释放。这些结果表明，在海星卵受精过程中，通过SH2结构域介导的过程激活磷脂酶Cγ会刺激产生导致细胞内Ca2+释放的IP3。
