Functional Coupling of Rat Myometrial α1-Adrenergic Receptors to Ghα/Tissue Transglutaminase 2 during Pregnancy

Abstract

Ghα protein, which exhibits both transglutaminase and GTPase activities, represents a new class of GTP-binding proteins. In the present study, we characterized Ghα in rat uterine smooth muscle (myometrium) and followed its expression during pregnancy by reverse transcription-PCR and Western blot. We also measured transglutaminase and GTP binding functions and used a smooth muscle cell line to evaluate the role of Ghα in cell proliferation. The results show that pregnancy is associated with an up-regulation of Ghα expression at both the mRNA and protein level. Ghα induced during pregnancy is preferentially localized to the plasma membrane. This was found associated with an increased ability of plasma membrane preparations to catalyze Ca2+-dependent incorporation of [3H]putrescine into casein in vitro. In the cytosol, significant changes in the level of immunodetected Ghα and transglutaminase activity were seen only at term. Activation of α1-adrenergic receptors (α1-AR) enhanced photoaffinity labeling of plasma membrane Ghα. Moreover, the level of α1-AR-coupled Ghα increased progressively with pregnancy, which parallels the active period of myometrial cell proliferation. Overexpression of wild type Ghα in smooth muscle cell line DDT1-MF2 increased α1-AR-induced [3H]thymidine incorporation. A similar response was obtained in cells expressing the transglutaminase inactive mutant (C277S) of Ghα. Together, these findings underscore the role of Ghα as signal transducer of α1-AR-induced smooth muscle cell proliferation. In this context, pregnant rat myometrium provides an interesting physiological model to study the mechanisms underlying the regulation of the GTPase function of Ghα.