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

Abstract

Gh␣ protein, which exhibits both transglutaminase and GTPase activities, represents a new class of GTPbinding proteins. In the present study, we characterized Gh␣ 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 Gh␣ in cell proliferation. The results show that pregnancy is associated with an up-regulation of Gh␣ expression at both the mRNA and protein level. Gh␣ induced during pregnancy is preferentially localized to the plasma membrane. This was found associated with an increased ability of plasma membrane preparations to catalyze Ca 2؉ -dependent incorporation of [ 3 H]putrescine into casein in vitro. In the cytosol, significant changes in the level of immunodetected Gh␣ and transglutaminase activity were seen only at term. Activation of ␣1-adrenergic receptors (␣1-AR) enhanced photoaffinity labeling of plasma membrane Gh␣. Moreover, the level of ␣1-ARcoupled Gh␣ increased progressively with pregnancy, which parallels the active period of myometrial cell proliferation. Overexpression of wild type Gh␣ in smooth muscle cell line DDT1-MF2 increased ␣1-AR-induced [ 3 H]thymidine incorporation. A similar response was obtained in cells expressing the transglutaminase inactive mutant (C277S) of Gh␣. Together, these findings underscore the role of Gh␣ as signal transducer of ␣1-ARinduced 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 Gh␣.

Increased potency of α1-adrenergic receptors to induce inositol phosphates production correlates with the up-regulation of α1d/Ghα/phospholipase Cδ1 signaling pathway in term rat myometrium

“…Indeed, Gha protein was shown to mediate a1-AR-induced hepatocyte proliferation (Wu et al 2000) and activation of extracellular signal-regulated kinases in neonatal rat cardiomyocytes (Lee et al 2003). In agreement with these results, we have recently shown that Gha protein participates to the a1-AR-mediated regulation of smooth muscle cell proliferation (Dupuis et al 2004). Alternatively, the a1/Gha/PLCd1 system may also play a role in the preparation of uterus to labor by regulating the expression of some contraction-associated proteins.…”

“…In line with these data, we have shown that the up-regulation of rat myometrial PLCb1 and PLCb3 and Gqa protein correlates well with the increased uterine responsiveness to oxytocin and carbachol at term (Houdeau et al 2005). In pregnant and parturient rat myometrium, we have also shown that stimulation of a1-AR results in the activation of Gha protein pathway (Dupuis et al 2004). Gha protein is a bifunctional enzyme with both transglutaminase and GTPase activities (Nakaoka et al 1994).…”

“…Indeed, both the level of a1-AR/Gha protein coupling and the amount of PLCd1 significantly increased at this period. The enhanced a1-AR/Gha interaction could be explained by the induction of Gha expression at both mRNAs and protein levels as reported previously (Dupuis et al 2004). In addition, we also showed that, among the Gha-protein-coupled a1-AR, the expression of a1b-AR was down-regulated, whereas that of a1d-AR was significantly enhanced at term.…”

“…We recently reported that Gha protein, a new class of GTPbinding proteins, functionally interacts with the rat myometrial a1-AR (Dupuis et al 2004). In order to determine whether Gha protein plays an important role in the transduction of rat myometrial a1-AR, we compared by photoaffinity labeling studies a1-AR/Gha coupling between mid-pregnancy and term.…”

“…PCR amplification was performed with 1/20 volume of each RT reaction using specific upstream and downstream primers for a1a-, a1b-, and a1d-AR subtypes and the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as described previously (Mhaouty-Kodja et al 2001, Dupuis et al 2004. For semi-quantitative analysis, PCR cycle profiles were conducted and the cycle number (25 cycles) was chosen from the linear portion of the curve.…”