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␣.

In the present study, we studied the potential regulation by rat myometrial a1-adrenergic receptors (a1-AR) of the newly identified Gha protein/phospholipase Cd1 (PLCd1) signaling pathway and compared myometrial inositol phosphates (InsP) production and activity of the uterine circular muscle in response to a1-AR activation between mid-pregnancy and term. For this, we quantified the level of rat myometrial a1-AR coupling to Gha protein by photoaffinity-labeling, the cytosolic amount of PLCd1 enzyme by immunoblotting, and the expression level of a1-AR subtypes by RT-PCR. The results showed an increased level of a1-AR/Gha protein coupling and the amount of PLCd1 at term (C147 and C65% respectively, versus mid-pregnancy). This was correlated with an up-regulation of a1d-AR subtype (C70% versus midpregnancy). Incubation of myometrial strips with phenylephrine (Phe), a global a1-agonist, increased InsP production in a dose-dependent manner at both mid-pregnancy and term, but with an enhanced potency (tenfold decrease in EC 50 value) at term. Phe also dose-dependently induced contraction of the circular muscle at both mid-pregnancy and term. However, unlike InsP response, no amelioration of potency was observed at term. Similar results were obtained with the endogenous agonist norepinephrine. Our results show, for the first time, that rat myometrial a1d-AR/Gha/PLCd1 signaling pathway is up-regulated at term. This is associated with an increased potency of a1-AR to elicit InsP production but not uterine contraction at this period. It is thus hypothesized that a1-AR, through activation of Gha/PLCd1 system, are not primarily involved in the initiation of labor but may rather regulate responses such as myometrial cell proliferation or hypertrophy. Reproduction (2008) 135 55-62