Regulation of meiotic prophase arrest in mouse oocytes by GPR3, a constitutive activator of the Gs G protein

The arrest of meiotic prophase in mouse oocytes within antral follicles requires the G protein Gs and an orphan member of the G protein–coupled receptor family, GPR3. To determine whether GPR3 activates Gs, the localization of Gαs in follicle-enclosed oocytes from Gpr3+/+ and Gpr3−/− mice was compared by using immunofluorescence and GαsGFP. GPR3 decreased the ratio of Gαs in the oocyte plasma membrane versus the cytoplasm and also decreased the amount of Gαs in the oocyte. Both of these properties indicate that GPR3 activates Gs. The follicle cells around the oocyte are also necessary to keep the oocyte in prophase, suggesting that they might activate GPR3. However, GPR3-dependent Gs activity was similar in follicle-enclosed and follicle-free oocytes. Thus, the maintenance of prophase arrest depends on the constitutive activity of GPR3 in the oocyte, and the follicle cell signal acts by a means other than increasing GPR3 activity

Increased Expression of αqFamily G-proteins during Oocyte Maturation and Early Development ofXenopus laevis

AbstractG-proteins of the αqfamily link extracellular stimulation of plasma membrane receptors to phospholipase C and consequently to intracellular Ca2+release. Because they might function in initiating Ca2+release at fertilization, we examined Gαqfamily proteins in oocytes and eggs ofXenopus laevis.Three members of this protein family were identified by immunoblotting and antisense depletion. These proteins are barely detectable in the immature oocyte, but undergo a 6-fold increase in amount during oocyte maturation. This increase in Gαqfamily protein expression correlates with the acquisition, during oocyte maturation, of the ability to release Ca2+at fertilization (Schlichter and Elinson, 1981,Dev. Biol.83, 33–41). In contrast, amounts of Gαsand Gαi3are constant during maturation. We also examined the amounts of Gαq, Gαs, and Gαi3proteins during early development. While amounts of Gαsand Gαi3show little or no change, Gαqfamily protein expression increases 27-fold between the egg and neurula stages, suggesting that these proteins may be important in initiating Ca2+release during early development

Crystal Structure of the Human Acyl Protein Thioesterase I from a Single X-Ray Data Set to 1.5 Å

AbstractBackground: Many proteins undergo posttranslational modifications involving covalent attachment of lipid groups. Among them is palmitoylation, a dynamic, reversible process that affects trimeric G proteins and Ras and constitutes a regulatory mechanism for signal transduction pathways. Recently, an acylhydrolase previously identified as lysophospholipase has been shown to function as an acyl protein thioesterase, which catalyzes depalmitoylation of Gα proteins as well as Ras. Its amino acid sequence suggested that the protein is evolutionarily related to neutral lipases and other thioesterases, but direct structural information was not available.Results: We have solved the crystal structure of the human putative Gα-regulatory protein acyl thioesterase (hAPT1) with a single data set collected from a crystal containing the wild-type protein. The phases were calculated to 1.8 Å resolution based on anomalous scattering from Br− ions introduced in the cryoprotectant solution in which the crystal was soaked for 20 s. The model was refined against data extending to a resolution of 1.5 Å to an R factor of 18.6%. The enzyme is a member of the ubiquitous α/β hydrolase family, which includes other acylhydrolases such as the palmitoyl protein thioesterase (PPT1).Conclusions: The human APT1 is closely related to a previously described carboxylesterase from Pseudomonas fluorescens. The active site contains a catalytic triad of Ser-114, His-203, and Asp-169. Like carboxylesterase, hAPT1 appears to be dimeric, although the mutual disposition of molecules in the two dimers differs. Unlike carboxylesterase, the substrate binding pocket and the active site of hAPT1 are occluded by the dimer interface, suggesting that the enzyme must dissociate upon interaction with substrate