Expression profiles of the Gα subunits during Xenopus tropicalis embryonic development.

Heterotrimeric G protein signaling plays major roles during different cellular events. However, there is a limited understanding of the molecular mechanisms underlying G protein control during embryogenesis. G proteins are highly conserved and can be grouped into four subfamilies according to sequence homology and function.

The CREB Transcription Factor Controls Transcriptional Activity of the Human RIC8B Gene.

Proper regulation of gene expression is essential for normal development, cellular growth, and differentiation. Differential expression profiles of mRNA coding for vertebrate Ric-8B during embryo and adult stages have been observed. In addition, Ric-8B is expressed in few cerebral nuclei subareas.

Ric-8A, a guanine nucleotide exchange factor for heterotrimeric G proteins, is critical for cranial neural crest cell migration.

The neural crest (NC) is a transient embryonic structure induced at the border of the neural plate. NC cells extensively migrate towards diverse regions of the embryo, where they differentiate into various derivatives, including most of the craniofacial skeleton and the peripheral nervous system. The Ric-8A protein acts as a guanine nucleotide exchange factor for several Gα subunits, and thus behaves as an activator of signaling pathways mediated by heterotrimeric G proteins.

The Ric-8B gene is highly expressed in proliferating preosteoblastic cells and downregulated during osteoblast differentiation in a SWI/SNF- and C/EBPbeta-mediated manner.

The Ric-8 gene encodes a guanine exchange factor (GEF) that modulates G protein-mediated signaling, exhibiting a relevant role during regulation of cell division. In mammals, two Ric-8 homologues have been reported (Ric-8A and Ric-8B), and recent studies indicate equivalent roles for each protein. Here, we show that the Ric-8B gene is negatively regulated during osteoblast differentiation by the transcription factor C/EBPβ.

Biophysical studies support a predicted superhelical structure with armadillo repeats for Ric-8.

Ric-8 is a highly conserved cytosolic protein (MW 63 KDa) initially identified in C. elegans as an essential factor in neurotransmitter release and asymmetric cell division. Two different isoforms have been described in mammals, Ric-8A and Ric-8B; each possess guanine nucleotide exchange activity (GEF) on heterotrimeric G-proteins, but with different Galpha subunits specificities.

xRic-8 is a GEF for Gsalpha and participates in maintaining meiotic arrest in Xenopus laevis oocytes.

Immature stage VI Xenopus oocytes are arrested at the G(2)/M border of meiosis I until exposed to progesterone, which induces meiotic resumption through a non-genomic mechanism. One of the earliest events produced by this hormone is inhibition of the plasma membrane enzyme adenylyl cyclase (AC), with the concomitant drop in intracellular cAMP levels and reinitiation of the cell cycle. Recently Gsalpha and Gbetagamma have been shown to play an important role as positive regulators of Xenopus oocyte AC, maintaining the oocyte in the arrested state.

1alpha,25-dihydroxy vitamin D3-enhanced expression of the osteocalcin gene involves increased promoter occupancy of basal transcription regulators and gradual recruitment of the 1alpha,25-dihydroxy vitamin D3 receptor-SRC-1 coactivator complex.

Binding of 1alpha,25-dihydroxy vitamin D(3) to the C-terminal ligand-binding domain (LBD) of its receptor (VDR) induces a conformational change that enables interaction of VDR with transcriptional coactivators such as members of the p160/SRC family or the DRIP (vitamin D receptor-interacting complex)/Mediator complex. These interactions are critical for VDR-mediated transcriptional enhancement of target genes. The p160/SRC members contain intrinsic histone acetyl transferase (HAT) activities that remodel chromatin at promoter regulatory regions, and the DRIP/Mediator complex may establish a molecular bridge between the VDR complex and the basal transcription machinery.

Nucleosome organization and targeting of SWI/SNF chromatin-remodeling complexes: contributions of the DNA sequence.

Chromatin organization within the nuclear compartment is a fundamental mechanism to regulate the expression of eukaryotic genes. During the last decade, a number of nuclear protein complexes with the ability to remodel chromatin and regulate gene transcription have been reported. Among these complexes is the SWI/SNF family, which alters chromatin structure in an ATP-dependent manner.

Galphaq negatively regulates the Wnt-beta-catenin pathway and dorsal embryonic Xenopus laevis development.

The non-canonical Wnt/Ca2+ signaling pathway has been implicated in the regulation of axis formation and gastrulation movements during early Xenopus laevis embryo development, by antagonizing the canonical Wnt/beta-catenin dorsalizing pathway and specifying ventral cell fate. However, the molecular mechanisms involved in this antagonist crosstalk are not known. Since Galphaq is the main regulator of Ca2+ signaling in vertebrates and from this perspective probably involved in the events elicited by the non-canonical Wnt/Ca2+ pathway, we decided to study the effect of wild-type Xenopus Gq (xGalphaq) in dorso-ventral axis embryo patterning.

Phosphorylation at serine 208 of the 1alpha,25-dihydroxy Vitamin D3 receptor modulates the interaction with transcriptional coactivators.

Upon ligand binding the 1alpha,25-dihydroxy Vitamin D3 receptor (VDR) undergoes a conformational change that allows interaction with coactivator proteins including p160/SRC family members and the multimeric DRIP complex through the DRIP205 subunit. Casein kinase II (CKII) phosphorylates VDR both in vitro and in vivo at serine 208 within the hinge domain. This phosphorylation does not affect the ability of VDR to bind DNA, but increases its ability to transactivate target promoters.

Classical Xenopus laevis progesterone receptor associates to the plasma membrane through its ligand-binding domain.

During the last decade, considerable evidence is accumulating that supports the view that the classic progesterone receptor (xPR-1) is mediating Xenopus laevis oocyte maturation through a non-genomic mechanism. Overexpression and depletion of oocyte xPR-1 have been shown to accelerate and to block progesterone-induced oocyte maturation, respectively. In addition, rapid inhibition of plasma membrane adenylyl cyclase (AC) by the steroid hormone, supports the idea that xPR-1 should be localized at the oocyte plasma membrane.

The 1alpha,25-dihydroxy Vitamin D3 receptor preferentially recruits the coactivator SRC-1 during up-regulation of the osteocalcin gene.

Binding of 1alpha,25-dihydroxy Vitamin D3 to the C-terminal domain (LBD) of its receptor (VDR), induces a conformational change that enables interaction of VDR with transcriptional coactivators such as the members of the p160/SRC family or the DRIP (Vitamin D interacting complex)/Mediator complex. These interactions are critical for VDR-mediated transcriptional enhancement of target genes. Recent reports indicate that nuclear receptors, including VDR, interact with p160/SRC members and the DRIP/Mediator complex in a sequential, cyclical, and mutually exclusive manner when bound to a target promoter, exhibiting also a high exchange rate.

Molecular determinants for G protein betagamma modulation of ionotropic glycine receptors.

The ligand-gated ion channel superfamily plays a critical role in neuronal excitability. The functions of glycine receptor (GlyR) and nicotinic acetylcholine receptor are modulated by G protein betagamma subunits. The molecular determinants for this functional modulation, however, are still unknown.

The classic receptor for 1alpha,25-dihydroxy vitamin D3 is required for non-genomic actions of 1alpha,25-dihydroxy vitamin D3 in osteosarcoma cells.

1alpha,25-dihydroxy vitamin D3 has a major role in the regulation of the bone metabolism as it promotes the expression of key bone-related proteins in osteoblastic cells. In recent years it has become increasingly evident that in addition to its well-established genomic actions, 1alpha,25-dihydroxy vitamin D3 induces non-genomic responses by acting through a specific plasma membrane-associated receptor. Results from several groups suggest that the classical nuclear 1alpha,25-dihydroxy vitamin D3 receptor (VDR) is also responsible for these non-genomic actions of 1alpha,25-dihydroxy vitamin D3.

Chromatin remodeling and transcriptional activity of the bone-specific osteocalcin gene require CCAAT/enhancer-binding protein beta-dependent recruitment of SWI/SNF activity.

Tissue-specific activation of the osteocalcin (OC) gene is associated with changes in chromatin structure at the promoter region. Two nuclease-hypersensitive sites span the key regulatory elements that control basal tissue-specific and vitamin D3-enhanced OC gene transcription. To gain understanding of the molecular mechanisms involved in chromatin remodeling of the OC gene, we have examined the requirement for SWI/SNF activity.

Plasma membrane destination of the classical Xenopus laevis progesterone receptor accelerates progesterone-induced oocyte maturation.

Xenopus laevis oocyte maturation is induced by the steroid hormone progesterone through a non-genomic mechanism initiated at the cell membrane. Recently, two Xenopus oocyte progesterone receptors have been cloned; one is the classical progesterone receptor (xPR-1) involved in genomic actions and the other a putative seven-transmembrane-G-protein-couple receptor. Both receptors are postulated to be mediating the steroid-induced maturation process in the frog oocyte.

Sodium vitamin C cotransporter SVCT2 is expressed in hypothalamic glial cells.

Kinetic analysis of vitamin C uptake demonstrated that different specialized cells take up ascorbic acid through sodium-vitamin C cotransporters. Recently, two different isoforms of sodium-vitamin C cotransporters (SVCT1/SLC23A1 and SVCT2/SLC23A2) have been cloned. SVCT2 was detected mainly in choroidal plexus cells and neurons; however, there is no evidence of SVCT2 expression in glial and endothelial cells of the brain.

Bone-specific transcription factor Runx2 interacts with the 1alpha,25-dihydroxyvitamin D3 receptor to up-regulate rat osteocalcin gene expression in osteoblastic cells.

Bone-specific transcription of the osteocalcin (OC) gene is regulated principally by the Runx2 transcription factor and is further stimulated in response to 1alpha,25-dihydroxyvitamin D3 via its specific receptor (VDR). The rat OC gene promoter contains three recognition sites for Runx2 (sites A, B, and C). Mutation of sites A and B, which flank the 1alpha,25-dihydroxyvitamin D3-responsive element (VDRE), abolishes 1alpha,25-dihydroxyvitamin D3-dependent enhancement of OC transcription, indicating a tight functional relationship between the VDR and Runx2 factors.

A Gbetagamma stimulated adenylyl cyclase is involved in Xenopus laevis oocyte maturation.

Xenopus laevis oocyte maturation is induced by the steroid hormone progesterone through a nongenomic mechanism that implicates the inhibition of the effector system adenylyl cyclase (AC). Recently, it has been shown that the G protein betagamma heterodimer is involved in oocyte maturation arrest. Since AC is the proposed target for Gbetagamma action, we considered of importance to identify and characterize the Gbetagamma regulated AC isoform(s) that are expressed in the Xenopus oocyte.

Mutation of the highly conserved Arg165 and Glu168 residues of human Gsalpha disrupts the alphaD-alphaE loop and enhances basal GDP/GTP exchange rate.

G protein signalling regulates a wide range of cellular processes such as motility, differentiation, secretion, neurotransmission, and cell division. G proteins consist of three subunits organized as a Galpha monomer associated with a Gbetagamma heterodimer. Structural studies have shown that Galpha subunits are constituted by two domains: a Ras-like domain, also called the GTPase domain (GTPaseD), and an helical domain (HD), which is unique to heterotrimeric G-proteins.

The Runx2 transcription factor plays a key role in the 1alpha,25-dihydroxy Vitamin D3-dependent upregulation of the rat osteocalcin (OC) gene expression in osteoblastic cells.

Bone-specific transcription of the osteocalcin (OC) gene is principally regulated by the Runx2 transcription factor and further stimulated in response to 1alpha,25-dihydroxy Vitamin D3 via its specific receptor (VDR). The rat OC gene promoter contains three recognition sites for Runx2 (sites A-C). Mutation of sites A and B, which flank the 1alpha,25-dihydroxy Vitamin D3-responsive element (VDRE), abolishes 1alpha,25-dihydroxy Vitamin D3-dependent enhancement of OC transcription, indicating a tight functional relationship between VDR and Runx2 factors.

Modulation of glycine-activated ion channel function by G-protein betagamma subunits.

Glycine receptors (GlyRs), together with GABA(A) and nicotinic acetylcholine (ACh) receptors, form part of the ligand-activated ion channel superfamily and regulate the excitability of the mammalian brain stem and spinal cord. Here we report that the ability of the neurotransmitter glycine to gate recombinant and native ionotropic GlyRs is modulated by the G protein betagamma dimer (Gbetagamma). We found that the amplitude of the glycine-activated Cl- current was enhanced after application of purified Gbetagamma or after activation of a G protein-coupled receptor.

Regulation of the bone-specific osteocalcin gene by p300 requires Runx2/Cbfa1 and the vitamin D3 receptor but not p300 intrinsic histone acetyltransferase activity.

p300 is a multifunctional transcriptional coactivator that serves as an adapter for several transcription factors including nuclear steroid hormone receptors. p300 possesses an intrinsic histone acetyltransferase (HAT) activity that may be critical for promoting steroid-dependent transcriptional activation. In osteoblastic cells, transcription of the bone-specific osteocalcin (OC) gene is principally regulated by the Runx2/Cbfa1 transcription factor and is stimulated in response to vitamin D(3) via the vitamin D(3) receptor complex.

Human brain synembryn interacts with Gsalpha and Gqalpha and is translocated to the plasma membrane in response to isoproterenol and carbachol.

Heterotrimeric G-proteins transduce signals from heptahelical transmembrane receptors to different effector systems, regulating diverse complex intracellular pathways and functions. In brain, facilitation of depolarization-induced neurotransmitter release for synaptic transmission is mediated by Gsalpha and Gqalpha. To identify effectors for Galpha-proteins, we performed a yeast two-hybrid screening of a human brain cDNA library, using the human Galphas protein as a bait.

G(alpha)s levels regulate Xenopus laevis oocyte maturation.

Progesterone, produced by follicular cells, induces Xenopus laevis oocyte maturation through a very early event that inhibits the activity of the adenylyl cyclase effector system. The participation of a G-protein has been implicated, based on the fact that the inhibitory effect of the steroid is GTP-dependent, and it has been proposed that progesterone acts interfering with G(alpha)s function at the plasma membrane. Here we investigate whether the change in oocyte G(alpha)s levels affects the maturation process induced by progesterone.