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. To further studies on G protein function during embryogenesis, the present analysis identified four Gα subunits representative of the different subfamilies and determined their spatiotemporal expression patterns during Xenopus tropicalis embryogenesis. Each of the Gα subunit transcripts was maternally and zygotically expressed, and, as development progressed, dynamic expression patterns were observed. In the early developmental stages, the Gα subunits were expressed in the animal hemisphere and dorsal marginal zone. While expression was observed at the somite boundaries, in vascular structures, in the eye, and in the otic vesicle during the later stages, expression was mainly found in neural tissues, such as the neural tube and, especially, in the cephalic vesicles, neural crest region, and neural crest-derived structures. Together, these results support the pleiotropism and complexity of G protein subfamily functions in different cellular events. The present study constitutes the most comprehensive description to date of the spatiotemporal expression patterns of Gα subunits during vertebrate development.

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. These facts point to a dynamic control of RIC8B gene expression. In order to understand the transcriptional regulation of this gene, we searched for cis-elements in the sequence of the human RIC8B promoter region, identifying binding sites for the basic/leucine zipper (bZip) CREB transcription factor family (CRE sites) and C/EBP transcription factor family (C/EBP sites). CRE sites were found clustered near the transcription start site, while the C/EBP sites were found clustered at around 300 bp upstream the CRE sites. Here, we demonstrate the ability of CREB1 and C/EBPß to bind their respective elements identified in the RIC8B promoter. Comparative protein-DNA interaction analyses revealed only the proximal elements as high affinity sites for CREB1 and only the distal elements as high affinity sites for C/EBPß. Chromatin immunoprecipitation analyses, carried out using a human neuroblastoma cell line, confirmed the preferential association of CREB to the proximal region of the RIC8B promoter. By performing luciferase reporter assays, we found the CRE sites as the most relevant elements for its transcriptional activity. Taken together, these data show the existence of functional CREB and C/EBP binding sites in the human RIC8B gene promoter, a particular distribution of these sites and demonstrate a relevant role of CREB in stimulating transcriptional activity of this gene. J. Cell. Biochem. 117: 1797-1805, 2016. © 2016 Wiley Periodicals, Inc.

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. Using in vivo transplantation assays, we demonstrate that Ric-8A levels are critical for the migration of cranial NC cells and their subsequent differentiation into craniofacial cartilage during Xenopus development. NC cells explanted from Ric-8A morphant embryos are unable to migrate directionally towards a source of the Sdf1 peptide, a potent chemoattractant for NC cells. Consistently, Ric-8A knock-down showed anomalous radial migratory behavior, displaying a strong reduction in cell spreading and focal adhesion formation. We further show that during in vivo and in vitro neural crest migration, Ric-8A localizes to the cell membrane, in agreement with its role as a G protein activator. We propose that Ric-8A plays essential roles during the migration of cranial NC cells, possibly by regulating cell adhesion and spreading.

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/EBPß. Only the larger C/EBPß isoform (C/EBPß-LAP*) downregulates Ric-8B gene promoter activity in osteoblastic cells. Accordingly, knockdown of C/EBPß expression by small intefering RNA in osteoblastic cells results in a significant increase of Ric-8B gene expression. Transient overexpression of Brg1 or Brm, the catalytic subunits of the SWI/SNF chromatin-remodeling complex, inhibits Ric-8B promoter activity. Also, the presence of inactive SWI/SNF complexes in osteoblastic cells results in increased endogenous Ric-8B transcription, indicating that SWI/SNF activity negatively regulates Ric-8B expression. During osteoblast differentiation, Ric-8B gene repression is accompanied by changes in nucleosome placement at the proximal Ric-8B gene promoter and reduced accessibility to regulatory sequences.

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. To gain insight on the mechanisms involved in Ric-8 cellular functions it is essential to obtain some information about its structure. Therefore, the aim of this work was to create a structural model for Ric-8. In this case, it was not possible to construct a model based on comparison with a template structure because Ric-8 does not present sequence similarity with any other protein. Consequently, different bioinformatics approaches that include protein folding and structure prediction were used. The Ric-8 structural model is composed of 10 armadillo folding motifs, organized in a right-twisted alpha-alpha super helix. In order to validate the structural model, a His-tag fusion construct of Ric-8 was expressed in E. coli, purified by affinity and anion exchange chromatography and subjected to circular dichroism analysis (CD) and thermostability studies. Ric-8 is approximately 80% alpha helix, with a Tm of 43.1 degrees C, consistent with an armadillo-type structure such as alpha-importin, a protein composed of 10 armadillo repeats. The proposed structural model for Ric-8 is intriguing because armadillo proteins are known to interact with multiple partners and participate in diverse cellular functions. These results open the possibility of finding new protein partners for Ric-8 with new cellular functions.

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. Here, we have analyzed the rate of recruitment of these coactivators to the bone-specific osteocalcin (OC) gene in response to short and long exposures to 1alpha,25-dihydroxy vitamin D3. We report that in intact osteoblastic cells VDR, in association with SRC-1, rapidly binds to the OC promoter in response to the ligand. The recruitment of SRC-1 correlates with maximal transcriptional enhancement of the OC gene at 4 h and with increased histone acetylation at the OC promoter. In contrast to other 1alpha,25-dihydroxy vitamin D3-enhanced genes, binding of the DRIP205 subunit, which anchors the DRIP/Mediator complex to the VDR, is detected at the OC promoter only after several hours of incubation with 1alpha,25-dihydroxy vitamin D(3), concomitant with the release of SRC-1. Together, our results support a model where VDR preferentially recruits SRC-1 to enhance bone-specific OC gene transcription.

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. However, a question that still remains unanswered, is how the activated state of Gsalpha and Gbetagamma is achieved in the immature oocyte, since no receptor or ligand have been found to be required. Here we provide evidence that xRic-8 can act in vitro and in vivo as a GEF for Gsalpha. Overexpression of xRic-8, through mRNA injection, greatly inhibits progesterone induced oocyte maturation and endogenous xRic-8 mRNA depletion, through siRNA microinjection, induces spontaneous oocyte maturation. These results suggest that xRic-8 is participating in the immature oocyte by keeping Gsalpha-Gbetagamma-AC signaling complex in an activated state and therefore maintaining G2 arrest.

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. Overexpression of xGalphaq or its endogenous activation at the dorsal animal region of Xenopus embryo both induced a strong ventralized phenotype and inhibited the expression of dorsal-specific mesoderm markers goosecoid and chordin. Dorsal expression of an xGalphaq dominant-negative mutant reverted the xGalphaq-induced ventralized phenotype. Finally, we observed that the Wnt8-induced secondary axis formation is reverted by endogenous xGalphaq activation, indicating that it is negatively regulating the Wnt/beta-catenin pathway.

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. A considerable effort has been made to understand the molecular mechanisms by which SWI/SNF catalyzes nucleosome remodeling. However, limited attention has been dedicated to studying the role of the DNA sequence in this remodeling process. Therefore, in this minireview, we discuss the contribution of nucleosome positioning and nucleosome excluding sequences to the targeting and activity of SWI/SNF complexes. This discussion includes results from our group using the rat osteocalcin gene promoter as a model. Based on these results, we postulate a model for chromatin remodeling and transcriptional activation of this gene in osteoblastic cells.

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. Here, we have analyzed whether phosphorylation of VDR by CKII modulates the ability of VDR to interact with coactivators in vitro. We find that both mutation of serine 208 to aspartic acid (VDRS208D) or phosphorylation of VDR by CKII enhance the interaction of VDR with DRIP205 in the presence of 1alpha,25-dihydroxy Vitamin D3. We also find that the mutation VDRS208D neither affects the ability of this protein to bind DNA nor to interact with SRC-1 and RXRalpha. Together, our results indicate that phosphorylation of VDR at serine 208 contributes to modulate the affinity of VDR for the DRIP complex and therefore may have a role in vivo regulating VDR-mediated transcriptional enhancement.

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. To test this hypothesis, we transiently transfected xPR-1 cDNA into Cos-7 cells and analyzed its subcellular distribution. Through Western blot and immunofluorescence analysis, we were able to detect xPR-1 associated to the plasma membrane of transfected Cos-7 cells. Additionally, using Progesterone-BSA-FITC, we identified specific progesterone-binding sites at the cell surface of xPR-1 expressing cells. Finally, we found that the receptor ligand-binding domain displayed membrane localization, in contrast to the N-terminal domain, which expressed in similar levels, remained cytosolic. Overall, these results indicate that a fraction of xPR-1 expressed in Cos-7 cells, associates to the plasma membrane through its LBD.

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. Here, we present an overview of how these coactivators are recruited to the bone-specific osteocalcin (OC) gene in response to short and long exposures to 1alpha,25-dihydroxy Vitamin D3. We find that in intact osteoblastic cells VDR and SRC-1 rapidly bind to the OC promoter in response to the ligand. This recruitment correlates with transcriptional enhancement of the OC gene and with increased histone acetylation at the OC promoter. In contrast, binding of the DRIP205 subunit, which anchors the DRIP/Mediator complex to the VDR, is detected at the OC promoter after several hours of incubation with 1alpha,25-dihydroxy Vitamin D3. Together, our results indicate that VDR preferentially recruits SRC-1 to enhance basal bone-specific OC gene transcription. We propose a model where specific protein-DNA and protein-protein interactions that occur within the context of the OC gene promoter in osteoblastic cells stabilize the preferential association of the VDR-SRC-1 complex.

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. Studying mutant receptors, we identified two basic amino acid motifs within the large intracellular loop of the GlyR alpha(1) subunit that are critical for binding and functional modulation by Gbetagamma. Mutations within these sequences demonstrated that all of the residues detected are important for Gbetagamma modulation, although both motifs are necessary for full binding. Molecular modeling predicts that these sites are alpha-helixes near transmembrane domains 3 and 4, near to the lipid bilayer and highly electropositive. Our results demonstrate for the first time the sites for G protein betagamma subunit modulation on GlyRs and provide a new framework regarding the ligand-gated ion channel superfamily regulation by intracellular signaling.

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. Here, we have used siRNA to suppress the expression of VDR in osteoblastic cells and assessed the role of VDR in the non-genomic response to 1alpha,25-dihydroxy vitamin D3. We report that expression of the classic VDR in osteoblasts is required to generate a rapid 1alpha,25-dihydroxy vitamin D3-mediated increase in the intracellular Ca(2+) concentration, a hallmark of the non-genomic actions of 1alpha,25-dihydroxy vitamin D3 in these cells.

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. We inducibly expressed an ATPase-defective BRG1 catalytic subunit that forms inactive SWI/SNF complexes that bind to the OC promoter. This interaction results in inhibition of both basal and vitamin D3-enhanced OC gene transcription and a marked decrease in nuclease hypersensitivity. We find that SWI/SNF is recruited to the OC promoter via the transcription factor CCAAT/enhancer-binding protein beta, which together with Runx2 forms a stable complex to facilitate RNA polymerase II binding and activation of OC gene transcription. Together, our results indicate that the SWI/SNF complex is a key regulator of the chromatin-remodeling events that promote tissue-specific transcription in osteoblasts.

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. In this study, we tested the hypothesis that the classical progesterone receptor, associated to the oocyte plasma membrane, is participating in the reinitiation of the cell cycle. Addition of a myristoilation and palmytoilation signal at the amino terminus of xPR-1 (mp xPR-1), increased the amount of receptor associated to the oocyte plasma membrane and most importantly, significantly potentiated progesterone-induced oocyte maturation sensitivity. These findings suggest that the classical xPR-1, located at the plasma membrane, is mediating through a non-genomic mechanism, the reinitiation of the meiotic cell cycle in the X. laevis oocyte.

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. Through biochemical studies, we found that stage VI plasma membrane oocyte AC activity showed attributes of an AC2 isoform. Furthermore, exogenous Gbetagamma was capable to activate oocyte AC only in the presence of the activated form of Galphas (Galphas-GTPgammaS), which is in agreement with the Ggammabeta conditional activation reported for the mammalian AC2 and AC4 isotypes. In order to study the functional role of AC in oocyte maturation we cloned from a Xenopus oocyte cDNA library a gene encoding an AC with high identity to AC7 (xAC7). Based on this sequence, we constructed a minigene encoding the AC-Gbetagamma interacting region (xAC7pep) to block, within the oocyte, this interaction. We found that microinjection of the xAC7pep potentiated progesterone-induced maturation, as did the AC2 minigene. From these results we can conclude that a Gbetagamma-activated AC is playing an important role in Xenopus oocyte meiotic arrest in a Galphas-GTP dependent manner.

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. Certain brain locations, including the hippocampus and hypothalamus, consistently show higher ascorbic acid values compared with other structures within the central nervous system. However, molecular and kinetic analysis addressing the expression of SVCT transporters in cells isolated from these specific areas of the brain had not been done. The hypothalamic glial cells, or tanycytes, are specialized ependymal cells that bridge the cerebrospinal fluid with different neurons of the region. Our hypothesis postulates that SVCT2 is expressed selectively in tanycytes, where it is involved in the uptake of the reduced form of vitamin C (ascorbic acid), thereby concentrating this vitamin in the hypothalamic area. In situ hybridization and optic and ultrastructural immunocytochemistry showed that the transporter SVCT2 is highly expressed in the apical membranes of mouse hypothalamic tanycytes. A newly developed primary culture of mouse hypothalamic tanycytes was used to confirm the expression and function of the SVCT2 isoform in these cells. The results demonstrate that tanycytes express a high-affinity transporter for vitamin C. Thus, the vitamin C uptake mechanisms present in the hypothalamic glial cells may perform a neuroprotective role concentrating vitamin C in this specific area 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. In contrast to most of the members of the nuclear receptor family, VDR possesses a very short N-terminal A/B domain, which has led to the suggestion that its N-terminal region does not contribute to transcriptional enhancement. Here, we have combined transient-overexpression, coimmunoprecipitation, in situ colocalization, chromatin immunoprecipitation, and glutathione S-transferase pull-down analyses to demonstrate that in osteoblastic cells expressing OC, VDR interacts directly with Runx2 bound to site B, which is located immediately adjacent to the VDRE. This interaction contributes significantly to 1alpha,25-dihydroxyvitamin D3-dependent enhancement of the OC promoter and requires a region located C terminal to the runt homology DNA binding domain of Runx2 and the N-terminal region of VDR. Together, our results indicate that Runx2 plays a key role in the 1alpha,25-dihydroxyvitamin D3-dependent stimulation of the OC promoter in osteoblastic cells by further stabilizing the interaction of the VDR with the VDRE. These studies demonstrate a novel mechanism for combinatorial control of bone tissue-specific gene expression. This mechanism involves the intersection of two major pathways: Runx2, a "master" transcriptional regulator of osteoblast differentiation, and 1alpha,25-dihydroxyvitamin D3, a hormone that promotes expression of genes associated with these terminally differentiated bone cells.

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 HD display significantly higher primary structure diversity than the GTPaseD. Regardless of this diversity, there are small regions of the HD which show high degree of identity with residues that are 100% conserved. One of such regions is the alpha helixD-alpha helixE loop (alphaD-alphaE) in the HD, which contains the consensus aminoacid sequence R*-[RSA]-[RSAN]-E*-[YF]-[QH]-L in all mammalian Galpha subunits. Interestingly, the highly conserved arginine (R*) and glutamic acid (E*) residues form a salt bridge that stabilizes the alphaD-alphaE loop, that is localized in the top of the cleft formed between the GTPaseD and HD. Because the guanine nucleotide binding site is deeply buried in this cleft and those interdomain interactions are playing an important role in regulating the basal GDP/GTP nucleotide exchange rate of Galpha subunits, we studied the role of these highly conserved R and E residues in Galpha function. In the present study, we mutated the human Gsalpha R165 and E168 residues to alanine (A), thus generating the R165--> A, E168--> A, and R165/E168--> A mutants. We expressed these human Gsalpha (hGsalpha) mutants in bacteria as histidine tagged proteins, purified them by niquel-agarose chromatography and studied their nucleotide exchange properties. We show that the double R165/E168--> A mutant exhibited a fivefold increased GTP binding kinetics, a higher GDP dissociation rate, and an augmented capacity to activate adenylyl cyclase. Structure analysis showed that disruption of the salt bridge between R165 and E168 by the introduced mutations, caused important structural changes in the HD at the alphaD-alphaE loop (residues 160-175) and in the GTPaseD at a region required for Gsalpha activation by the receptor (residues 308-315). In addition, other two GTPaseD regions that surround the GTP binding site were also affected.