María Victoria Hinrichs

Human brain synembryn interacts with Gsα and Gqα 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 Gsα and Gqα. To identify effectors for Gα‐proteins, we performed a yeast two‐hybrid screening of a human brain cDNA library, using the human Gαs protein as a bait. We identified a protein member of the synembryn family as one of the interacting proteins. Extending the study to other Gα subunits, we found that Gqα also interacts with synembryn, and these interactions were confirmed by in vitro pull down studies and by in vivo confocal laser microscopy analysis. Furthermore, synembryn was shown to translocate to the plasma membrane in response to carbachol and isoproterenol. This study supports recent findings in C. elegans where, through genetic studies, synembryn was shown to act together with Gqα regulating neuronal transmitter release. Based on these observations, we propose that synembryn is playing a similar role in human neuronal cells.

xRic‐8 is a GEF for Gsα and participates in maintaining meiotic arrest in Xenopus laevis oocytes

Immature stage VI Xenopus oocytes are arrested at the G2/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 Gsα and Gβγ 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 Gsα and Gβγ 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 Gsα. 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 Gsα‐Gβγ‐AC signaling complex in an activated state and therefore maintaining G2 arrest. J. Cell. Physiol. 214: 673–680, 2008.

Mutagenesis in the switch IV of the helical domain of the human Gsα reduces its GDP/GTP exchange rate

The Gα subunits of heterotrimeric G proteins are constituted by a conserved GTPase “Ras‐like” domain (RasD) and by a unique α‐helical domain (HD). Upon GTP binding, four regions, called switch I, II, III, and IV, have been identified as undergoing structural changes. Switch I, II, and III are located in RasD and switch IV in HD. All Gα known functions, such as GTPase activity and receptor, effector, and Gβγ interaction sites have been found to be localized in RasD, but little is known about the role of HD and its switch IV region. Through the construction of chimeras between human and Xenopus Gsα we have previously identified a HD region, encompassing helices αA, αB, and αC, that was responsible for the observed functional differences in their capacity to activate adenylyl cyclase (Antonelli et al. [1994]: FEBS Lett 340:249–254). Since switch IV is located within this region and contains most of the nonconservative amino acid differences between both Gsα proteins, in the present work we constructed two human Gsα mutant proteins in which we have changed four and five switch IV residues for the ones present in the Xenopus protein. Mutants M15 (hGsααS133N, M135P, P138K, P143S) and M17 (hGsααS133N, M135P, V137Y, P138K, P143S) were expressed in Escherichia coli, purified, and characterized by their ability to bind GTPγS, dissociate GDP, hydrolyze GTP, and activate adenylyl cyclase. A decreased rate of GDP release, GTPγS binding, and GTP hydrolysis was observed for both mutants, M17 having considerably slower kinetics than M15 for all functions tested. Reconstituted adenylyl cyclase activity with both mutants showed normal activation in the presence of AlF4−, but a decreased activation with GTPγS, which is consistent with the lower GDP dissociating rate they displayed. These data provide new evidence on the role that HD is playing in modulating the GDP/GTP exchange of the Gsα subunit. J. Cell. Biochem. 76:368–375, 2000.

Cloning and spatiotemporal expression of RIC-8 in Xenopus embryogenesis

RIC-8 is a highly conserved protein that promotes G protein signaling as it acts as a Guanine nucleotide Exchanging Factor (GEF) over a subset of Gα subunits. In invertebrates, RIC-8 plays crucial roles in synaptic transmission as well as in asymmetric cell division. As a first step to address further studies on RIC-8 function in vertebrates, here we have cloned a ric-8 gene from Xenopus tropicalis (xtric-8) and determined its spatiotemporal expression pattern throughout embryogenesis. The xtric-8 transcript is expressed maternally and zygotically and, as development proceeds, it shows a dynamic expression pattern. At early developmental stages, xtric-8 is expressed in the animal hemisphere, whereas its expression is later restricted to neural tissues, such as the neural tube and the brain, as well as in the eye and neural crest-derived structures, including those of the craniofacial region. Together, our findings suggest that RIC-8 functions are related to the development of the nervous system.

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. J. Cell. Physiol. 211: 560–567, 2007.

The classic receptor for 1α,25‐dihydroxy vitamin D3 is required for non‐genomic actions of 1α,25‐dihydroxy vitamin D3 in osteosarcoma cells

1α,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, 1α,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 1α,25‐dihydroxy vitamin D3 receptor (VDR) is also responsible for these non‐genomic actions of 1α,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 1α,25‐dihydroxy vitamin D3. We report that expression of the classic VDR in osteoblasts is required to generate a rapid 1α,25‐dihydroxy vitamin D3‐mediated increase in the intracellular Ca2+ concentration, a hallmark of the non‐genomic actions of 1α,25‐dihydroxy vitamin D3 in these cells. J. Cell. Biochem. 99: 995–1000, 2006.

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 Gα 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 α‐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°C, consistent with an armadillo‐type structure such as α‐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.

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.

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. J. Cell. Biochem. 99: 853–859, 2006.

Molecular cloning and expression of an adenylyl cyclase from Xenopus laevis oocytes

© 1997 Federation of European Biochemical Societies.