Rin, a neuron-specific and calmodulin-binding small G-protein, and Rit define a novel subfamily of ras proteins (original) (raw)
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Proceedings of the National Academy of Sciences, 1991
A neuron-specific Ca2_/calmodulin-dependent protein kinase, CaM kinase Gr, phosphorylates selectively a Ras-related GTP-binding protein (Rap-lb) that is enriched in brain tissue. The phosphorylation reaction achieves a stoichiometry of about 1 and involves a serine residue near the carboxyl terminus of the substrate. Both CaM kinase Gr and cAMP-dependent protein kinase, but not CaM kinase II, phosphorylate identical or contiguous serine residues in Raplb. The rate of phosphorylation of Rap-lb by CaM kinase Gr is enhanced following autophosphorylation of the protein kinase. Other low molecular weight GTP-binding proteins belonging to the Ras superfamily, including Rab-3A, Rap-2b, and c-Ha-ras p21, are not phosphorylated by CaM inase Gr. The phosphorylation of Rap-lb itself can be reversed by an endogenous brain phosphoprotein phosphatase. These observations provide a potential connection between a neuronal Ca2+signaling pathway and a specific low molecular weight GTPbinding protein that may regulate neuronal trnnsmembrane signaling, vesicle transport, or neurotransmitter release. Neuronal Ca2+-signaling is partly mediated by two neuronspecific Ca2_/calmodulin-dependentprotein kinases, CaM
The Role of the Ras Guanyl-Nucleotide Exchange Factor Rasgrp1 in Synaptic Transmission
Official Symbol Name Other designations, notes Calb1 Calbindin D-28K, calbindin D28 Calb2 Calretinin, Calbindin2 CR Camk2a calcium/calmodulin-dependent protein kinase II alpha CamkII subunit alpha Canx Calnexin Cnx Creb1 cAMP responsive element binding protein 1 Dab2ip disabled homolog 2 (Drosophila) interacting protein Dgkz diacylglycerol kinase zeta Diap1 diaphanous homolog 1 Dia1 Dlg2 disks large homolog 2 PSD-93, Psd93, Chapsyn-110 Dlg4 disks large homolog 4 PSD-95, Psd95 Eea1 early endosome antigen 1 Egf Epidermal growth factor Gad1 glutamic acid decarboxylase 1 EP10, GAD25, GAD44, GAD67 Gad2 glutamic acid decarboxylase 2 GAD65 Golgb1 Giantin, golgi autoantigen, golgin subfamily b, macrogolgin 1 Gm6840 Grb2 growth factor receptor-bound protein 2 Gria1 glutamate receptor, ionotropic, AMPA1 (alpha 1) GluR1, GluA1, GluRA Gria2 glutamate receptor, ionotropic, AMPA2 (alpha 2) GluR2, GluA2, GluR-B, Glur-Diese Studie enthält die ersten Beweise für eine spezifische neuronale Funktion von Rasgrp1. Sie zeigt, dass Rasgrp1 selektiv die postsynaptische Sensitivität an glutamatergen Synapsen reguliert. Diese Studie zeigt, dass die selektive Veränderung der Regulation von Ras eine hilfreiche Methode ist, um die vielfältigen Effekte der Ras Signaltransduktion in Neuronen verstehen zu können. represent the postsynapse. Upon binding of Glutamate, the receptors open and allow Sodium to enter the cell. Upon this influx of cations, the cell depolarizes locally. This electrical signal propagates as the depolarization passively spreads through the dendrites. Spine shape, dendrite caliber and branching influence signal propagation as it travels to the soma. In the soma, final integration of all incoming signals takes place. If the resulting depolarization passes a certain threshold, a new action potential is generated in an all-or-none fashion. Postsynaptic spines, dendrites, the soma, the axon and presynaptic boutons represent highly specialized compartments that are part of the complex morphology of neurons. In fact, without knowledge of ion channels or biophysical properties of the Rho family G proteins regulate cytoskeletal rearrangements through actin binding proteins such as Wasl (Wiskott-Aldrich syndrome-like, also known as N-WASP) and Diap1 (diaphanous homolog 1, also known as Dia1). In this way, they function in the formation of stress fibers, lamellipodia and other morphological processes. In addition, they also regulate gene expression and signal via Pi3k (Takai et al., 2001). Rab family G proteins function in protein sorting, intracellular vesicle trafficking, targeting, docking and fusion. Their mechanism is to activate effectors that directly influence vesicular membrane shape, vesicle tethering or vesicle motility. (Stenmark, 2009; Takai et al., 2001). By similar mechanisms, Arf G proteins function in intracellular trafficking, in particular in vesicle budding from endomembranes, in II.2.2. The Classical Ras Signaling Cascade The first findings indicating a neuronal involvement of the classical Ras G proteins came from experiments using the pheochromocytoma cell line (PC12). Overexpression of Hras, Nras or infection with the Kirsten murine sarcoma virus led to neuronal differentiation of these cells, which was recognized by the outgrowth of II.3. Controversies in the Research of Neuronal Ras Signaling Since publication of the findings described above, research on neuronal Ras signaling has led to many controversies. In this regard, one experimental system in particular appears to have given rise to most of the controversies in the field. Extensive research on Ras signaling is conducted using activated mutants of Ras proteins. The activated mutated protein is able to bind GTP, but unable to hydrolyze it to GDP and therefore remains in a constitutively active state (Karnoub and Weinberg, 2008). The corresponding frequently used Glycine to Valine mutation at amino acid (aa) position 12 (G12V) is normally found in oncogenic Hras. II.5.1. Specificity of Ras GEFs GEFs that activate classical Ras G proteins in the brain belong to three families, the Sos, Rasgrf and Rasgrp family. In case of the Rasgrf and Rasgrp proteins, an activity towards the Rras/Mras subfamily besides the classical Ras G proteins seems to be a general pattern of specificity. The activity of Sos GEFs seems to be restricted Rasgrp1, Rasgrp2a, Rasgrp2b and Rasgrp3. Rasgrp1 mRNA and protein are highly expressed in the brain. It is found in the olfactory bulb, cortex, caudo-putamen (including striatum), hippocampus and thalamus, but only at very low levels in midbrain, cerebellum, pons and medulla
Small GTPase RIT1 in Mouse Retina; Cellular and Functional Analysis
Current eye research, 2018
Ras-like without CAAX 1 (RIT1/Rit) is a member of the Ras subfamily of small GTP-binding proteins with documented roles in regulating neuronal function, including contributions to neurotrophin signaling, neuronal survival, and neurogenesis. The aim of the study was to (1) examine the expression of RIT1 protein in mouse retina and retinal cell types and (2) determine whether RIT1 contributes to retinal ganglion cell (RGC) survival and synaptic stability following excitotoxic stress. Gene expression and immunohistochemical analysis were used to examine RIT1 expression in the mouse retina. Primary RGC and Müller glia cultures were used to validate novel RIT1 lentiviral RNAi silencing reagents, and to demonstrate that RIT1 loss does not alter RGC morphology. Finally, in vitro glutamate exposure identified a role for RIT1 in the adaptation of RGCs to excitotoxic stress. Gene expression analysis and immunohistochemical studies in whole eyes and primary cell culture demonstrate RIT1 expres...
R-Ras1 and R-Ras2 Expression in Anatomical Regions and Cell Types of the Central Nervous System
International Journal of Molecular Sciences, 2022
Since the optic nerve is one of the most myelinated tracts in the central nervous system (CNS), many myelin diseases affect the visual system. In this sense, our laboratory has recently reported that the GTPases R-Ras1 and R-Ras2 are essential for oligodendrocyte survival and maturation. Hypomyelination produced by the absence of one or both proteins triggers axonal degeneration and loss of visual and motor function. However, little is known about R-Ras specificity and other possible roles that they could play in the CNS. In this work, we describe how a lack of R-Ras1 and/or R-Ras2 could not be compensated by increased expression of the closely related R-Ras3 or classical Ras. We further studied R-Ras1 and R-Ras2 expression within different CNS anatomical regions, finding that both were more abundant in less-myelinated regions, suggesting their expression in non-oligodendroglial cells. Finally, using confocal immunostaining colocalization, we report for the first time that R-Ras2 is...
The Journal of biological chemistry, 2002
Post-translational modifications of GTPases from the Ras superfamily enable them to associate with membrane compartments where they exert their biological activities. However, no protein acting like Rho and Rab dissociation inhibitor (GDI) that regulate the membrane association of Rho and Rab GTPases has been described for Ras and closely related proteins. We report here that the delta subunit of retinal rod phosphodiesterase (PDEdelta) is able to interact with prenylated Ras and Rap proteins, and to solubilize them from membranes, independently of their nucleotide-bound (GDP or GTP) state. We show that PDEdelta exhibits striking structural similarities with RhoGDI, namely conservation of the Ig-like fold and presence of a series of hydrophobic residues which could act as in RhoGDI to sequester the prenyl group of its target proteins, thereby providing structural support for the biochemical activity of PDEdelta. We observe that the overexpression of PDEdelta interferes with Ras traf...
Proceedings of the National Academy of Sciences, 1987
Several oligonucleotide mixtures corresponding to a 6-amino acid sequence that is strictly conserved in all the ras and ras-related proteins (from various organisms) were tested for their ability to hybridize to 11 cloned members of the ras gene superfamily. Among these mixtures, a combination of two sets of partially complementary oligomers were able to hybridize to all the tested sequences. To identify members of the ras superfamily, we screened a rat brain cDNA library with these probes and isolated four genes, denoted rabl, -2, -3, and 4, encoding proteins homologous to the yeast YPT protein.
Biochemical Characterization of the Ras-Related GTPases Rit and Rin
Archives of Biochemistry and Biophysics, 1999
We report the biochemical characterization of Rit and Rin, two members of the Ras superfamily identified by expression cloning. Recombinant Rit and Rin bind GTP and exhibit intrinsic GTPase activity. Conversion of Gln to Leu at position 79 (for Rit) or 78 (for Rin) (equivalent to position 61 in Ras) resulted in a complete loss of GTPase activity. Surprisingly, significant differences were found when the guanine nucleotide dissociation constants of Rit and Rin were compared with the majority of Ras-related GTPases. Both proteins display higher k off values for GTP than GDP in the presence of 10 mM Mg 2؉ . These GTP dissociation rates are 5-to 10-fold faster than most Ras-like GT-Pases. Despite these unique biochemical properties, our data support the notion that both Rit and Rin function as nucleotide-dependent molecular switches. To begin to address whether these proteins act as regulators of distinct signaling pathways, we examined their interaction with a series of known Ras-binding proteins by yeast two-hybrid analysis. Although Rit, Rin, and Ras have highly related effector domain sequences, Rit and Rin were found to interact with the known Ras binding proteins RalGDS, Rlf, and AF-6/ Canoe but not with the Raf kinases, RIN1, or the p110 subunit of phosphatidylinositol 3-kinase. These interactions were GTP and effector domain dependent and suggest that RalGDS, Rlf, and AF-6 are Rit and Rin effectors. Their biochemical properties and interaction with a subset of known Ras effector proteins sug-gest that Rit and Rin may play important roles in the regulation of signaling pathways and cellular processes distinct from those controlled by Ras.
Proceedings of the National Academy of Sciences, 1994
For identification of Rab, Rac, Rho, Ral, Rap, and Arfproteins on two-dimensional polyacrylamide gels, we have expressed full-length cDNAs of members of these protein families with the T7 RNA polymerase-recombinant vaccinia virus expression system. Membrane preparations from cells expressing the cDNAs were subjected to highresolution two-dimensional polyacrylamide gel electrophoresis followed by [a-32P]GTP ligand blotting. We have mapped 28 small GTP-binding proteins relative to their isoelectric points and according to their molecular weights and by immunoblotting with specific antibodies. Rab and Rho proteins could be specifically identified by extraction of streptolysin O-permeabilized Madin-Darby canine kidney (MDCK) cells with Raband Rho-GDP dissociation inhibitor. We applied the reference mapping to analyze the GTP-binding patterns of synaptosome fractions from rat brain. The purified synaptosomes exhibited specific enrichment of Rab3a, Rab5a, Ral, and several other GTPases. This approach and the map we have produced should provide a useful aid for the analysis of the expression and localization of members of all families of small GTP-binding proteins in various cell types and subcellular fractions.