Several findings suggest that signals from tyrosine kinases are transduced, at least in part, through ras proteins. These findings include (i) blockage of the transforming activity of constitutively active tyrosine kinases by inhibiting ras function and (ii) genetic screens in Caenorhabditis elegans and in Drosophila that identified ras genes as downstream effectors of tyrosine kinases. The recently isolated Drosophila gene Son of sevenless (Sos) is postulated to act as a positive regulatory link between tyrosine kinase and ras proteins by catalyzing exchange of GDP for GTP on ras protein. Such exchange proteins have been reported in extracts of mammalian cells but have not been previously characterized at a molecular level. As Sos appears to function in this role in Drosophila, attempts were made to isolate a vertebrate counterpart. Two widely expressed murine genes have been characterized with a high degree of homology to Sos. Hybridization with human DNA and RNA indicates a high degree of conservation of these genes in other vertebrates (Bowtell, 1992).
Many tyrosine kinases, including the receptors for hormones such as epidermal growth factor (EGF), nerve growth factor and insulin, transmit intracellular signals through Ras proteins. Ligand binding to such receptors stimulates Ras guanine-nucleotide-exchange activity and increases the level of GTP-bound Ras, suggesting that these tyrosine kinases may activate a guanine-nucleotide releasing protein (GNRP). In C. elegans and Drosophila, genetic studies have shown that Ras activation by tyrosine kinases requires the protein Sem-5/drk, which contains a single Src-homology (SH) 2 domain and two flanking SH3 domains. Sem-5 is homologous to the mammalian protein Grb2, which binds the autophosphorylated EGF receptor and other phosphotyrosine-containing proteins such as Shc through its SH2 domain. In rodent fibroblasts, the SH3 domains of Grb2 are bound to the proline-rich carboxy-terminal tail of mSos1, a protein homologous to Drosophila Sos. Sos is required for Ras signalling and contains a central domain related to known Ras-GNRPs. EGF stimulation induces binding of the Grb2-mSos1 complex to the autophosphorylated EGF receptor, and mSos1 phosphorylation. Grb2 therefore appears to link tyrosine kinases to a Ras-GNRP in mammalian cells (Rozakis-Adcock, 1993).
Activation of receptor tyrosine kinases such as those for epidermal growth factor (EGF), platelet-derived growth factor, or nerve growth factor converts the inactive, GDP-bound form of Ras to the active, GTP-bound form, and a dominant negative mutant of Ras interferes with signalling from such receptors. The mechanisms by which receptor tyrosine kinases and Ras are coupled, however, are not well understood. Many cytoplasmic proteins regulated by such receptors contain Src-homology (SH) 2 and 3 domains, and the SH2- and SH3-containing protein Grb2, like its homologue from C. elegans, Sem-5, appears to play an important role in the control of Ras by receptor tyrosine kinases. Overexpression of Grb2 potentiates the EGF-induced activation of Ras and mitogen-activated protein kinase by enhancing the rate of guanine nucleotide exchange on Ras. Cellular Grb2 appears to form a complex with a guanine-nucleotide-exchange factor for Ras, which binds to the ligand-activated EGF receptor, allowing the tyrosine kinase to modulate Ras activity (Gale, 1993).
A number of growth factors, including insulin and epidermal growth factor (EGF), induce accumulation of the GTP-bound form of p21ras. This accumulation could be caused either by an increase in guanine nucleotide exchange on p21ras or by a decrease in the GTPase activity of p21ras. To investigate whether insulin and EGF affect nucleotide exchange on p21ras, binding of [alpha-32P]GTP to p21ras was measured in cells permeabilized with streptolysin O. For this purpose, a cell line was used which expressed elevated levels of p21 H-ras and which was highly responsive to insulin and EGF. Stimulation with insulin or EGF resulted in an increase in the rate of nucleotide binding to p21ras. To determine whether this increased binding rate is due to the activation of a guanine nucleotide exchange factor, use was made of the inhibitory properties of a dominant negative mutant of p21ras, p21ras (Asn-17). Activation of p21ras by insulin and EGF in intact cells was abolished in cells infected with a recombinant vaccinia virus expressing p21ras (Asn-17). In addition, the enhanced nucleotide binding to p21ras in response to insulin and EGF in permeabilized cells was blocked upon expression of p21ras (Asn-17). From these data, it is conclude that the activation of a guanine nucleotide exchange factor is involved in insulin- and EGF-induced activation of p21ras (Medema, 1993).
The human protein Grb2 binds to ligand-activated growth factor receptors and downstream effector proteins through its respective Src-homology (SH) domains SH2 and SH3. Like its homolog from Caenorhabditis elegans, Sem-5, Grb2 apparently forms part of a highly conserved pathway by which these receptors can control Ras activity. The SH3 domains of Grb2 bind to the carboxy-terminal part of hSos1, the human homolog of the Drosophila guanine-nucleotide-releasing factor for Ras, which is essential for control of Ras activity by Epidermal growth factor receptor and Sevenless. A synthetic 10-amino-acid peptide containing the sequence PPVPPR specifically blocks the interaction. These results indicate that the Grb2/hSos1 complex couples activated EGF receptor to Ras signaling (Li, 1993).
Antisera against murine Son of sevenless (Sos) recognize a protein of M(r) 155,000 in rat-1 fibroblasts with specific guanine nucleotide exchange activity toward p21c-Ha-ras. Epidermal growth factor (EGF) receptor coimmunoprecipitates with Sos from EGF-stimulated, but not quiescent, cells. The SH2 and SH3 domain-containing 'adapter' protein Grb2 is also found in Sos immunoprecipitates in an EGF-inducible manner. In vitro reconstitution shows that Grb2 is required for the binding of activated EGF receptor to Sos. A phosphopeptide corresponding to tyrosine 1068 of the EGF receptor blocks both the assembly of the complex and EGF stimulation of nucleotide exchange on p21ras in a permeabilized cell system. These results suggest that EGF-induced activation of nucleotide exchange on p21ras proceeds through the recruitment of cytosolic Sos to a complex with EGF receptor and Grb2 at the plasma membrane (Buday, 1993).
The three proteins Grb2-Sem-5, Shc and Sos have been implicated in the signaling pathway that extends from tyrosine kinase receptors to Ras. Grb2-Sem-5 binds directly to murine Sos1, a Ras exchange factor, through two SH3 domains. Sos is also associated with ligand-activated tyrosine kinase receptors that bind Grb2-Sem-5, and with the Grb2-Sem-5 binding protein, Shc. Ectopic expression of Drosophila Sos stimulates morphological transformation of rodent fibroblasts. These data define a pathway by which tyrosine kinases act through Ras to control cell growth and differentiation (Egan, 1993).
It has been suggested that a key event in growth factor-induced p21Ras activation by the guanine nucleotide exchange factor Sos, is the recruitment of Sos to the plasma membrane by its interaction with the adaptor protein Grb2. However, other evidence argues that the sub cellular localization of Sos is independent of Grb2, and that the Sos/Grb2 interaction can be dispensed with for p21Ras activation. To clarify the role of the Sos/Grb2 interaction in ligand-stimulated p21Ras activation, the observation that overexpression of the Sos C-terminal domain can effectively inhibit p21Ras-dependent signaling in three different mammalian systems has been utilized. Concurrent expression of Grb2, but not SH2 or SH3 domain mutants of Grb2, or the alternative adaptor protein Nck (Drosophila homolog: Dreadlocks) can rescue this inhibitory effect of the C-terminus. This shows that the Grb2/Sos interaction is required to mediate growth factor-dependent activation of p21Ras, and requires the presence of intact SH2 and SH3 domains of Grb2. This approach was also used for a functional analysis of Sos which reveals that growth factor dependent signals are transmitted through both the N-terminal and C-terminal domains (Byrne, 1996).
The crystal structure of human H-Ras complexed with the Ras guanine-nucleotide-exchange-factor region of the Son of sevenless (Sos) protein has been determined at 2.8 A resolution. The normally tight interaction of nucleotides with Ras is disrupted by Sos in two ways. (1) The insertion into Ras of an alpha-helix from Sos results in the displacement of the Switch 1 region of Ras, opening up the nucleotide-binding site. (2) Side chains presented by this helix and by a distorted conformation of the Switch 2 region of Ras alter the chemical environment of the binding site for the phosphate groups of the nucleotide and the associated magnesium ion, so that their binding is no longer favoured. Sos does not impede the binding sites for the base and the ribose of GTP or GDP, so the Ras-Sos complex adopts a structure that allows nucleotide release and rebinding (Boriack-Sjodin, 1998).
Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. This study characterized the specific activity of individual SOS molecules catalyzing nucleotide exchange in H-Ras. Single-molecule kinetic traces revealed that SOS samples a broad distribution of turnover rates through stochastic fluctuations between distinct, long-lived (more than 100 seconds), functional states. The expected allosteric activation of SOS by Ras-guanosine triphosphate (GTP) was conspicuously absent in the mean rate. However, fluctuations into highly active states were modulated by Ras-GTP. This reveals a mechanism in which functional output may be determined by the dynamical spectrum of rates sampled by a small number of enzymes, rather than the ensemble average (Iversen, 2014).
The guanine nucleotide exchange factor Sos mediates the coupling of receptor tyrosine kinases to Ras activation. To investigate the mechanisms that control Sos activity, the contribution of various domains to its catalytic activity has been analyzed. Using human Sos1 (hSos1) truncation mutants, it has been shown that Sos proteins lacking either the amino or the carboxyl terminus domain, or both; they also display a guanine nucleotide exchange activity that is significantly higher compared with that of the full-length protein. The amino terminal domain of Sos is approximately 600 amino acids long and contains regions of homology to the Dbl (DH) and pleckstin (PH) domains. PH domains in Sos proteins have been implicated in the regulation of Sos guanine nucleotide exchange activity and ligand-dependent membrane targeting. The function of the DH domain is unknown. The catalytic activity of Sos is mediated by a central domain of approximately 420 amino acids that is highly conserved among different Ras exchange factors. The C-terminus domain of Sos proteins is characterized by the presence of multiple proline-rich SH3 binding sites, which mediate interaction with the adaptor molecule Grb2. Both the amino and the carboxyl terminus domains of Sos are involved in the negative regulation of Sos's catalytic activity. In vitro Ras binding experiments suggest that the amino and carboxyl terminus domains exert negative allosteric control on the interaction of the Sos catalytic domain with Ras. The guanine nucleotide exchange activity of hSos1 is not augmented by growth factor stimulation, indicating that Sos activity is constitutively maintained in a downregulated state. Deletion of both the amino and the carboxyl terminus domains is sufficient to activate the transforming potential of Sos. These findings suggest a novel negative regulatory role for the amino terminus domain of Sos and indicate a cooperation between the amino and the carboxyl terminus domains in the regulation of Sos activity (Corbalan-Garcia, 1998).
A fragment consisting of residues 584-1071 of the mouse Son-of-sevenless 1 (mSos1) protein was found to be sufficient for stimulation of the guanine nucleotide exchange of Ras in vitro, which defines the CDC25 homology (CDC25H) domain of mSos1. Furthermore, it was found that the CDC25H-domain fragment activates the extracellular signal-regulated protein kinases (ERKs), and is mainly membrane localized, when expressed in unstimulated human embryonic kidney 293 cells. Then, the roles of other mSos1 domains were examined in autoinhibition of the CDC25H-domain functions in unstimulated cellular environments. Longer fragments that have the CDC25H domain and the following proline-rich Grb2-binding domain exhibited negligible membrane localization, and accordingly much lower ERK-activation activities, under serum-starved conditions. In contrast, the preceding Pleckstrin-homology (PH) domain affects neither the ERK-activation activity nor the membrane-localization activity of the CDC25H domain. By contrast, the cells expressing a fragment containing the Dbl homology (DH) domain in addition to the PH and CDC25H domains exhibit remarkably low ERK activities under serum-starved conditions. This autoinhibitory effect of the DH domain on the CDC25H-domain function is relieved when cells are stimulated with epidermal growth factor. The DH-domain extension affects neither the in vitro guanine nucleotide exchange activity nor the membrane-localization activity of the CDC25H domain. Therefore, one of the roles of the DH domain is to exert an autoinhibition over the CDC25H-domain function on the cell membrane, in the absence, but not in the presence, of extracellular stimuli (Kim, 1998).
Guanine nucleotide exchange factors (GEFs) activate Ras proteins by stimulating the exchange of GTP for GDP in a multistep mechanism which involves binary and ternary complexes between Ras, guanine nucleotide, and GEF. Fluorescence measurements are presented to define the kinetic constants that characterize the interactions between Ras, GEF, and nucleotides. The dissociation constant for the binary complex between nucleotide-free Ras and the catalytic domain of mouse Cdc25, Cdc25(Mm285), was 4.6 nM, i.e., a 500-fold lower affinity than the Ras.GDP interaction. The affinities defining the ternary complex Ras. nucleotide.Cdc25(Mm285) are several orders of magnitude lower. The maximum acceleration by Cdc25(Mm285) of the GDP dissociation from Ras was more than 10(5)-fold. Kinetic measurements of the association of nucleotide to nucleotide-free Ras and to the binary complex Ras. Cdc25(Mm285) show that these reactions are practically identical: a fast binding step is followed by a reaction of the first order which becomes rate limiting at high nucleotide concentrations. The second reaction is thought to be a conformational change from a low- to a high-affinity nucleotide binding conformation in Ras. Taking into consideration all experimental data, the reverse isomerization reaction from a high- to a low-affinity binding conformation in the ternary complex Ras. GDP.Cdc25(Mm285) is postulated to be the rate-limiting step of the GEF-catalyzed exchange. Furthermore, the disruption of the Mg2+-binding site is not the only factor in the mechanism of GEF-catalyzed nucleotide exchange on Ras (Lenzen, 1998).
Guanine nucleotide exchange factors are essential components of the mode of action of GTPases involved in signal transduction. Their fundamental mechanism is generally accepted to derive from stabilization of the nucleotide-free form of GTPases, which is reflected in an increase in the rate of GDP dissociation when such an exchange factor is bound to a GTPase. The known kinetic properties of exchange factors can be explained on the basis of this simple allosteric competitive mechanism. This study describes experiments designed to distinguish this mechanism from a newer model, which invokes an active role for the incoming (i.e., displacing) nucleotide, implying the transient formation of a quaternary complex consisting of an exchange factor, a GTPase, and two nucleotides, one which is being displaced while the other stimulates this displacement. For a well-known system (the small GTPase Ras and its exchange factor Cdc25) there is no evidence for an effect of the concentration or the nature (i.e., GDP or GTP) of the displacing nucleotide on the rate constant of GDP release from the Cdc25.Ras.GDP complex, consistent with the simple allosteric competitive model, and in disagreement with the newer suggestion. In addition, arguments are presentedthat demonstrate how the erroneous conclusions leading to the alternative model were derived (Guo, 2005).
Ras GTPases function as binary switches in signaling pathways controlling cell growth and differentiation. The guanine nucleotide exchange factor Sos mediates the activation of Ras in response to extracellular signals. The crystal structure of nucleotide-free Ras in complex with the catalytic domain of Sos demonstrates that Sos induces conformational changes in two loop regions of Ras known as switch 1 and switch 2. Site-directed mutagenesis has been used to investigate the functional significance of the conformational changes for the catalytic function of Sos. Switch 2 of Ras is held in a very tight embrace by Sos, with almost every external side chain coordinated by Sos. Mutagenesis of contact residues at the switch 2-Sos interface shows that only a small set of side chains affect binding, with the most important contact being mediated by tyrosine 64, which is buried in a hydrophobic pocket of Sos in the Ras.Sos complex. Substitutions of Ras and Sos side chains that are inserted into the Mg(2+)- and nucleotide phosphate-binding site of switch 2 (Ras Ala(59) and Sos Leu(938) and Glu(942)) have no effect on the catalytic function of Sos. These results indicate that the interaction of Sos with switch 2 is necessary for tight binding, but is not the critical driving force for GDP displacement. The structural distortion of switch 1 induced by Sos is mediated by a small number of specific contacts between highly conserved residues on both Ras and Sos. Mutations of a subset of these residues (Ras Tyr(32) and Tyr(40)) result in an increase in the intrinsic rate of nucleotide dissociation from Ras and impair the binding of Ras to Sos. Based on this analysis, it is proposed that the interactions of Sos with the switch 1 and switch 2 regions of Ras have distinct functional consequences: the interaction with switch 2 mediates the anchoring of Ras to Sos, whereas the interaction with switch 1 leads to disruption of the nucleotide-binding site and GDP dissociation (Hall, 2001).
NCK, an SH2- and SH3 domain-containing protein, becomes phosphorylated and associated with tyrosine kinase receptors upon growth factor stimulation. The sequence of NCK suggests that NCK functions as a linker between receptors and a downstream signaling molecule. To determine if NCK can mediate growth factor-stimulated responses, the ability of NCK to activate the fos promoter was measured. In NIH 3T3 cells, NCK strongly activates this promoter. The effect of NCK on the fos promoter is enhanced by c-ras and blocked by dominant negative ras. NCK binds directly to the guanine nucleotide exchange factor SOS. This interaction is mediated by the SH3 domains of NCK. These findings suggest that NCK can regulate p21ras-dependent gene transcription through interaction with SOS protein (Hu, 1995).
In response to stimulation with epidermal growth factor (EGF), the guanine nucleotide exchange factor human SOS1 (hSOS1) promotes the activation of Ras by forming a complex with Grb2 and the human EGF receptor (hEGFR). hSOS1 is phosphorylated in cells stimulated with EGF or phorbol 12-myristate 13-acetate or following co-transfection with activated Ras or Raf. Co-transfection with dominant negative Ras results in a decrease of EGF-induced hSOS1 phosphorylation. The mitogen-activated protein kinase (MAPK) phosphorylates hSOS1 in vitro within the carboxyl-terminal proline-rich domain. The same region of hSOS1 is phosphorylated in vivo, in cells stimulated with EGF. Tryptic phosphopeptide mapping shows that MAPK phosphorylates hSOS1 in vitro on sites that were also phosphorylated in vivo. Phosphorylation by MAPK does not affect hSOS1 binding to Grb2 in vitro. However, reconstitution of the hSOS1-Grb2-hEGFR complex shows that phosphorylation by MAPK markedly reduced the ability of hSOS1 to associate with the hEGFR through Grb2. Similarly, phosphorylated hSOS1 is unable to form a complex with Shc through Grb2. Thus phosphorylation of hSOS1 down-regulates signal transduction from the hEGFR to the Ras pathway, by affecting hSOS1's interaction with the hEGFR or Shc (Porfiri, 1996).
The Son of Sevenless (Sos) proteins control receptor-mediated activation of Ras by catalyzing the exchange of guanosine diphosphate for guanosine triphosphate on Ras. The NH2-terminal region of Sos contains a Dbl homology (DH) domain in tandem with a pleckstrin homology (PH) domain. In COS-1 cells, the DH domain of Sos stimulated guanine nucleotide exchange on Rac but not Cdc42 in vitro and in vivo. The tandem DH-PH domain of Sos (DH-PH-Sos) is defective in Rac activation but regains Rac stimulating activity when it is coexpressed with activated Ras. Ras-mediated activation of DH-PH-Sos does not require activation of mitogen-activated protein kinase but it is dependent on activation of phosphoinositide 3-kinase. These results reveal a potential mechanism for coupling of Ras and Rac signaling pathways (Nimnual, 1998).
Vulval induction in C. elegans has helped define an evolutionarily conserved signal transduction pathway from receptor tyrosine kinases (RTKs) through the adaptor protein SEM-5 to RAS. One component present in other organisms, a guanine nucleotide exchange factor for Ras, has been missing in C. elegans. To understand the regulation of this pathway it is crucial to have all positive-acting components in hand. The identification, cloning and genetic characterization of C. elegans SOS-1, a putative guanine nucleotide exchanger for LET-60 RAS, is described. RNA interference experiments suggest that SOS-1 participates in RAS-dependent signaling events downstream of LET-23 EGFR, EGL-15 FGFR and an unknown RTK. The previously identified let-341 gene encodes SOS-1. Analyzing vulval development in a let-341 null mutant, an SOS-1-independent pathway has been found that is involved in the activation of RAS signaling. This SOS-1-independent signaling is not inhibited by SLI-1/Cbl and is not mediated by PTP-2/SHP, raising the possibility that there could be another RasGEF (Chang, 2000).
Phospholipase C-gamma1 (PLC-gamma1: see Drosophila Small wing) hydrolyzes phosphatidylinositol 4,5-bisphosphate to the second messengers inositol 1,4,5-trisphosphate and diacylglycerol (DAG). PLC-gamma1 is implicated in a variety of cellular signalings and processes including mitogenesis and calcium entry. However, numerous studies demonstrate that the lipase activity is not required for PLC-gamma1 to mediate these events. The phospholipase activity of PLC-gamma1 plays an essential role in nerve growth factor (NGF)-triggered Raf/MEK/MAPK pathway activation in PC12 cells. Employing PC12 cells stably transfected with an inducible form of wild-type PLC-gamma1 or lipase inactive PLC-gamma1 with histidine 335 mutated into glutamine in the catalytic domain, it is shown that NGF provokes robust activation of MAP kinase in wild-type but not in lipase inactive cells. Both Ras/C-Raf/MEK1 and Rap1/B-Raf/MEK1 pathways are intact in the wild-type cells. By contrast, these signaling cascades are diminished in the mutant cells. Pretreatment with cell permeable DAG analog 1-oleyl-2-acetylglycerol rescues the MAP kinase pathway activation in the mutant cells. These observations indicate that the lipase activity of PLC-gamma1 mediates NGF-regulated MAPK signaling upstream of Ras/Rap1 activation probably through second messenger DAG-activated Ras and Rap-GEFs (Rong, 2004).
Growth factor receptors activate Ras by recruiting the nucleotide exchange factor son of sevenless (SOS) to the cell membrane, thereby triggering the production of GTP-loaded Ras. Crystallographic analyses of Ras bound to the catalytic module of SOS have led to the unexpected discovery of a highly conserved Ras binding site on SOS that is located distal to the active site and is specific for Ras-GTP. The crystal structures suggest that Ras-GTP stabilizes the active site of SOS allosterically, Ras-GTP is shown to form ternary complexes with SOScat in solution and increases significantly the rate of SOScat-stimulated nucleotide release from Ras. These results demonstrate the existence of a positive feedback mechanism for the spatial and temporal regulation of Ras (Margarit, 2003; full text of article).
The sequence of human SOS1 was compared to the sequences of SOS from two insects, Drosophila melanogaster, and Anopheles gambiae, and to that of the worm Caenorhabditis elegans. The SOS protein is highly conserved throughout the REM and cdc25 domains. The REM (Ras exchanger motif) domain and cdc25 domain (so named because of sequence similarity to cdc25, the Ras-specific nucleotide exchange factor in Saccharomyces cerevisiae) of human SOS1 are 52% and 61% identical in sequence, respectively, to the corresponding domains in the D. melanogaster SOS protein. For the human-A. gambiae and human-C. elegans comparisons, the levels of sequence identity are 54% and 63% (A. gamabiae) and 34% and 42% (C. elegans), respectively, for the two domains. Examination of sequence variation on the surface of SOScat reveals that in addition to a patch of highly conserved residues at the active site of SOScat, there is also a patch of conserved residues on the surface extending out of the active site and into the REM domain, surrounding the distal surface of SOScat. At the levels of sequence identity which pertain here (35%-55% overall identity in the pairwise comparisons), it is expected that residues in the hydrophobic core and the active site of SOScat will be highly conserved but that residues on the surface will not be conserved unless they have functional importance. In particular, it is significant that there is a striking conservation in the surface-exposed residues of SOScat that make contact with the distal Ras-GTP (Margarit, 2003).
Several of the residues in SOS that interact with the distal Ras are invariant across all four species. Arg 688, which forms hydrogen bonds with Glu-37 in the Switch 1 region of Ras, is invariant. The two cis-prolines in the hairpin base that forms the core of the cdc25 interface with distal Ras are part of a sequence motif (921SINPPC926 in human SOS1) that is invariant across all four sequences, suggesting that the unusual turn structure is conserved. The conservation of these features of the distal binding site raises the possibility that the distal interaction with Ras-GTP may also be conserved (Margarit, 2003).
A striking feature of the ternary Ras-GTP:SOScat:Ras complex is a network of tightly linked interactions, which span the REM and cdc25 domains and Ras-GTP and are suggestive of an allosteric mechanism whereby Ras-GTP stabilizes SOScat and stimulates its exchange factor activity. The rate of release of fluorescent nucleotide derivatives bound to Ras was measured in the presence of SOScat and if was found that the addition of Ras-GTP significantly accelerates the rate of SOScat-stimulated GDP release from Ras, whereas the addition of Ras-GDP does not. Taken together, these results point to the presence of a hitherto unsuspected positive feedback mechanism in the activation of Ras by SOS (Margarit, 2003).
The classical model for the activation of the nucleotide exchange factor Son of sevenless (SOS) involves its recruitment to the membrane, where it engages Ras. The recent discovery that RasGTP is an allosteric activator of SOS indicates that the regulation of SOS is more complex than originally envisaged. Crystallographic and biochemical analyses are presented of a construct of SOS that contains the Dbl homology-pleckstrin homology (DH-PH) and catalytic domains; the DH-PH unit blocks the allosteric binding site for Ras and suppresses the activity of SOS. SOS is dependent on Ras binding to the allosteric site for both a lower level of activity, which is a result of RasGDP binding, and maximal activity, which requires RasGTP. The action of the DH-PH unit gates a reciprocal interaction between Ras and SOS, in which Ras converts SOS from low activity form to high activity form as RasGDP is converted to RasGTP by SOS (Sondermann, 2004).
The signaling protein Ras is a molecular switch that cycles between inactive GDP bound and active GTP bound states. Receptors that signal through tyrosine kinases activate Ras by recruiting the Ras-specific nucleotide exchange factor Son of sevenless (SOS) to the plasma membrane, where SOS and Ras form a complex that results in the expulsion of otherwise tightly bound nucleotides from Ras. Ras is kept under strict control in the cell, and the unregulated activation of Ras is a consistent hallmark of many cancers (Sondermann, 2004 and references therein).
SOS is a complex multidomain protein of about 1330 residues. The N-terminal domain (200 residues) contains two tandem histone folds of unknown function and is followed by a Dbl homology (DH) domain (200 residues) and a pleckstrin homology (PH) domain (150 residues) that together are implicated in the activation of the small GTPase Rac1. The next two domains are both required for the Ras-specific nucleotide exchange activity of SOS and are always found together in other Ras-specific nucleotide exchange factors. The first of these is the Ras exchanger motif (Rem) domain (200 residues), which is followed by the Cdc25 domain (300 residues; named for homology to Cdc25, the Ras activator protein in yeast). These two domains together are referred to as SOScat. Finally, the 250 residues in the C-terminal region provide docking sites for adaptor proteins such as Grb2 (Sondermann, 2004 and references therein).
The structure of nucleotide-free Ras in complex with SOScat shows that Ras is bound in such a way that its nucleotide binding site is almost completely disrupted. The interaction between Ras and SOS is localized entirely to the Cdc25 domain, and the position and function of the Rem domain, which interacts with a surface of the Cdc25 domain that is distal to the active site, was puzzling at first. A recent crystallographic study uncovered a role for the Rem domain in a previously unsuspected allosteric mechanism in SOS. RasGTP, the product of the exchange reaction, interacts with a distal binding site on SOScat that is between the Rem and Cdc25 domains, thereby forming a bridge between these two domains. Binding of RasGTP to this distal allosteric site results in increased Ras exchange activity, indicating the presence of a positive feedback loop in the activation of Ras by SOS (Sondermann, 2004).
Initial models for the regulation of SOS emphasized its recruitment to the membrane as the key step of activation, since Ras is membrane bound. The regulation of SOS is likely to be more complex than simple membrane recruitment. In addition to the Grb2-mediated recruitment of SOS to the plasma membrane, early experiments have suggested a role for the N-terminal segment of SOS in its activation. Deletion of the C-terminal docking segment or the N-terminal 550 amino acids (including the histone domain and the DH-PH unit) increases SOS activity in cellular assays. These results, as well as a recent genetic study of Drosophila SOS (Silver, 2004), suggest that there is a complex but poorly characterized interplay between the domains of SOS that results in modulation of the ability of SOS to activate Ras (Sondermann, 2004).
The present study investigates a construct of SOS (SOSDH-PH-cat) that contains the DH-PH unit in addition to the catalytic unit (SOScat). By determining the structure of SOSDH-PH-cat, it has been shown that the DH-PH unit inhibits SOS by blocking the distal allosteric RasGTP binding site of SOS. Surprisingly, blockage of the allosteric Ras binding site suppresses both the unstimulated (by RasGTP) and the allosterically stimulated levels of activity of SOS, leading to the discovery that the basal level of SOS activity is dependent on the binding of RasGDP to the distal site. It appears that the SOS protein has evolved to have its nucleotide exchange activity be masked until as yet undiscovered signals trigger the displacement of the DH-PH unit and the opening of the allosteric site, allowing Ras itself to stimulate SOS to first low and then high levels of activity (Sondermann, 2004).
Co-immunoprecipitation of whole cell extracts demonstrated that the guanylnucleotide exchange factor SOS is associated with the small adapter proteins Grb2, CrkII, and Nck. In vitro binding indicates a similar binding affinity of SOS for all three adapter proteins but with a slightly lower Kd for Grb2 (approximately 2.5-fold) compared with Nck and CrkII. Insulin stimulation results in co-immunoprecipitation of tyrosine-phosphorylated IRS1 with Grb2 and to a lesser extent CrkII. Although Grb2 also associates with tyrosine-phosphorylated Shc, there was no detectable interaction of CrkII with Shc. In contrast, EGF stimulation results in the predominant co-immunoprecipitation of Grb2 with the EGF receptor, whereas CrkII primarily associates with an unidentified 120-130-kDa protein. Similar to the ability of insulin to induce the dissociation of the Grb2-SOS complex, there is a concomitant time-dependent dissociation of the CrkII-SOS and Nck-SOS complexes. However, EGF stimulation has no effect on the association state of the Grb2-SOS or the Nck-SOS complexes but does result in a time-dependent dissociation of the CrkII from SOS. Together, these data demonstrate that different cellular pools of SOS associate with different adapter proteins forming various signaling complexes, each undergoing distinct patterns of assembly/disassembly following growth factor stimulation (Okada, 1996).
Intersectin is a member of a growing family of adaptor proteins that possess conserved Eps15 homology (EH) domains as well as additional protein recognition motifs. In general, EH domain-containing proteins play an integral role in clathrin-mediated endocytosis. Indeed, intersectin functions in the intermediate stages of clathrin-coated vesicle assembly. However, recent evidence suggests that components of the endocytic machinery also regulate mitogenic signaling pathways. Evidence is provided that intersectin has the capacity to activate mitogenic signaling pathways. (1) Intersectin overexpression activates the Elk-1 transcription factor in an MAPK-independent manner. This ability resides within the EH domains, since expression of the tandem EH domains is sufficient to activate Elk-1. (2) Intersectin cooperates with epidermal growth factor to potentiate Elk-1 activation; however, a similar level of Elk-1 activation is obtained by expression of the tandem EH domains suggesting that the coiled-coil region and SH3 domains act to regulate the EH domains. (3) Intersectin expression is sufficient to induce oncogenic transformation of rodent fibroblasts. And (4), intersectin cooperates with progesterone to accelerate maturation of Xenopus laevis oocytes. Together, these data suggest that intersectin links endocytosis with regulation of pathways important for cell growth and differentiation (Adams, 2000).
Intersectin is a multiple EH and SH3 domain-containing protein, that serves as a component of the endocytic machinery. Overexpression of the SH3 domains of intersectin blocks transferrin receptor endocytosis, possibly by disrupting targeting of accessory proteins of clathrin-coated pit formation. Mammalian Sos, a guanine-nucleotide exchange factor for Ras, is an intersectin SH3 domain-binding partner. Overexpression of intersectin's SH3 domains blocks activation of Ras and MAP kinase in various cell lines. Several studies suggest that activation of MAP kinase downstream of multiple receptor types is dependent on endocytosis. Thus, the dominant-negative effect of the SH3 domains on Ras/MAP kinase activation may be indirectly mediated through a block in endocytosis. Consistent with this idea, incubating cells at 4°C or with phenylarsine oxide, a treatment that inhibits EGF receptor endocytosis, blocks EGF-dependent activation of MAP kinase. However, under these conditions, Ras activity is unaffected and overexpression of the SH3 domains of intersectin is still able to block Ras activation. Thus, intersectin SH3 domain overexpression can effect EGF-mediated MAP kinase activation directly through a block in Ras, consistent with a functional role for intersectin in Ras activation (Tong, 2000a).
Intersectin, a protein containing two EH and five SH3 domains, has been idnetified as a component of the endocytic machinery. The N-terminal SH3 domain (SH3A), unlike other SH3 domains from intersectin or various endocytic proteins, specifically inhibits intermediate events leading to the formation of clathrin-coated pits. A brain-enriched, 170 kDa protein (p170) interacts specifically with SH3A. Screening of combinatorial peptides reveals the optimal ligand for SH3A as Pp(V/I)PPR. The 170 kDa mammalian son-of-sevenless (mSos1) protein, a guanine-nucleotide exchange factor for Ras, contains two copies of the matching sequence, PPVPPR. Immunodepletion studies confirm that p170 is mSos1. Intersectin and mSos1 are co-enriched in nerve terminals and are co-immunoprecipitated from brain extracts. SH3A competes with the SH3 domains of Grb2 in binding to mSos1, and the intersectin-mSos1 complex can be separated from Grb2 by sucrose gradient centrifugation. Overexpression of the SH3 domains of intersectin blocks epidermal growth factor-mediated Ras activation. These results suggest that intersectin functions in cell signaling in addition to its role in endocytosis and may link these cellular processes (Tong, 2000b).
Given that intersectin is involved in the formation of clathrin-coated pits, and that the intersectin SH3A domain interacts specifically with cellular targets that function early in the formation of a clathrin-coated bud, it is interesting to speculate that mSos1 may also play a role in clathrin-coated pit formation, possibly through activation of Ras. Many vesicular budding events that are mediated by coat proteins are initiated by the activation of small GTP-binding proteins through the actions of guanine-nucleotide exchange factors. Overexpression of mutant forms of Ras, as well as the small GTP-binding protein Ral, which plays a major role in mediating downstream Ras function, blocks the internalization of the EGF receptor. Furthermore, mSos1 can activate Rac, which has been implicated in transferrin receptor endocytosis. Finally, it should be noted that the long form of intersectin, which is generated by alternative splicing in neuronal tissues, contains DH, PH and C2 domains. Comparison of the primary structure of the DH and PH domains with other proteins suggests that the long form of intersectin may be a guanine-nucleotide exchange factor for Rho. Further work is necessary to clarify the involvement of GTP-binding proteins in clathrin-mediated endocytosis (Tong, 2000b and references therein).
Another possible function of the intersectin-mSos1 complex is to couple the molecular machineries for endocytosis and signal transduction. For example, it has been demonstrated that dynamin-dependent endocytosis of the EGF receptor is necessary for EGF-dependent activation of the MAP-kinase pathway. The ability of insulin-like growth factor-1 (IGF-1) to activate the SHC/MAP-kinase pathway, but not the insulin receptor substrate 1 pathway, is also dependent on clathrin-mediated endocytosis of the IGF receptor. Furthermore, endocytosis of the beta2-adrenergic receptor is necessary for coupling to MAP-kinase activation. Specifically, overexpression of a mutant form of beta-arrestin, which prevents the beta2-adrenergic receptor from targeting to clathrin-coated pits, blocks agonist activation of MAP kinase. Thus, it is possible that the clathrin-coated pit can function as a membrane microdomain, directing the assembly of signaling complexes, much as has been proposed for caveoli. In fact, activation of the EGF receptor can lead to the formation of signaling complexes that include mSos1. Such signaling complexes are localized largely in endosomes (Tong, 2000b and references therein).
Given the evidence for a link between endocytosis and signaling, it is interesting to speculate that intersectin could play an important role in bringing together endocytic proteins such as dynamin, with signaling molecules such as mSos1. In fact, the data demonstrating that overexpression of the SH3 domains of intersectin functions in a dominant-negative manner to block EGF-dependent Ras activation strongly support a role for intersectin in cell signaling. Moreover, human intersectin has been found to interact by yeast two-hybrid screening with the proto-oncogene product, c-Cbl, a tyrosine kinase substrate with ubiquitin ligase activity, and transfection experiments have revealed that full-length intersectin functions in cell signaling pathways leading to activation of the Elk-1 transcription factor. Finally, in a genetic screen in Drosophila, Dap160, the Drosophila homolog of intersectin, was selected as a negatively regulating component of the Sevenless receptor tyrosine kinase/MAP-kinase pathway (Rintelen and Hafen, personal communication to Tong, 2000b). Thus, intersectin appears to have a dual function in both endocytosis as well as signal transduction pathways, and it may play a role as an interface between these two important cellular processes (Tong, 2000b and references therein).
Endocytosis is a regulated physiological process by which cell surface proteins are internalized along with extracellular factors such as nutrients, pathogens, peptides, toxins, etc. The process begins with the invagination of small regions of the plasma membrane that ultimately form intracellullar vesicles. These internalized vesicles may shuttle back to the plasma membrane to recycle the membrane components or they may be targeted for degradation. One role for endocytosis is in the attenuation of receptor signaling. For example, desensitization of activated membrane bound receptors such as G-protein coupled receptors (GPCRs) or receptor tyrosine kinases (RTKs) occurs, in part, through endocytosis of the activated receptor. However, accumulating evidence suggests that endocytosis also mediates intracellular signaling. Endocytosis is a critical component in cellular signal transduction, both in the initiation of a signal as well as in the termination of a signal. The adaptor protein, intersectin (ITSN) provides a link to both the endocytic and the mitogenic machinery of a cell. ITSN functions at a crossroad in the biochemical regulation of cell function (O'Bryan, 2001).
ITSN (also known as Ese-1, EHSH-1, Dap-160) has been isolated by a number of groups based on its ability to bind proline-rich peptides (ITSN), to form a complex with dynamin. ITSN is a 145 kDa adaptor protein consisting of two amino-terminal Eps15 homology domains (EH), a central coiled-coil domain (CC) and has five tandem Src homology 3 domains (SH3). EH domains promote the interaction with Asp-Pro-Phe sequences and are present in numerous endocytic accessory proteins. CC domains promote both homo- and hetero-typic interactions with other CC-containing proteins and are also widely distributed. SH3 domains recognize Pro-rich sequences within target proteins and are present in a variety of cytoskeletal and signaling proteins. ITSN is conserved throughout evolution with homologues present in humans, rodents, Xenopus, Drosophila and likely C. elegans. ITSN proteins are present predominantly in the nervous system with lower expression elsewhere. In addition, there is a larger (~200 kDa) isoform of ITSN, termed intersectin-long (ITSN-L), that is predominantly expressed in the nervous system. This iso-form is derived by alternative RNA splicing. ITSN-L possesses a carboxy-terminal extension encoding a Dbl homology domain (DH), a pleckstrin homology domain (PH) and a C2 domain. DH domains function as guanine nucleotide exchange factors (GEFs) for the Rho subfamily of Ras-like GTPases, whose members include Rho, Rac and Cdc42. These domains function in concert with PH domains which direct interaction with lipids and membrane. Thus, ITSN-L may serve to regulate Rho family activation within the nervous system. C2 domains bind phospholipid membranes, proteins or soluble inositol polyphosphates using both Ca 2+ -dependent and -independent mechanisms. Interestingly, EH domains also bind Ca2+ although an importance for this activity in the function of this domain has not been demonstrated. An ITSN-related protein, termed ITSN-2/Ese-2, has also been identified. ITSN-2 shares a similar structural architecture with ITSN-1 possessing both long and short isoforms. In contrast to ITSN-1L, ITSN-2L appears to be more widely expressed suggesting that this isoform is a more general regulator of Rho family members (O'Bryan, 2001).
The domain structure of ITSN-1 suggests that this protein may act as a scaffolding or adaptor protein that regulates various biochemical pathways. ITSN localizes to clathrin coated pits (CCPs) via the EH region, suggesting that it may serve to assemble multiprotein complexes at sites of CCP formation. Indeed, ITSN associates directly with a number of proteins including epsin, secretory carrier membrane protein 1 (SCAMP1), HIV Rev binding protein, Eps15, SNAP-23/25, dynamin, synaptojanin and Sos, a Ras GEF. Several lines of evidence suggest that ITSN does indeed regulate cellular signaling pathways. (1) ITSN, through its SH3 domains, forms a stable complex with Sos both in vitro and in vivo. (2) ITSN directly activates mitogenic signaling pathways. (3) Overexpression of ITSN is sufficient to induce morphological transformation of rodent fibroblast as well as accelerate hormone-induced Xenopus oocyte maturation in culture. (4) ITSN-L possesses an exchange factor domain for the Rho family of GTPases suggesting that ITSN may regulate Rho, Rac or Cdc42 as well as Ras. Together these data provide compelling evidence that ITSN directly participates in cellular signaling (O'Bryan, 2001).
The non-receptor tyrosine kinase Abl participates in receptor tyrosine kinase (RTK)-induced actin cytoskeleton remodelling, a signalling pathway in which the function of Rac is pivotal. More importantly, the activity of Rac is indispensable for the leukaemogenic ability of the BCR-Abl oncoprotein. Thus, Rac might function downstream of Abl and be activated by it. This study elucidates the molecular mechanisms through which Abl signals to Rac in RTK-activated pathways. Sos-1, a dual guanine nucleotide-exchange factor (GEF), is phosphorylated on tyrosine, after activation of RTKs, in an Abl-dependent manner. Sos-1 and Abl interact in vivo, and Abl-induced tyrosine phosphorylation of Sos-1 is sufficient to elicit its Rac-GEF activity in vitro. Genetic or pharmacological interference with Abl (and the related kinase Arg) results in a marked decrease in Rac activation induced by physiological doses of growth factors. Thus, these data identify the molecular connections of a pathway RTKs-Abl-Sos-1-Rac that is involved in signal transduction and actin remodelling (Sini, 2004).
Signaling from receptor tyrosine kinases (RTKs) requires the sequential activation of the small GTPases Ras and Rac. Son of sevenless (Sos-1), a bifunctional guanine nucleotide exchange factor (GEF), activates Ras in vivo and displays Rac-GEF activity in vitro, when engaged in a tricomplex with Eps8 and E3b1-Abi-1, a RTK substrate and an adaptor protein, respectively. A mechanistic understanding of how Sos-1 coordinates Ras and Rac activity is, however, still missing. This study demonstrate that (a) Sos-1, E3b1, and Eps8 assemble into a tricomplex in vivo under physiological conditions; (b) Grb2 and E3b1 bind through their SH3 domains to the same binding site on Sos-1, thus determining the formation of either a Sos-1-Grb2 (S/G) or a Sos-1-E3b1-Eps8 (S/E/E8) complex, endowed with Ras- and Rac-specific GEF activities, respectively; (c) the Sos-1-Grb2 complex is disrupted upon RTKs activation, whereas the S/E/E8 complex is not; and (d) in keeping with the previous result, the activation of Ras by growth factors is short-lived, whereas the activation of Rac is sustained. Thus, the involvement of Sos-1 at two distinct and differentially regulated steps of the signaling cascade allows for coordinated activation of Ras and Rac and different duration of their signaling within the cell (Innocenti, 2002).
Class I phosphoinositide 3-kinases (PI3Ks) are implicated in many cellular responses controlled by receptor tyrosine kinases (RTKs), including actin cytoskeletal remodeling. Within this pathway, Rac is a key downstream target/effector of PI3K. However, how the signal is routed from PI3K to Rac is unclear. One possible candidate for this function is the Rac-activating complex Eps8-Abi1-Sos-1, which possesses Rac-specific guanine nucleotide exchange factor (GEF) activity. Abi1 (also known as E3b1) recruits PI3K, via p85, into a multimolecular signaling complex that includes Eps8 and Sos-1. The recruitment of p85 to the Eps8-Abi1-Sos-1 complex and phosphatidylinositol 3, 4, 5 phosphate (PIP3), the catalytic product of PI3K, concur to unmask its Rac-GEF activity in vitro. Moreover, they are indispensable for the activation of Rac and Rac-dependent actin remodeling in vivo. On growth factor stimulation, endogenous p85 and Abi1 consistently colocalize into membrane ruffles, and cells lacking p85 fail to support Abi1-dependent Rac activation. These results define a mechanism whereby propagation of signals, originating from RTKs or Ras and leading to actin reorganization, is controlled by direct physical interaction between PI3K and a Rac-specific GEF complex (Innocetti, 2003).
Tyrosine kinase receptors stimulate the Ras signaling pathway by enhancing the activity of the SOS nucleotide-exchange factor. This occurs, at least in part, by the recruitment of an SOS-GRB2 complex to Ras in the plasma membrane. A different signaling pathway to Ras exists that involves activation of the Ras-GRF exchange factor in response to Ca2+ influx. The ability of Ras-GRF to activate Ras in vivo is markedly enhanced by raised Ca2+ concentrations. Activation is mediated by calmodulin binding to an IQ motif in Ras-GRF, because substitutions in conserved amino acids in this motif prevent both calmodulin binding to Ras-GRF and Ras-GRF activation in vivo. So far, full-length Ras-GRF has been detected only in brain neurons. These findings implicate Ras-GRF in the regulation of neuronal functions that are influenced by Ca2+ signals (Farnsworth, 1995).
Members of the Ras subfamily of small guanine-nucleotide-binding proteins are essential for controlling normal and malignant cell proliferation as well as cell differentiation. The neuronal-specific guanine-nucleotide-exchange factor, Ras-GRF/CDC25Mm, induces Ras signaling in response to Ca2+ influx and activation of G-protein-coupled receptors in vitro, suggesting that it plays a role in neurotransmission and plasticity in vivo. Ras-GRF is exclusively expressed in neurons of the postnatal and adult central nervous system and is mainly localized in the synaptosomal fraction. Following activation of muscarinic M1 and M2 receptors, Ras-GRF becomes phosphorylated; this increases its exchange activity. Instead of presenting a Grb2-binding domain, Ras-GRF contains an ilimaquinone domain. When intracellular calcium is increased, this domain is necessary for binding to Ca2+ calmodulin and for Ras-GRF-dependent activation of the Ras/MAPK pathway. Mice lacking Ras-GRF are impaired in the process of memory consolidation, as revealed by emotional conditioning tasks that require the function of the amygdala; learning and short-term memory are intact. Electrophysiological measurements in the basolateral amygdala reveal that long-term plasticity is abnormal in mutant mice. In contrast, Ras-GRF mutants do not reveal major deficits in spatial learning tasks, such as the Morris water maze, a test that requires hippocampal function. Consistent with apparently normal hippocampal functions, Ras-GRF mutants show normal NMDA (N-methyl-D-aspartate) receptor-dependent long-term potentiation in this structure. These results implicate Ras-GRF signaling via the Ras/MAP kinase pathway in synaptic events leading to formation of long-term memories (Brambilla, 1997).
Ras and Rac are membrane-associated GTPases that function as molecular switches activating intracellular mitogen-activated protein kinase (MAPK) cascades and other effector pathways in response to extracellular signals. Activation of Ras and Rac into their GTP-bound conformations is directly controlled by specific guanine-nucleotide exchange factors (GEFs), which catalyze GDP release. Several Ras-specific GEFs that are related to the budding yeast protein Cdc25p have been described, whereas GEFs for Rac-related GTPases contain a region that is homologous to the oncoprotein DbI. The Ras-GRF1 and Ras-GRF2 proteins, which couple Ras activation to serpentine receptors and calcium signals, contain both Cdc25 and DbI homology (DH) regions. Ras-GRF2 is a bifunctional signaling protein that is able to bind and activate Ras and Rac, and thereby coordinate the activation of the extracellular-signal-regulated kinase (ERK) and stress-activated protein kinase (SAPK) pathways (Fan, 1998).
p140 Ras-GRF1 and p130 Ras-GRF2 constitute a family of calcium/calmodulin-regulated guanine-nucleotide exchange factors that activate the Ras GTPases. Studies on mice lacking these exchange factors revealed that both p140 Ras-GRF1 and p130 Ras-GRF2 couple NMDA glutamate receptors (NMDARs) to the activation of the Ras/Erk signaling cascade and to the maintenance of CREB transcription factor activity in cortical neurons of adult mice. Consistent with this function for Ras-GRFs and the known neuroprotective effect of CREB activity, ischemia-induced CREB activation is reduced in the brains of adult Ras-GRF knockout mice and neuronal damage is enhanced. Interestingly, in cortical neurons of neonatal animals NMDARs signal through Sos rather than Ras-GRF exchange factors, implying that Ras-GRFs endow NMDARs with functions unique to mature neurons (Tian, 2004).
Two important Ras guanine nucleotide exchange factors, Son of sevenless (Sos) and Ras guanine nucleotide releasing protein (RasGRP), have been implicated in controlling Ras activation when cell surface receptors are stimulated. To address the specificity or redundancy of these exchange factors, Sos1/Sos2 double- or RasGRP3-deficient B cell lines were generated and their ability to mediate Ras activation upon B cell receptor (BCR) stimulation was determined. The BCR requires RasGRP3; in contrast, epidermal growth factor receptor is dependent on Sos1 and Sos2. Furthermore, BCR-induced recruitment of RasGRP3 to the membrane and the subsequent Ras activation are significantly attenuated in phospholipase C-gamma2-deficient B cells. This defective Ras activation is suppressed by the expression of RasGRP3 as a membrane-attached form, suggesting that phospholipase C-gamma2 regulates RasGRP3 localization and thereby Ras activation (Oh-hora, 2003).
The PDZ domain-containing proteins, such as PSD-95 and GRIP, have been suggested to be involved in the targeting of glutamate receptors, a process that plays a critical role in the efficiency of synaptic transmission and plasticity. To address the molecular mechanisms underlying AMPA receptor synaptic localization, several GRIP-associated proteins (GRASPs) have been identified that bind to distinct PDZ domains within GRIP. GRASP-1 is a neuronal rasGEF associated with GRIP and AMPA receptors in vivo. Overexpression of GRASP-1 in cultured neurons specifically reduces the synaptic targeting of AMPA receptors. In addition, the subcellular distribution of both AMPA receptors and GRASP-1 is rapidly regulated by the activation of NMDA receptors. These results suggest that GRASP-1 may regulate neuronal ras signaling and contribute to the regulation of AMPA receptor distribution by NMDA receptor activity (Ye, 2000).
LTP and LTD have been proposed to be mediated, in part, by changes in AMPA receptor function. Increases in AMPA receptor responses have been observed during the expression of LTP. Recently, it has been shown that a high proportion of synapses in hippocampal CA1 region contains only NMDA receptors and acquires AMPA receptors only after the induction of LTP. This emergence of AMPA receptor current seems due to the appearance of synaptic AMPA receptors. Moreover, NMDA receptor-dependent LTD in cultured neurons has recently been observed to correlate with a decrease in the levels of synaptic AMPA receptors. Previous studies have suggested that AMPA receptor-associated proteins, such as GRIP, are involved in the synaptic targeting of AMPA receptors. In this study, GRASP-1 has been added to this complex and evidence is provided that GRASP-1 may also be important in regulation of AMPA receptor function and may play a role in AMPA receptor synaptic targeting. Overexpression of GRASP-1 in neurons downregulates synaptic AMPA receptor clusters, while it has no effect on synaptic NMDA receptor synaptic targeting. Both the rasGEF catalytic domain and the C-terminal 'regulatory' domain were required for this activity. Activation of NMDA receptors dramatically induces the redistribution of both GRASP-1 and AMPA receptors from punctate membrane structures to a more diffuse pattern. Together with the GRASP-1 overexpression data, these results suggest that the overall spatial distribution of GRASP-1, as well as the absolute levels, may be important for AMPA receptor targeting. These results suggest that GRASP-1 and possibly ras signaling may play a role in the regulation of AMPA receptor synaptic targeting and its regulation by NMDA receptor activity (Ye, 2000).
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