Insulin-induced desensitization of extracellular signal-regulated kinase activation results from an inhibition of Raf activity independent of Ras activation and dissociation of the Grb2-SOS complex.

Previous studies have suggested that the interaction between the small adaptor protein Grb2 with the Ras guanyl nucleotide exchange factor SOS is functionally important in the regulation of the Ras activation/inactivation cycle. To examine the relationship between the Grb2-SOS complex and Ras activation, we observed that insulin stimulation results in a rapid but transient activation of Ras and the extracellular-signal regulated kinase (ERK) followed by dissociation of the Grb2-SOS complex. Although treatment with the phorbol myristate acetate resulted in ERK activation and complete dissociation of the Grb2-SOS complex, there was no effect on subsequent insulin-stimulated Ras activation. Similarly, insulin stimulation followed by insulin removal resulted in a time-dependent restoration of the Grb2-SOS complex but which was significantly slower than the recovery of insulin-stimulated Ras activation. In addition, although insulin was able to activate Ras under these conditions, there was a complete desensitization of Raf and ERK activation. This apparent homologous desensitization of insulin action was specific for Raf and ERK as the insulin re-stimulation of insulin receptor autophosphorylation and protein kinase B activation were unaffected. Together, these data demonstrate the presence of a pathway independent of the Grb2-SOS complex that can lead to Ras activation but that the desensitization of Raf accounts for the homologous desensitization of ERK.     

Activation of Rac1 by paxillin-Crk-DOCK180 signaling complex is antagonized by Rap1 in migrating NBT-II cells

Induction of epithelial cell motility is a fundamental morphogenetic event that is recapitulated during carcinoma metastasis. Random motility of NBT-II carcinoma cells on collagen critically depends on paxillin phosphorylation at Tyr-31 and Tyr-118, the binding sites for the adapter protein CrkII. Two constitutive partners of CrkII are the exchange factors DOCK180 and C3G. CrkII bound to DOCK180 formed a signaling complex with phosphorylated paxillin that was necessary for cell migration as inferred from the inhibition caused by a DOCK180-interfering mutant. DOCK180, which acts predominantly on the Rho family GTPase Rac1, restored cell locomotion in cells expressing Phe-31/118 paxillin mutants deficient in Rac1 GTP-loading, suggesting that formation of paxillin-Crk-DOCK180 signaling complex controls collagen-dependent migration mainly through Rac1 activation. In migrating cells, CrkII constitutive association with C3G was not sufficient to stimulate its GDP/GTP exchange activity toward the Ras family GTPase Rap1. However, when constitutively active RapV12 was overexpressed, it negatively regulated cell motility. Activation of the C3G/Rap1 signaling pathway resulted in down-regulation of the paxillin-Crk-DOCK180 complex and reduction of Rac1-GTP, suggesting that Rap1 activation could suppress the Rac1 signaling pathway in epithelial cells.     

Critical binding and regulatory interactions between Ras and Raf occur through a small, stable N-terminal domain of Raf and specific Ras effector residues.

Genetic and biochemical evidence suggests that the Ras protooncogene product regulates the activation of the Raf kinase pathway, leading to the proposal that Raf is a direct mitogenic effector of activated Ras. Here we report the use of a novel competition assay to measure in vitro the relative affinity of the c-Raf-1 regulatory region for Ras-GTP, Ras-GDP, and 10 oncogenic and effector mutant Ras proteins. c-Raf-1 associates with normal Ras and the oncogenic V12 and L61 forms of Ras with equal affinity. The moderately transforming mutant Ras[E30K31] also bound to the c-Raf-1 regulatory region with normal affinity. Transformation-defective Ras effector mutants Ras[N33], Ras[S35], and Ras[N38] bound poorly. In contrast, the transformation defective Ras[G26I27] and Ras[E45] mutants bound to the c-Raf-1 regulatory region with nearly wild-type affinity. A stable, high-affinity Ras-binding region of c-Raf-1 was mapped to a 99-amino-acid subfragment of the first 257 residues. The smallest Ras-binding region identified consisted of N-terminal residues 51 to 131, although stable expression of the domain and high-affinity binding were improved by the presence of residues 132 to 149. Deletion of the Raf zinc finger region did not reduce Ras-binding affinity, while removal of the first 50 amino acids greatly increased affinity. Phosphorylation of Raf[1-149] by protein kinase A on serine 43 resulted in significant inhibiton of Ras binding. demonstrating that the mechanism of cyclic AMP downregulation results through structural changes occurring exclusively in this small Ras-binding domain.     

Stimulation of the ERK pathway by GTP-loaded Rap1 requires the concomitant activation of Ras,protein kinase C,and protein kinase A in neuronal cells

The small GTPases Ras or Rap1 were suggested to mediate the stimulatory effect of some G protein-coupled receptors on ERK activity in neuronal cells. Accordingly, we reported here that pituitary adenylate cyclase-activating polypeptide (PACAP), whose G protein-coupled receptor triggers neuronal differentiation of the PC12 cell line via ERK1/2 activation, transiently activated Ras and induced the sustained GTP loading of Rap1. Ras mediated peak stimulation of ERK by PACAP, whereas Rap1 was necessary for the sustained activation phase. However, PACAP-induced GTP-loading of Rap1 was not sufficient to account for ERK activation by PACAP because 1) PACAP-elicited Rap1 GTP-loading depended only on phospholipase C, whereas maximal stimulation of ERK by PACAP also required the activity of protein kinase A (PKA), protein kinase C (PKC), and calcium-dependent signaling; and 2) constitutively active mutants of Rap1, Rap1A-V12, and Rap1B-V12 only minimally stimulated the ERK pathway compared with Ras-V12. The effect of Rap1A-V12 was dramatically potentiated by the concurrent activation of PKC, the cAMP pathway, and Ras, and this potentiation was blocked by dominant-negative mutants of Ras and Raf. Thus, this set of data indicated that GPCR-elicited GTP loading of Rap1 was not sufficient to stimulate efficiently ERK in PC12 cells and required the permissive co-stimulation of PKA, PKC, or Ras.     

Activation of a signaling cascade by cytoskeleton stretch

Cells sense and respond to mechanical force. However, the mechanisms of transduction of extracellular matrix (ECM) forces to biochemical signals are not known. After removing the cell membrane and soluble proteins by Triton X-100 extraction, we found that the remaining complex (Triton cytoskeletons) activated Rap1 upon stretch. Rap1 guanine nucleotide exchange factor, C3G, was required for this activation; C3G as well as the adaptor protein, CrkII, in cell extract bound to Triton cytoskeletons in a stretch-dependent manner. CrkII binding, which was Cas dependent, correlated with stretch-dependent tyrosine phosphorylation of proteins in Triton cytoskeletons including Cas at the contacts with ECM. These in vitro findings were compatible with in vivo observations of stretch-enhanced phosphotyrosine signals, accumulation of CrkII at cell-ECM contacts, and CrkII-Cas colocalization. We suggest that mechanical force on Triton cytoskeletons activates local tyrosine phosphorylation, which provides docking sites for cytosolic proteins, and initiates signaling to activate Rap1.     

Thermodynamic and kinetic characterization of the interaction between the Ras binding domain of AF6 and members of the Ras subfamily

Cellular signaling downstream of Ras is highly diversified and may involve many different effector molecules. A potential candidate is AF6 which was originally identified as a fusion to ALL-1 in acute myeloid leukemia. In the present work the interaction between Ras and AF6 is characterized and compared with other effectors. The binding characteristics are quite similar to Raf and RalGEF, i.e. nucleotide dissociation as well as GTPase-activating protein activity are inhibited, whereas the intrinsic GTPase activity of Ras is unperturbed by AF6 binding. Particularly, the dynamics of interaction are similar to Raf and RalGEF with a lifetime of the Ras. AF6 complex in the millisecond range. As probed by 31P NMR spectroscopy one of two major conformational states of Ras is stabilized by the interaction with AF6. Looking at the affinities of AF6 to a number of Ras mutants in the effector region, a specificity profile emerges distinct from that of other effector molecules. This finding may be useful in defining the biological function of AF6 by selectively switching off other pathways downstream of Ras using the appropriate effector mutant. Notably, among the Ras-related proteins AF6 binds most tightly to Rap1A which could imply a role of Rap1A in AF6 regulation.     

Associations of B- and C-Raf with cholesterol, phosphatidylserine, and lipid second messengers: preferential binding of Raf to artificial lipid rafts

The serine/threonine kinase C-Raf is a key mediator in cellular signaling. Translocation of Raf to membranes has been proposed to be facilitated by Ras proteins in their GTP-bound state. In this study we provide evidence that both purified B- and C-Raf kinases possess lipophilic properties and associate with phospholipid membranes. In the presence of phosphatidylserine and lipid second messengers such as phosphatidic acid and ceramides these associations were very specific with affinity constants (K(D)) in the range of 0.5-50 nm. Raf association with liposomes was accompanied by displacement of 14-3-3 proteins and inhibition of Raf kinase activities. Interactions of Raf with cholesterol are of particular interest, since cholesterol has been shown to be involved, together with sphingomyelin and glycerophospholipids in the formation of specialized lipid microdomains called rafts. We demonstrate here that purified Raf proteins have moderate binding affinity for cholesterol. However, under conditions of lipid raft formation, Raf association with cholesterol (or rafts) increased dramatically. Since ceramides also support formation of rafts and interact with Raf we propose that Raf may be present at the plasma membrane in two distinct microdomains: in raft regions via association with cholesterol and ceramides and in non-raft regions due to interaction with phosphatidylserine and phosphatidic acid. At either location Raf kinase activity was inhibited by lipid binding in the absence or presence of Ras. Ras-Raf interactions with full-length C-Raf were studied both in solution and in phospholipid environment. Ras association with Raf was GTP dependent as previously demonstrated for C-Raf-RBD fragments. In the presence of liposomes the recruitment of C-Raf by reconstituted Ras-farnesyl was only marginal, since almost 70% of added C-Raf was bound by the lipids alone. Thus Ras-Raf binding in response to activation of Ras-coupled receptors may utilize Raf protein that is already present at the membrane.     

Identification of Rap1 as a target for the Crk SH3 domain-binding guanine nucleotide-releasing factor C3G.

C3G, which was identified as a Crk SH3 domain-binding guanine nucleotide-releasing factor, shows sequence similarity to CDC25 and Sos family proteins (S. Tanaka, T. Morishita, Y. Hashimoto, S. Hattori, S. Nakamura, M. Shibuya, K. Matuoka, T. Takenawa, T. Kurata, K. Nagashima, and M. Matsuda, Proc. Natl. Acad. Sci. USA 91:3443-3447, 1994). The substrate specificity of C3G was examined by in vitro and in vivo experiments. C3G markedly stimulated dissociation of bound GDP from Rap1B but marginally affected the same reaction of other Ras family proteins (Ha-Ras, N-Ras, and RalA). C3G also stimulated binding of GTP-gamma S [guanosine 5'-3-O-(thio)triphosphate] to Rap1B. When C3G and Rap1A were expressed in COS7 cells, marked accumulation of the active GTP-bound form of Rap1A was observed, while Sos was not effective in the activation of Rap1A. These results clearly show that C3G is an activator for Rap1. Furthermore, expression of C3G with a membrane localization signal in a v-Ki-ras transformant, DT, induced a reversion of the cells to the flat form, possibly through the activation of endogenous Rap1.     

The activation of RalGDS can be achieved independently of its Ras binding domain. Implications of an activation mechanism in Ras effector specificity and signal distribution.

Small GTPases of the Ras family are major players of signal transduction in eukaryotic cells. They receive signals from a number of receptors and transmit them to a variety of effectors. The distribution of signals to different effector molecules allows for the generation of opposing effects like proliferation and differentiation. To understand the specificity of Ras signaling, we investigated the activation of RalGDS, one of the Ras effector proteins with guanine-nucleotide exchange factor activity for Ral. We determined the GTP level on RalA and showed that the highly conserved Ras binding domain (RBD) of RalGDS, which mediates association with Ras, is important but not sufficient to explain the stimulation of the exchange factor. Although a point mutation in the RBD of RalGDS, which abrogates binding to Ras, renders RalGDS independent to activated Ras, an artificially membrane-targeted version of RalGDS lacking its RBD could still be activated by Ras. The switch II region of Ras is involved in the activation, because the mutant Y64W in this region is impaired in the RalGDS activation. Furthermore, it is shown that Rap1, which was originally identified as a Ras antagonist, can block Ras-mediated RalGDS signaling only when RalGDS contains an intact RBD. In addition, kinetic studies of the complex formation between RalGDS-RBD and Ras suggest that the fast association between RalGDS and Ras, which is analogous to the Ras/Raf case, achieves signaling specificity. Conversely, the Ras x RalGDS complex has a short lifetime of 0.1 s and Rap1 forms a long-lived complex with RalGDS, possibly explaining its antagonistic effect on Ras.     

The development of insulin receptors and responses in the differentiating nonfusing muscle cell line BC3H-1

We have studied the development of high affinity insulin receptors and insulin-stimulated responses in the differentiating nonfusing muscle cell line BC3H-1. In the logarithmic growth phase, these myoblasts exhibit very low levels of insulin binding and no detectable insulin-stimulated glucose or amino acid uptake. Following the cessation of cell division and subsequent spontaneous differentiation, the resulting myocytes develop a 5-fold increase in specific 125I-insulin binding and demonstrate physiologic insulin-stimulated glucose and amino acid uptake (100% increase above baseline) with half-maximum stimulation at 1-3 nM in agreement with the known in vivo and in vitro insulin sensitivity of muscle tissue. Insulin stimulation of 2-deoxyglucose uptake is detectable within 3 min, becomes maximal within 15 min, and is mediated by a rapid increase of plasma membrane transport units, as determined by D-glucose-inhibitable cytochalasin B binding, resulting in a 2-fold increase in the Vmax for 2-deoxyglucose transport with no change in Km. Myocyte insulin binding is specific, reversible, and saturable, yielding equilibrium within 18 h at 4 degrees C. Scatchard analysis identified the high affinity insulin receptor with a Kd of 0.5 nM at 4 degrees C. The myocytes also demonstrate sensitive down-regulation of cell surface insulin receptors, with a maximum decrease of 50% in cell surface insulin binding following exposure to 20 nM insulin for 18 h at 37 degrees C. Since the differentiation of this muscle cell line from myoblasts to nonfusing myocytes is accompanied by the development of high affinity insulin receptors and physiologic insulin-stimulated glucose and alpha-methylaminoisobutyric acid uptake, this continuously cultured system provides an excellent model for the study of differentiation and mechanism of insulin action in muscle, its quantitatively most significant target tissue.     

Galpha and Gbeta gamma require distinct Src-dependent pathways to activate Rap1 and Ras

The Src tyrosine kinase is necessary for activation of extracellular signal-regulated kinases (ERKs) by the beta-adrenergic receptor agonist, isoproterenol. In this study, we examined the role of Src in the stimulation of two small G proteins, Ras and Rap1, that have been implicated in isoproterenol's signaling to ERKs. We demonstrate that the activation of isoproterenol of both Rap1 and Ras requires Src. In HEK293 cells, isoproterenol activates Rap1, stimulates Rap1 association with B-Raf, and activates ERKs, all via PKA. In contrast, the activation by isoproterenol of Ras requires Gbetagamma subunits, is independent of PKA, and results in the phosphoinositol 3-kinase-dependent activation of AKT. Interestingly, beta-adrenergic stimulation of both Rap1 and ERKs, but not Ras and AKT, can be blocked by a Src mutant (SrcS17A) that is incapable of being phosphorylated and activated by PKA. Furthermore, a Src mutant (SrcS17D), which mimics PKA phosphorylation at serine 17, stimulates Rap1 activation, Rap1/B-Raf association, and ERK activation but does not stimulate Ras or AKT. These data suggest that Rap1 activation, but not that of Ras, is mediated through the direct phosphorylation of Src by PKA. We propose that the beta(2)-adrenergic receptor activates Src via two independent mechanisms to mediate distinct signaling pathways, one through Galpha(s) to Rap1 and ERKs and the other through Gbetagamma to Ras and AKT.     

Biochemical characterization of baculovirus-expressed rap1A/Krev-1 and its regulation by GTPase-activating proteins.

Normal human rap1A and 35A rap1A (which encodes a protein with a Thr-35----Ala mutation) were cloned into a baculovirus transfer vector and expressed in Sf9 insect cells. The resulting proteins were purified, and their nucleotide binding, GTPase activities, and responsiveness to GTPase-activating proteins (GAPs) were characterized and compared with those of Rap1 purified from human neutrophils. Recombinant wild-type Rap1A bound GTP gamma S, GTP, and GDP with affinities similar to those observed for neutrophil Rap1 protein. The rate of exchange of GTP by Rap1 without Mg2+ was much slower than that by Ras. The basal GTPase activities by both recombinant proteins were lower than that observed with the neutrophil Rap1, but the GTPase activity of the neutrophil and wild-type recombinant Rap1 proteins could be stimulated to similar levels by Rap-GAP activity in neutrophil cytosol. In contrast to wild-type Rap1A, the GTPase activity of 35A Rap was unresponsive to Rap-GAP stimulation. Neither recombinant Rap1A nor neutrophil Rap1 protein GTPase activity could be stimulated by recombinant Ras-GAP at a concentration 25-fold higher than that required to hydrolyze 50% of H-Ras-bound GTP under similar conditions. These results suggest that the putative effector domains (amino acids 32 to 40) shared between Rap1 and Ras are functionally similar and interact with their respective GAPs. However, although Rap1 and Ras are identical in this region, secondary structure or additional regions must confer the ability to respond to GAPs.     

Interaction of insulin receptor substrate 3 with insulin receptor, insulin receptor-related receptor, insulin-like growth factor-1 receptor, and downstream signaling proteins.

Insulin receptor substrates (IRS) mediate biological actions of insulin, growth factors, and cytokines. All four mammalian IRS proteins contain pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains at their N termini. However, the molecules diverge in their C-terminal sequences. IRS3 is considerably shorter than IRS1, IRS2, and IRS4, and is predicted to interact with a distinct group of downstream signaling molecules. In the present study, we investigated interactions of IRS3 with various signaling molecules. The PTB domain of mIRS3 is necessary and sufficient for binding to the juxtamembrane NPXpY motif of the insulin receptor in the yeast two-hybrid system. This interaction is stronger if the PH domain or the C-terminal phosphorylation domain is retained in the construct. As determined in a modified yeast two-hybrid system, mIRS3 bound strongly to the p85 subunit of phosphatidylinositol 3-kinase. Although high affinity interaction required the presence of at least two of the four YXXM motifs in mIRS3, there was not a requirement for specific YXXM motifs. mIRS3 also bound to SHP2, Grb2, Nck, and Shc, but less strongly than to p85. Studies in COS-7 cells demonstrated that deletion of either the PH or the PTB domain abolished insulin-stimulated phosphorylation of mIRS3. Insulin stimulation promoted the association of mIRS3 with p85, SHP2, Nck, and Shc. Despite weak association between mIRS3 and Grb2, this interaction was not increased by insulin, and may not be mediated by the SH2 domain of Grb2. Thus, in contrast to other IRS proteins, mIRS3 appears to have greater specificity for activation of the phosphatidylinositol 3-kinase pathway rather than the Grb2/Ras pathway.     

beta 2-adrenergic receptor activates extracellular signal-regulated kinases (ERKs) via the small G protein rap1 and the serine/threonine kinase B-Raf

G protein-coupled receptors can induce cellular proliferation by stimulating the mitogen-activated protein (MAP) kinase cascade. Heterotrimeric G proteins are composed of both alpha and betagamma subunits that can signal independently to diverse intracellular signaling pathways including those that activate MAP kinases. In this study, we examined the ability of isoproterenol, an agonist of the beta(2)-adrenergic receptor (beta(2)AR), to stimulate extracellular signal-regulated kinases (ERKs). Using HEK293 cells, which express endogenous beta(2)AR, we show that isoproterenol stimulates ERKs via beta(2)AR. This action of isoproterenol requires cAMP-dependent protein kinase and is insensitive to pertussis toxin, suggesting that Galpha(s) activation of cAMP-dependent protein kinase is required. Interestingly, beta(2)AR activates both the small G proteins Rap1 and Ras, but only Rap1 is capable of coupling to Raf isoforms. beta(2)AR inhibits the Ras-dependent activation of both Raf isoforms Raf-1 and B-Raf, whereas Rap1 activation by isoproterenol recruits and activates B-Raf. beta(2)AR activation of ERKs is not blocked by expression of RasN17, an interfering mutant of Ras, but is blocked by expression of either RapN17 or Rap1GAP1, both of which interfere with Rap1 signaling. We propose that isoproterenol can activate ERKs via Rap1 and B-Raf in these cells.     

RNAi-based analysis of CAP, Cbl, and CrkII function in the regulation of GLUT4 by insulin

Stimulation of glucose transport by insulin in cultured adipocytes through translocation of intracellular GLUT4 glucose transporters to the plasma membrane has been suggested to require phosphatidylinositol (PI) 3-kinase-dependent and independent mechanisms. To test the involvement of a PI 3-kinase-independent pathway leading to activation of the TC10 GTPase, the putative intermediates CAP, c-Cbl, Cbl-b, and CrkII were selectively depleted in 3T3-L1 adipocytes using highly efficient small interfering (si) RNAs. Simultaneous depletion of the ubiquitination factors c-Cbl plus Cbl-b in cultured adipocytes had the expected effect of delaying dephosphorylation of EGF receptors upon removal of EGF. However, siRNA-mediated gene silencing of both Cbl isoforms or CAP or CrkII in these cells failed to attenuate insulin-stimulated deoxyglucose transport or Myc-tagged GLUT4-GFP translocation at either sub-maximal or maximal concentrations of insulin. The dose-response relationship for insulin stimulation of deoxyglucose transport in primary adipocytes derived from c-Cbl knock-out mice was also identical to insulin action on adipocytes from wild type mice. These data are consistent with the hypothesis that CAP, Cbl iso-forms, and CrkII are not required components of insulin signaling to GLUT4 transporters.     

Recognizing and defining true Ras binding domains I: biochemical analysis

Common domain databases contain sequence motifs which belong to the ubiquitin fold family and are called Ras binding (RB) and Ras association (RalGDS/AF6 Ras associating) (RA) domains. The name implies that they bind to Ras (or Ras-like) GTP-binding proteins, and a few of them have been documented to qualify as true Ras effectors, defined as binding only to the activated GTP-bound form of Ras. Here we have expressed a large number of these domains and investigated their interaction with Ras, Rap and M-Ras. While their (albeit weak) sequence homology suggest that the domains adopt a common fold, not all of them bind to Ras proteins, irrespective of whether they are called RB or RA domains. We used fluorescence spectroscopy and isothermal titration calorimetry to show that the binding affinities vary over a large range, and are usually specific for either Ras or Rap. Moreover, the specificity is dictated by a set of key residues in the interface. Stopped-flow kinetic analysis showed that the association rate constants determine the different affinities of effector binding, while the dissociation rate constants are in a similar range. Manual sequence analysis allowed us to define positively charged sequence epitopes in certain secondary structure elements of the ubiquitin fold (beta1, beta2 and alpha1) which are located at similar positions and comprise the hot spots of the binding interface. These residues are important to qualify an RA/RB domain as a true candidate Ras or Rap effector.     

Ras-dependent ERK activation by the human G(s)-coupled serotonin receptors 5-HT4(b) and 5-HT7(a)

Receptor tyrosine kinases activate mitogen-activated protein (MAP) kinases through Ras, Raf-1, and MEK. Receptor tyrosine kinases can be transactivated by G protein-coupled receptors coupling to G(i) and G(q). The human G protein-coupled serotonin receptors 5-HT(4(b)) and 5-HT(7(a)) couple to G(s) and elevate intracellular cAMP. Certain G(s)-coupled receptors have been shown to activate MAP kinases through a protein kinase A- and Rap1-dependent pathway. We report the activation of the extracellular signal-regulated kinases (ERKs) 1 and 2 (p44 and p42 MAP kinase) through the human serotonin receptors 5-HT(4(b)) and 5-HT(7(a)) in COS-7 and human embryonic kidney HEK293 cells. In transfected HEK293 cells, 5-HT-induced activation of ERK1/2 is sensitive to H89, which indicates a role for protein kinase A. The observed activation of ERK1/2 does not require transactivation of epidermal growth factor receptors. Furthermore, 5-HT induced activation of both Ras and Rap1. Whereas the presence of Rap1GAP1 did not influence the 5-HT-mediated activation of ERK1/2, the activation of ERK1/2 was abolished in the presence of dominant negative Ras (RasN17). ERK1/2 activation was reduced in the presence of "dominant negative" Raf1 (RafS621A) and slightly reduced by dominant negative B-Raf, indicating the involvement of one or more Raf isoforms. These findings suggest that activation of ERK1/2 through the human G(s)-coupled serotonin receptors 5-HT(4(b)) and 5-HT(7(a)) in HEK293 cells is dependent on Ras, but independent of Rap1.     

Improved Binding of Raf to Ras��GDP Is Correlated with Biological Activity

The GTP-binding protein Ras plays a central role in the regulation of various cellular processes, acting as a molecular switch that triggers signaling cascades. Only Ras bound to GTP is able to interact strongly with effector proteins like Raf kinase, phosphatidylinositol 3-kinase, and RalGDS, whereas in the GDP-bound state, the stability of the complex is strongly decreased, and signaling is interrupted. To determine whether this process is only controlled by the stability of the complex, we used computer-aided protein design to improve the interaction between Ras and effector. We challenged the Ras·Raf complex in this study because Raf among all effectors shows the highest Ras affinity and the fastest association kinetics. The proposed mutations were characterized as to their changes in dynamics and binding strength. We demonstrate that Ras-Raf interaction can only be improved at the cost of a loss in specificity of Ras·GTP versus Ras·GDP. As shown by NMR spectroscopy, the Raf mutation A85K leads to a shift of Ras switch I in the GTP-bound as well as in the GDP-bound state, thereby increasing the complex stability. In a luciferase-based reporter gene assay, Raf A85K is associated with higher signaling activity, which appears to be a mere matter of Ras-Raf affinity.