Rho guanine nucleotide exchange factors: regulators of Rho GTPase activity in development and disease.

The aberrant activity of Ras homologous (Rho) family small GTPases (20 human members) has been implicated in cancer and other human diseases. However, in contrast to the direct mutational activation of Ras found in cancer and developmental disorders, Rho GTPases are activated most commonly in disease by indirect mechanisms. One prevalent mechanism involves aberrant Rho activation via the deregulated expression and/or activity of Rho family guanine nucleotide exchange factors (RhoGEFs). RhoGEFs promote formation of the active GTP-bound state of Rho GTPases. The largest family of RhoGEFs is comprised of the Dbl family RhoGEFs with 70 human members. The multitude of RhoGEFs that activate a single Rho GTPase reflects the very specific role of each RhoGEF in controlling distinct signaling mechanisms involved in Rho activation. In this review, we summarize the role of Dbl RhoGEFs in development and disease, with a focus on Ect2 (epithelial cell transforming squence 2), Tiam1 (T-cell lymphoma invasion and metastasis 1), Vav and P-Rex1/2 (PtdIns(3,4,5)P3 (phosphatidylinositol (3,4,5)-triphosphate)-dependent Rac exchanger).

Molecular basis for Rac1 recognition by guanine nucleotide exchange factors.

Rho GTPases are activated by a family of guanine nucleotide exchange factors (GEFs) known as Dbl family proteins. The structural basis for how GEFs recognize and activate Rho GTPases is presently ill defined. Here, we utilized the crystal structure of the DH/PH domains of the Rac-specific GEF Tiam1 in complex with Rac1 to determine the structural elements of Rac1 that regulate the specificity of this interaction. We show that residues in the Rac1 beta2-beta3 region are critical for Tiam1 recognition. Additionally, we determined that a single Rac1-to-Cdc42 mutation (W56F) was sufficient to abolish Rac1 sensitivity to Tiam1 and allow recognition by the Cdc42-specific DH/PH domains of Intersectin while not impairing Rac1 downstream activities. Our findings identified unique GEF specificity determinants in Rac1 and provide important insights into the mechanism of DH/PH selection of GTPase targets.

Quantitative analysis of the effect of phosphoinositide interactions on the function of Dbl family proteins.

Normally, Rho GTPases are activated by the removal of bound GDP and the concomitant loading of GTP catalyzed by members of the Dbl family of guanine nucleotide exchange factors (GEFs). This family of GEFs invariantly contain a Dbl homology (DH) domain adjacent to a pleckstrin homology (PH) domain, and while the DH domain usually is sufficient to catalyze nucleotide exchange, possible roles for the conserved PH domain remain ambiguous. Here we demonstrate that the conserved PH domains of three distinct Dbl family proteins, intersectin, Dbs, and Tiam1, selectively bind lipid vesicles only when phosphoinositides are present. While the PH domains of intersectin and Dbs promiscuously bind several multiphosphorylated phosphoinositides, Tiam1 selectively interacts with phosphatidylinositol 3-phosphate (K(D) approximately 5-10 microm). In addition, and in contrast to recent reports, catalysis of nucleotide exchange on nonprenylated Rac1 provided by various extended portions of Tiam1 is not influenced by (a) soluble phosphoinositide head groups, (b) dibutyl versions of phosphoinositides, or (c) lipid vesicles containing phosphoinositides. Likewise, GEF activity afforded by DH/PH fragments of intersectin and Dbs are also not altered by phosphoinositide interactions. These results strongly suggest that unless all relevant components are localized to a lipid membrane surface, Dbl family GEFs generally are not intrinsically modulated by binding phosphoinositides.

Crystal structure of Rac1 in complex with the guanine nucleotide exchange region of Tiam1.

The principal guanine nucleotide exchange factors for Rho family G proteins contain tandem Dbl-homology (DH) and pleckstrin-homology (PH) domains that catalyse nucleotide exchange and the activation of G proteins. Here we have determined the crystal structure of the DH and PH domains of the T-lymphoma invasion and metastasis factor 1 (Tiam1) protein in complex with its cognate Rho family G protein, Rac1. The two switch regions of Rac1 are stabilized in conformations that disrupt both magnesium binding and guanine nucleotide interaction. The resulting cleft in Rac1 is devoid of nucleotide and highly exposed to solvent. The PH domain of Tiam1 does not contact Rac1, and the position and orientation of the PH domain is markedly altered relative to the structure of the uncomplexed, GTPase-free DH/PH element from Sos1. The Tiam1/Rac1 structure highlights the interactions that catalyse nucleotide exchange on Rho family G proteins, and illustrates structural determinants dictating specificity between individual Rho family members and their associated Dbl-related guanine nucleotide exchange factors.

Vav2 is an activator of Cdc42, Rac1, and RhoA.

Vav and Vav2 are members of the Dbl family of proteins that act as guanine nucleotide exchange factors (GEFs) for Rho family proteins. Whereas Vav expression is restricted to cells of hematopoietic origin, Vav2 is widely expressed. Although Vav and Vav2 share highly related structural similarities and high sequence identity in their Dbl homology domains, it has been reported that they are active GEFs with distinct substrate specificities toward Rho family members. Whereas Vav displayed GEF activity for Rac1, Cdc42, RhoA, and RhoG, Vav2 was reported to exhibit GEF activity for RhoA, RhoB, and RhoG but not for Rac1 or Cdc42. Consistent with their distinct substrate targets, it was found that constitutively activated versions of Vav and Vav2 caused distinct transformed phenotypes when expressed in NIH 3T3 cells. In contrast to the previous findings, we found that Vav2 can act as a potent GEF for Cdc42, Rac1, and RhoA in vitro. Furthermore, we found that NH(2)-terminally truncated and activated Vav and Vav2 caused indistinguishable transforming actions in NIH 3T3 cells that required Cdc42, Rac1, and RhoA function. In addition, like Vav and Rac1, we found that Vav2 activated the Jun NH(2)-terminal kinase cascade and also caused the formation of lamellipodia and membrane ruffles in NIH 3T3 cells. Finally, Vav2-transformed NIH 3T3 cells showed up-regulated levels of Rac-GTP. We conclude that Vav2 and Vav share overlapping downstream targets and are activators of multiple Rho family proteins. Therefore, Vav2 may mediate the same cellular consequences in nonhematopoietic cells as Vav does in hematopoietic cells.

Dependence of Dbl and Dbs transformation on MEK and NF-kappaB activation.

Dbs was identified initially as a transforming protein and is a member of the Dbl family of proteins (>20 mammalian members). Here we show that Dbs, like its rat homolog Ost and the closely related Dbl, exhibited guanine nucleotide exchange activity for the Rho family members RhoA and Cdc42, but not Rac1, in vitro. Dbs transforming activity was blocked by specific inhibitors of RhoA and Cdc42 function, demonstrating the importance of these small GTPases in Dbs-mediated growth deregulation. Although Dbs transformation was dependent upon the structural integrity of its pleckstrin homology (PH) domain, replacement of the PH domain with a membrane localization signal restored transforming activity. Thus, the PH domain of Dbs (but not Dbl) may be important in modulating association with the plasma membrane, where its GTPase substrates reside. Both Dbs and Dbl activate multiple signaling pathways that include activation of the Elk-1, Jun, and NF-kappaB transcription factors and stimulation of transcription from the cyclin D1 promoter. We found that Elk-1 and NF-kappaB, but not Jun, activation was necessary for Dbl and Dbs transformation. Finally, we have observed that Dbl and Dbs regulated transcription from the cyclin D1 promoter in a NF-kappaB-dependent manner. Previous studies have dissociated actin cytoskeletal activity from the transforming potential of RhoA and Cdc42. These observations, when taken together with those of the present study, suggest that altered gene expression, and not actin reorganization, is the critical mediator of Dbl and Rho family protein transformation.

Increasing complexity of Ras signaling.

The initial discovery that ras genes endowed retroviruses with potent carcinogenic properties and the subsequent determination that mutated ras genes were present in a wide variety of human cancers, prompted a strong suspicion that the growth-promoting actions of mutated Ras proteins contribute to their aberrant regulation of growth stimulatory signaling pathways. In 1993, a remarkable convergence of experimental observations from genetic analyses of Drosophila, S. cerevisiae and C. elegans as well as biochemical and biological studies in mammalian cells came together to define a clear role for Ras in signal transduction. What emerged was an elegant linear signaling pathway where Ras functions as a relay switch that is positioned downstream of cell surface receptor tyrosine kinases and upstream of a cytoplasmic cascade of kinases that included the mitogen-activated protein kinases (MAPKs). Activated MAPKs in turn regulated the activities of nuclear transcription factors. Thus, a signaling cascade where every component between the cell surface and the nucleus was defined and conserved in worms, flies and man. This was a remarkable achievement in our efforts to appreciate how the aberrant function of Ras proteins may contribute to the malignant growth properties of the cancer cell. However, the identification of this pathway has proven to be just the beginning, rather than the culmination, of our understanding of Ras in signal transduction. Instead, we now appreciate that this simple linear pathway represents but a minor component of a very complex signaling circuitry. Ras signaling has emerged to involve a complex array of signaling pathways, where cross-talk, feedback loops, branch points and multi-component signaling complexes are recurring themes. The simplest concept of a signaling cascade, where each component simply relays the same message to the next, is clearly not the case. In this review, we summarize our current understanding of Ras signal transduction with an emphasis on new complexities associated with the recognition and/or activation of cellular effectors, and the diverse array of signaling pathways mediated by interaction between Ras and Ras-subfamily proteins with multiple effectors.

Rho family proteins and Ras transformation: the RHOad less traveled gets congested.

The Rho family of small GTPases has attracted considerable research interest over the past 5 years. During this time, we have witnessed a remarkable increase in our knowledge of the biochemistry and biology of these Ras-related proteins. Thus, Rho family proteins have begun to rival, if not overshadow, interest in their more celebrated cousins, the Ras oncogene proteins. The fascination in Rho family proteins is fueled primarily by two major observations. First, like Ras, Rho family proteins serve as guanine nucleotide-regulated binary switches that control signaling pathways that in turn regulate diverse cellular processes. Rho family proteins are key components in cellular processes that control the organization of the actin cytoskeleton, activate kinase cascades, regulate gene expression, regulate membrane trafficking, promote growth transformation and induce apoptosis. Second, at least five Rho family proteins have been implicated as critical regulators of oncogenic Ras transformation. Thus, it is suspected that Rho family proteins contribute significantly to the aberrant growth properties of Ras-transformed cells. Rho family proteins are also critical mediators of the transforming actions of other transforming proteins and include Dbl family oncogene proteins, G protein-coupled receptors and G protein alpha subunits. Thus, Rho family proteins may be key components for the transforming actions of diverse oncogene proteins. Major aims of Rho family protein studies are to define the molecular mechanism by which Rho family proteins regulate such a diverse spectrum of cellular behavior. These efforts may reveal novel targets for the development of anti-Ras and anti-cancer drugs.

The timing of congenital brachial plexus injury: a study of electromyography findings in the newborn piglet.

OBJECTIVE: Permanent congenital brachial plexus palsy is a recognized serious complication associated with shoulder dystocia. The timing and etiology of this injury remains controversial. Previous authorities have used adult-derived, non-brachial plexus data to extrapolate the anticipated timing for electromyographic denervation changes to date such injuries in the newborn. With use of a domestic swine model, this investigation tests the hypothesis that electromyographic evidence of brachial plexus denervation in the newborn is temporally different than that in the adult. STUDY DESIGN: Five healthy 2-day-old and two adult pigs underwent unilateral sharp transection of the brachial plexus. Daily electromyographic studies were performed in brachial plexus innervated muscle groups on the involved and contralateral (control) front limbs. Postmortem measurements of the transected nerve segments were obtained in one piglet and one adult animal. Representative hard copy recordings of individual electromyographic studies were collected. RESULTS: Immediately after surgical transection of the brachial plexus, no electromyographic evidence of denervation was observed. Uniformly in the newborn piglets, at 24 hours after transection, denervation in the form of fibrillation potentials, positive sharp waves, and complex repetitive discharges was seen. Serial testing demonstrated proximal to distal gradients of denervation over the next 24 to 48 hours. A delay in electromyographic evidence of denervation was observed in the two adult pigs until days 5 and 8, respectively. Control limb studies remained normal throughout the study period. Nerve length measurements for individual muscle groups were as follows for the adult and newborn pigs, respectively: deltoid 11.4 cm, 2.5 cm; cleidobrachialis 16.0 cm, 4.0 cm; triceps 15.5 cm, 4.5 cm; forelimb flexors 26.0 cm, 6.5 cm; and extensor carpi radialis 31.0 cm, 9.0 cm. CONCLUSION: Electromyographic evidence of brachial plexus denervation after surgical transection differs between the newborn and the adult pig. Consistent with wallerian degeneration, a correlation exists between length of the distal nerve segment and timing for electromyographic signs of denervation. These findings suggest it would be inappropriate to extrapolate the anticipated timing for electromyographic changes in the newborn on the basis of previously established adult non-brachial plexus data.

14-3-3 zeta negatively regulates raf-1 activity by interactions with the Raf-1 cysteine-rich domain.

Although Raf-1 is a critical effector of Ras signaling and transformation, the mechanism by which Ras promotes Raf-1 activation is complex and remains poorly understood. We recently reported that Ras interaction with the Raf-1 cysteine-rich domain (Raf-CRD, residues 139-184) may be required for Raf-1 activation. The Raf-CRD is located in the NH2-terminal negative regulatory domain of Raf-1 and is highly homologous to cysteine-rich domains found in protein kinase C family members. Recent studies indicate that the structural integrity of the Raf-CRD is also critical for Raf-1 interaction with 14-3-3 proteins. However, whether 14-3-3 proteins interact directly with the Raf-CRD and how this interaction may mediate Raf-1 function has not been determined. In the present study, we demonstrate that 14-3-3 zeta binds directly to the isolated Raf-CRD. Moreover, mutation of Raf-1 residues 143-145 impairs binding of 14-3-3, but not Ras, to the Raf-CRD. Introduction of mutations that impair 14-3-3 binding resulted in full-length Raf-1 mutants with enhanced transforming activity. Thus, 14-3-3 interaction with the Raf-CRD may serve in negative regulation of Raf-1 function by facilitating dissociation of 14-3-3 from the NH2 terminus of Raf-1 to promote subsequent events necessary for full activation of Raf-1.

The effect of femoral lengthening on knee articular cartilage: the role of apparatus extension across the joint.

Loss of joint motion is a common complication of limb lengthening despite newer methods of incremental bone elongation. A pilot canine study has demonstrated that 30% femoral lengthening causes reproducible knee cartilage injury manifest by frank loss of cartilage substance or fibrillation. This study was undertaken to examine the potential of knee joint protection by apparatus extension to the tibia. Four dogs underwent application of a modified Ilizarov apparatus to the femur and tibia with coaxial hinges at the knee. After osteotomy, 30% lengthening was undertaken at 0.75 mm daily in three increments. At the completion of lengthening, experimental and contralateral knee joints were harvested, assessed grossly, decalcified, sagittally sectioned, and stained with safranin-O. All control joints were normal histologically. All experimental joints demonstrated a decrease in proteoglycan staining without evidence of fibrillation or necrosis. These findings suggest a protective effect of the tibial apparatus by avoiding joint compression.

Interstitial irradiation versus interstitial thermoradiotherapy for supratentorial malignant gliomas: a comparative survival analysis.

PURPOSE: To compare the survival of two groups of patients with supratentorial malignant gliomas who were treated on two sequential protocols with either interstitial thermoradiotherapy or with interstitial irradiation without hyperthermia. METHODS AND MATERIALS: Between 1988-1992, patients with anaplastic astrocytoma or glioblastoma multiforme were treated at the University of Arizona on a Phase I/II protocol of interstitial thermoradiotherapy with ferro-magnetic seeds. The treatment protocol consisted of debulking surgery, a course of external beam radiotherapy and hyperthermia given immediately before and after brachytherapy. The survival of patients so treated was compared with that of a similar group of patients treated with interstitial brachytherapy alone at the Barrows Neurological Institute between 1982-1990. RESULTS: Twenty-five patients with primary tumors treated at the time of initial presentation with thermoradiotherapy were compared with a control group of 37 patients treated with interstitial brachytherapy alone. All primary patients were followed for a minimum of 34 months post implant. Multivariate analysis based on proportional hazards models showed that hyperthermia (p = 0.027), patient age (p < or = 0.00001) and histology (anaplastic astrocytoma vs. glioblastoma multiforme, p = 0.0017) were the only factors significantly associated with survival in this data set. From the fitted model, the hazard of dying when treated with hyperthermia was .53 times (95% confidence intervals 0.29-0.94) than that of the control group. In addition, we treated a small group of patients with recurrent tumors (13 with brachytherapy alone, and eight with thermoradiotherapy) and found no survival difference (p = 0.62). CONCLUSION: Within the constraints of the selection factors and the different treatment parameters used in these studies, we conclude that an interstitial thermoradiotherapy boost confers a statistically significant survival benefit to patients with primary high grade gliomas when compared to interstitial brachytherapy alone.

Hydrolaparoscopy in the rabbit: a fine model for the development of operative fetoscopy.

OBJECTIVE: Our goal was to become adept at performing laparoscopic procedures within a fluid medium and at using miniature instruments in a small animal model. STUDY DESIGN: Adult female New Zealand rabbits underwent carbon dioxide laparoscopy while they were under general anesthesia with the use of 2 mm instruments. The abdominal cavity was filled with lactated Ringer's solution. Visualization of the intraabdominal organs and surgical procedures were performed below the fluid level. RESULTS: Excellent visualization of the abdominal organs below the fluid level was obtained. Several surgical tasks were accomplished, including cutting and coagulation of the uterine horns with monopolar electrocautery, creation of a defect in the mesovarium and mesometrium, extracorporeal knot tying, and intraabdominal cutting of suture material. Relatively high amounts of energy were needed during electrocautery within the liquid medium (> 25 W). Occasional fluid and gas leakage through the skin punctures was prevented with accessory clamps. CONCLUSION: Hydrolaparoscopy can be performed in the rabbit with miniature instruments. As it simulates the human intraamniotic environment, it is a useful model for the development of operative fetoscopy.

A comparison of survival between radiosurgery and stereotactic implants for malignant astrocytomas.

The purpose of this paper is to compare the survival of three groups of patients with high grade supratentorial gliomas who were treated on three sequential protocols with surgical resection, external beam fractionated radiotherapy and a boost to the residual contrasting enhancing mass by either interstitial brachytherapy (IB, n = 33), by interstitial thermoradiotherapy (IT, n = 25) or by stereotactic radiosurgery (SRS, n = 19). The primary aim of this study was to evaluate the role of different boosting techniques in the initial management of primary brain tumors. External beam radiotherapy doses were escalated from one study to the next so that the median doses given to the IB, the IT, and the SRS groups were 41.4 Gy, 48.4 Gy, and 59.4 Gy, respectively. The median dose of interstitial irradiation or stereotactic radiosurgery, were 40 Gy, 32.2 Gy and 10 Gy, respectively, for the same groups. Follow-up was such that all living patients had been followed for a minimum of 30, 27, 4 months in the IB, IT, and SRS groups, respectively; hence, twelve-month survival was 52% (95% CI: 34%-69%), 80% (95% CI: 64%-96%), and 51% (95% CI: 24%-78%) in the same respective groups. Using a multivariate Cox proportional hazards model, treatment with IT conferred a survival advantage over IB (p = 0.029). Furthermore, survival of patients treated with SRS did not significantly differ from that of patients treated with an implant with or without hyperthermia.(ABSTRACT TRUNCATED AT 250 WORDS)

Local effects of ibogaine on extracellular levels of dopamine and its metabolites in nucleus accumbens and striatum: interactions with D-amphetamine.

Systemic administration of ibogaine (40 mg/kg, i.p.) has been reported to induce both acute (1-3 h) and persistent (19-20 h) changes in extracellular levels of dopamine and its metabolites in the nucleus accumbens and striatum. In the present study, local administration of ibogaine to the striatum and nucleus accumbens produced effects that mimicked both the acute and persistent effects of systemic administration: perfusion with high concentrations (200 and 400 microM) of ibogaine mimicked the acute effects (decreased extracellular dopamine levels and increased extracellular metabolite levels) whereas perfusion with a low concentration (10 microM) of ibogaine mimicked the persistent effects (decreased extracellular levels of DOPAC). These results indicate that ibogaine acts directly in brain regions containing dopaminergic nerve terminals and that long-lasting effects of systemically administered ibogaine might be mediated by persisting low levels of ibogaine. Locally administered ibogaine (10 microM) was also found to enhance the effects of systemically administered D-amphetamine (1.25 mg/kg, i.p.) on extracellular dopamine levels, and conversely, systemically administered ibogaine (40 mg/kg, i.p.; 19 h pretreatment) enhanced the effects of locally administered D-amphetamine (1-10 microM). These results indicate that, in addition to a metabolic mechanism implicated previously, a pharmacodynamic mechanism contributes to the interaction between ibogaine and D-amphetamine. The relevance of such mechanisms to claims regarding ibogaine's anti-addictive properties is unclear.

Differential effects of ibogaine pretreatment on brain levels of morphine and (+)-amphetamine.

Previous studies in rats have shown that ibogaine inhibits neurochemical and behavioral effects of morphine yet potentiates similar effects of (+)-amphetamine. To assess whether these different functional interactions have a metabolic basis, brain levels of morphine and (+)-amphetamine were measured by gas chromatography-mass spectrometry after ibogaine pretreatment (19 h before injection of morphine or (+)-amphetamine). Ibogaine pretreatment had no effect on brain morphine levels, either at 30 min or 2 h after morphine injection; however, ibogaine significantly increased brain amphetamine levels at 30 min and, to a greater extent, at 2 h after (+)-amphetamine injection. These and other data suggest that ibogaine irreversibly inhibits an amphetamine-metabolizing enzyme. The functional interactions between ibogaine and (+)-amphetamine, but not those between ibogaine and morphine, may result from a hepatic drug-drug interaction.

Effects of ibogaine on acute signs of morphine withdrawal in rats: independence from tremor.

Because of the claim that ibogaine suppresses the symptoms of "narcotic withdrawal" in humans, the effect of ibogaine on naltrexone-precipitated withdrawal signs in morphine-dependent rats was assessed. Morphine was administered subcutaneously through implanted silicone reservoirs for 5 days. Ibogaine (20, 40 or 80 mg/kg, i.p.) or saline was administered 30 min prior to challenge with naltrexone (1 mg/kg, i.p.) and withdrawal signs were counted for the following 2 hr. Ibogaine (40 and 80 mg/kg) significantly reduced the occurrence of four signs (wet-dog shakes, grooming, teeth chattering and diarrhea) during naltrexone-precipitated withdrawal; three other signs (weight loss, burying and flinching) were unaffected. Ibogaine induces head and body tremors lasting for 2-3 hr and the tremors might have interfered with the expression of opioid withdrawal. To examine this issue, another experiment was conducted in which ibogaine (40 mg/kg) or saline was administered 4 hr prior to challenge with naltrexone. Although there was a complete absence of tremors, ibogaine still significantly reduced the occurrence of the same four signs of withdrawal.