Nutrition Guide

There are more than a hundred proteins in the Ras superfamily. Based on structure, sequence and function, the Ras superfamily is divided into eight main families, each of which is further divided into subfamilies: Ras, Rho, Rab, Rap, Arf, Ran, Rheb, Rad and Rit. Miro is a recent contributor to the superfamily.

Each subfamily shares the common core G domain, which provides essential GTPase and nucleotide exchange activity.

The surrounding sequence helps determine the functional specificity of the small GTPase, for example the ‘Insert Loop’, common to the Rho subfamily, specifically contributes to binding to effector proteins such as IQGAP and WASP.

The Ras family is generally responsible for cell proliferation, Rho for cell morphology, nuclear transport for Ran and vesicle transport for Rab and Arf:[1]

In biology, small GTPases are small (20-25 kDa) proteins that bind to guanosine triphosphate (GTP). This family of proteins is homologous to Ras GTPases and also called the Ras superfamily GTPases. Together with heterotrimeric G-proteins they constitute the G-proteins. They are all GTPases and share common features, but small GTPases have slightly different structures and mechanisms of action.

A typical G-protein is active when bound to GTP and inactive when bound to GDP (i.e. when the GTP is hydrolyzed to GDP). The GDP can be then replaced by free GTP. Therefore, a G-protein can be switched on and off. GTP hydrolysis is accelerated by GTPase activating proteins (GAPs), while GTP exchange is catalyzed by Guanine nucleotide exchange factors (GEFs). Activation of a GEF typically activates its cognate G-protein, while activation of a GAP results in inactivation of the cognate G-protein.

Small GTPases regulate a wide variety of processes in the cell, including growth, cellular differentiation, cell movement and lipid vesicle transport.

After finding that Ras proteins are mutated in 30% of human cancers, it was suspected that mutated rho proteins are also involved in cancer reproduction, as the signaling pathways involving rho proteins are widely known to play an important role in cancer development[8]. However, Ellenbroek et al. reported in their review that, as of August 2007, no mutations have been found in rho proteins, and only one has been found to be genetically altered[8]. To explain the role of rho pathways without mutation, researchers have now turned to the regulators of rho activity and the levels of expression of the rho proteins for answers.

One way to explain altered signaling in the absence of mutation is through increased expression. Overexpression of RhoA, RhoB, RhoC, Rac1, Rac2, Rac3, RhoE, RhoG, RhoH, and Cdc42 has been shown in multiple types of cancer[8]. This increased presence of so many signaling molecules implies that these proteins promote the cellular functions that become overly active in cancerous cells.

A second target to explain the role of the rho proteins in cancer is their regulatory proteins. Rho proteins are very tightly controlled by a wide variety of sources, and over 60 activators and 70 inactivators have been identified[9]. Multiple GAPs, GDIs, and GEFs have been shown to undergo overexpression, downregulation, or mutation in different types of cancer[8]. As one can imagine, once an upstream signal is changed, the activity of its targets downstream, i.e. the rho proteins, will change in activity.

Ellenbroek et al. outlined a number of different effects of rho activation in cancerous cells. First, in the initiation of the tumor modification of rho activity can suppress apoptosis and therefore contribute to artificial cell longevity. After natural apoptosis is suppressed, abnormal tumor growth can be observed through the loss of polarity in which rho proteins play an integral role. Next, the growing mass can invade across its normal boundaries through the alteration of adhesion proteins potentially caused by rho proteins[8]. Finally, after inhibition of apoptosis, cell polarity and adhesion molecules, the cancerous mass is free to metastasize and spread to other regions of the body.

Rho proteins have also been implicated in mental retardation. Mental retardation occurs in approximately 3% of the population and is characterized by having an IQ of less than 70. Multiple sources have noticed that mental retardation in some cases shows malformation of the dendritic spines, which form the post-synaptic connections between neurons. As expected, the misshapen dendritic spines are sometimes the result of rho protein-signaling modulation. After cloning of various genes implicated in X-linked mental retardation, three genes that have effects on rho signaling were identified, including oligophrenin-1 (GAP protein that stimulates GTPase activity of Rac1, Cdc42, and RhoA), PAK3 (involved with the effects of Rac and Cdc42 on the actin cytoskeleton) and ?PIX (a GEF that helps activate Rac1 and Cdc42)[17]. Because of the effect of rho signaling on the actin cytoskeleton, genetic malfunctions of a rho protein could explain the irregular morphology of neuronal dendrites seen in many cases of mental retardation.

Because of their implications in cellular motility and shape, rho proteins became a clear target in the study of the growth cones that form during axonal generation and re-generation in the nervous system. Some consider rho proteins to be a potential target for delivery into spinal cord lesions after traumatic injury. Following injury to the spinal cord, the extracellular space becomes inhibitory to the natural efforts neurons undergo to regenerate.

These ‘natural efforts’ include the formation of a growth cone at the proximal end of an injured axon. Newly-formed growth cones subsequently attempt to ‘crawl’ across the lesion and are quite sensitive to chemical cues in the extracellular environment. One of the many inhibitory cues includes chondroitin sulfate proteoglycans or CSPGs. Neurons growing in culture increase in their ability to cross over inhibitory CSPG lanes after administration of constituently-active Cdc42, Rac1 and RhoA[16]. This is partly due to the exogenous rho proteins driving cellular locomotion despite the extracellular cues promoting apoptosis and growth cone collapse. It is situations like these that make intracellular modulation of rho proteins the subject of a significant amount of spinal cord research.

Yet another major aspect of cellular behavior that is thought to include rho protein signaling is the process of cell division, mitosis. While it was thought for years that rho GTPase activity is restricted only to actin polymerization and therefore only to cytokinesis, new evidence that shows some activity in microtubule formation and the overall process of mitosis has arisen. This topic is still debated, and there is evidence both for and against for the importance of rho in mitosis[15].

Another cellular behavior that is affected by rho proteins is phagocytosis. As with most other types of cell membrane modulation, phagocytosis requires the actin cytoskeleton in order to engulf other items. The actin filaments control the formation of the phagocytic cup, and active Rac1 and Cdc42 have been implicated in this signaling cascade[14].

One example of behavior that is modulated by Rho GTPase proteins is in the healing of wounds. Wounds heal differently between young chicks and adult chickens. In young chicks, wounds heal by contraction, much like a draw-string being pulled to close a bag. In older chickens, cells crawl across the wound through locomotion. The actin formation required to close the wounds in young chicks is controlled by Rho GTPase proteins, since, after injection of a bacterial exoenzyme used to block rho and rac activity, the actin polymers do not form, and thus the healing completely fails[13].

In addition to the formation of lamellipodia and filopodia, it has been shown that intracellular concentration and cross-talk between different rho proteins drives the extensions and contractions that cause cellular locomotion. Sakumura et al. proposed a model based on differential equations, which helps explain the activity of rhos and their relationship to motion. This model encompassed the three proteins Cdc42, RhoA, and Rac. Cdc42 was assumed to encourage filopodia elongation and block actin depolymerization. RhoA was considered to encourage actin retraction. Rac was treated to encourage lamellipodia exentsion but block actin depolymerization. These three proteins, although significantly simplified, covered the key steps in cellular locomotion. Through various mathematical techniques, solutions to the differential equations that described various regions of activity based on intracellular activity were found. The paper concludes by showing that the model predicts that there are a few threshold concentrations that cause interesting effects on the activity of the cell. Below a certain concentration, there is very little activity, causing no extension of the arms and feet of the cell. Above a certain concentration, the rho protein causes a sinusoidal oscillation to occur, much like the extensions and contractions of the lamellipodia and filopodia. In essence, this model predicts that increasing the intracellular concentration of these three key active rho proteins causes an out-of-phase activity of the cell, resulting in extensions and contractions that are also out of phase[12].

Much of what is known about cellular morphology changes, and the effects of Rho proteins comes from the creation of a constituently-active mutation of the protein, e.g., by injecting active rho protein into Swiss 3T3 cells. The proteins is made to be constituently active using recombinant techniques. In essence, by changing one codon of the protein’s DNA, one amino acid is changed, and, therefore, the conformation of the entire protein is altered into one that resembles the GTP-bound state[6]. After injection into the 3T3 cells, morphological changes ensue - contractions and filopodia [6].

Because rho proteins are G-proteins and plasma-membrane-bound, their location can be easily controlled. In each situation, whether it be wound-healing, cytokinesis, or budding, the location of the rho activation can be imaged and identified. For example, if a circular hole is inflicted in a spherical cell, Cdc42 and other active rhos are seen in highest concentration around the circumference of the circular injury.[11] One methods of maintaining the spatial zones of activation is, e.g., through anchoring to the actin cytoskeleton, keeping the membrane-bound protein from diffusing away from the region where it is most needed.[11] Another method of maintenance is through the formation of a large complex which is resistant to diffusion and more rigidly bound to the membrane than the rho itself [11].