Rho GTPase

Overview
A Rho GTPase is a small (~21 kDa) signaling G protein (more specifically a GTPase) of the Ras superfamily. Rho GTPases have been shown to regulate many aspects of intracellular actin dynamics. Rho GTPases are found in all eukaryotic organisms as well as in yeasts and some plants. Three members of the family have been particularly well-studied: RhoA, Rac1 and Cdc42. Rho GTPase proteins have been described as ‘molecular switches’ and have been described to play a role in cell proliferation, apoptosis, cell division, gene expression and multiple other common cellular functions.

History
Identification of the Rho family of GTPases began in the late 1980’s. The first report of the cloning and expression of Cdc42 was in 1990 with the Munemitsu et al. paper entitled, “Molecular Cloning and Expression of a G25K cDNA, the human homolog of the yeast cell cycle gene CDC42”. Since the early 1990’s numerous Rho GTPases have been identified and today 22 Rho GTPases have been identified in mammals.

Categorization
Rho proteins are a member of the ‘Rho’ family. The superfamily, named ‘Ras-like’ proteins, consists of over 150 varieties in mammals. The ‘Ras-like’ superfamily is divided into five categories: Ras, Rho, Rab, Arf and Ran according to intracellular function. The Ras family is generally responsible for cell proliferation, Rho for cell morphology, nuclear transport for Ran and vesicle transport for Rab and Arf.

In mammals, the Rho family contains 22 members. Almost all research involves the three most common members of the Rho family: Rac1, RhoA and Cdc42.

The current 22 members of the Rho family include RhoA, RhoB, RhoC, RhoD, Rif, Rnd1, Rnd2, Rnd3/RhoE, RhoH/TTF, Rac1, Rac2, Rac3, RhoG, Cdc42, TC10 (RhoQ), TCL (RhoJ), Wrch1 (RhoV), Chp/Wrch2 (RhoU), RhoBTB1, RhoBTB2, Miro1 (RhoT1), Miro2 (RhoT2).

Regulation & Effectors
Three general classes of regulators of rho protein signaling have been identified: guanine nucleotide exchange factor (GEFs), GTPase-activating proteins (GAPs) and guanine nucleotide dissociation inhibitors (GDIs). GEFs control the release of GDP from the rho protein and the replacement with GTP. GAPs control the ability of the GTPase to hydrolyze GTP to GDP, controlling the natural rate of movement from the active conformation to the inactive conformation. GDI proteins form a large complex with the rho protein helping to prevent diffusion within the membrane and into the cytosol, thus acting as an anchor and allowing for very specific spatial control of rho activation.

Each Rho protein affects numerous proteins downstream, all which have roles in various cell processes. In fact, over 60 targets of the three common Rho GTPases have been found. Two molecules which directly stimulate actin polymerization are the WASP/WAVE proteins and the Diaphanous-related formins.

Functions
Rho/Rac proteins are involved in a wide variety of cellular functions such as cell polarity, vesicular trafficking, the cell cycle and transcriptomal dynamics.

Morphology
Animal cells form many different shapes based on their function and location in the body. Rho proteins help cells regulate changes in shape throughout their life cycle. Before cells can undergo key processes such as budding, mitosis or locomotion, a certain degree of polarity is required. A ‘polar’ cell is one which has some sort of shape or direction rather than existing as an amorphous, symmetrical shape. For instance, an amoeba becomes polar when it undergoes locomotion and travels from one point to another.

One example of Rho GTPases’ role in cell polarity is seen in the well-studied yeast cell. Before the cell can bud, Cdc42 is used to locate the region of the cell’s membrane which will begin to bulge into the new cell. When Cdc42 is removed from the cell, the cell’s outgrowths still form but form in an unorganized manner.

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. As early as 1990, Paterson et al. began injecting active rho protein into Swiss 3T3 cells. The proteins were made to be constituently active using recombinant techniques. Essentially 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 which resembles the GTP bound state. After injection into the 3T3 cells, morphological changes were observed and described as ‘contractions, leaving finger-like projections’.

In the 2006 review article released by Bement et al, the spatial zones of rho activation were explained. 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. Bement also mentions two methods of maintaining the spatial zones of activation. The first is through anchoring to the actin cytoskeleton, keeping the membrane bound protein from diffusing away from the region where it is most needed. A second 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.

One of the most obvious changes to cell morphology controlled by rho proteins is the formation of lamellipodia and filopodia, the processes that look like ‘fingers’ or ‘feet’ and often propel cells across surfaces. As early as the mid-1990’s these processes and the effects of the rho proteins were observed in fibroblasts. Dr. Alan Hall, one of the front-runners in rho protein research, compiled evidence in his 1998 review which showed that it was not only fibroblasts which formed processes based on rho activation, but virtually all eukaryotic cells.

Movement
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 which 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 were found which described various regions of activity based on intracellular activity. The paper concludes by showing that the model predicts that there are a few threshold concentrations which 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. Essentially 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 which are also out of phase.

Behavior
One example of behavior which is modulated by Rho GTPase proteins is in the healing of wounds. In 1996, Brock et al. demonstrated this characteristic in chick embryos. 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. Brock et al. hypothesized that the actin formation required to close the wounds in young chicks was controlled by Rho GTPase proteins. As they expected, after injection of a bacterial exoenzyme used to block rho and rac activity, the actin polymers did not form and a complete failure of healing was observed.

Another cellular behavior which 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.

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

Nervous System Regeneration
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. In 2004, Jain et al. showed that neurons growing in culture increased in their ability to cross over inhibitory CSPG lanes after administration of constituently active Cdc42, Rac1 and RhoA. 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.

Mental Retardation
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 were identified that have effects on rho signaling, 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 αPIX (a GEF which helps activate Rac1 and Cdc42). Because of the effect of rho signaling on the actin cytoskeleton, genetic malfunctions of a rho protein could certainly explain the irregular morphology of neuronal dendrites seen in many cases of mental retardation.

Cancer
After finding that Ras proteins are mutated in 30% of human cancers, it was suspected that mutated rho proteins were also involved in cancer reproduction, as the signaling pathways involving rho proteins are widely known to play an important role in cancer development. 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. 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.

Again, 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. This increased presence of so many signaling molecules certainly implies that these proteins promote the cellular functions which 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. Multiple GAPs, GDIs and GEFs have been shown to undergo overexpression, downregulation or mutation in different types of cancer. 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.

More specifically, 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 roll. Next, the growing mass can invade across its normal boundaries through the alteration of adhesion proteins potentially caused by rho proteins. 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.