Rho family of GTPases

A Rho GTPase is a small (~21 kDa) signaling G proteins 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 of proteins. 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 factors (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.

GTPase	Effector
RhoA	Cit, Cnksr1, Diaph1, Diaph2, DgkQ, FlnA, KcnA2, Ktn1, Rtkn1, Rtkn2, Rhpn1, Rhpn2, Itpr1, PlcG1, PI-5-p5K, Pld1, Pkn1, Pkn2, Rock1, Rock2, PrkcA, Ppp1r12A
Rac1
Cdc42

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.

Regulations

Since Rho/Rac proteins are crucial in many cellular functions, they are regulated in a number of ways such as activation and inactivation by GEFs and GAPS as well as the regulation of their protein expression levels and subcellular localization .

Many Rho/Rac GTPases are specific to cell types. Stimulus such as UV irradiation, growth factors , and cytokines.