Rho family of GTPases
The Rho family of GTPases is a family of small signaling G proteins, and is a subfamily of the Ras superfamily. The members of the Rho GTPase family have been shown to regulate many aspects of intracellular actin dynamics, and are found in all eukaryotic kingdoms, including yeasts and some plants. Three members of the family have been studied in detail: Cdc42, Rac1, and RhoA. All G proteins are "molecular switches", and Rho proteins play a role in organelle development, cytoskeletal dynamics, cell movement, and other common cellular functions.
History
Identification of the Rho family of GTPases began in the mid-1980s. The first identified Rho member was RhoA, isolated serendipitously in 1985 from a low stringency cDNA screening. Rac1 and Rac2 were identified next, in 1989 followed by Cdc42 in 1990. Eight additional mammalian Rho members were identified from biological screenings until the late 1990s, a turning point in biology where availability of complete genome sequences allowed full identification of gene families. All eukaryote cells contain Rho GTPase. In mammals, the Rho family is thus made of 20 members distributed in 8 subfamilies: Rho, Rnd, RhoD/F, RhoH, Rac, Cdc42, RhoU/V and RhoBTB.As early as 1990, Paterson et al. began expressing activated Rho protein in Swiss 3T3 fibroblasts.
By the mid-1990s, Rho proteins had been observed to affect the formation of cellular projections in fibroblasts. In a 1998 review article, Alan Hall compiled evidence showing that not only do fibroblasts form processes upon Rho activation, but so do virtually all eukaryotic cells.
A 2006 review article by Bement et al. explored the significance of spatial zones of Rho activation.
Categorization
The Rho family of GTPases belong to the Ras superfamily of proteins, which consists of over 150 varieties in mammals. Rho proteins sometimes denote some members of the Rho family, and sometimes refers to all members of the family. This article is about the family as a whole.In mammals, the Rho family contains 20 members. Almost all research involves the three most common members of the Rho family: Cdc42, Rac1 and RhoA.
| Rho family member | Action on actin filaments |
| Cdc42 | affects filopodia |
| Rac1 | affects lamellipodia |
| RhoA | affects stress fibres |
These 20 mammalian members are subdivided in the Rac subfamily, Cdc42 subfamily, the RhoUV family, RhoA subfamily, the Rnd subfamily, the RhoD subfamily, RhoBTB and RhoH/TTF.
| Subclass | Cytoskeletal effect | Rho family members |
| Cdc42 subclass | filopodia | Cdc42 |
| Cdc42 subclass | filopodia | RhoQ |
| Cdc42 subclass | filopodia | RhoJ |
| RhoUV subclass | filopodia and lamellipodia | RhoU |
| RhoUV subclass | filopodia and lamellipodia | RhoV |
| Rac | lamellipodia | Rac1 |
| Rac | lamellipodia | Rac2 |
| Rac | lamellipodia | Rac3 |
| Rac | lamellipodia | RhoG |
| RhoBTB | protein stability | RhoBTB1 |
| RhoBTB | protein stability | RhoBTB2 |
| RhoBTB | protein stability | RhoBTB3 |
| RhoH | Rac agonist? | RhoH |
| Rho | ↑stress fibres and ↑focal adhesions | RhoA |
| Rho | ↑stress fibres and ↑focal adhesions | RhoB |
| Rho | ↑stress fibres and ↑focal adhesions | RhoC |
| Rnd | ↓stress fibres and ↓focal adhesions | Rnd1 |
| Rnd | ↓stress fibres and ↓focal adhesions | Rnd2 |
| Rnd | ↓stress fibres and ↓focal adhesions | Rnd3 |
| RhoF | Vesicle transport, filopodia | RhoD |
| RhoF | Vesicle transport, filopodia | RhoF |
Regulators
Three general classes of regulators of Rho protein signaling have been identified: guanine nucleotide exchange factor, GTPase-activating proteins and guanine nucleotide dissociation inhibitors. GEFs activate Rho proteins by catalyzing the exchange of GDP for 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 and thus acting as an anchor and allowing tight spatial control of Rho activation. In human, 82 GEF control positively the activity of Rho members, while 66 GAP proteins control it negatively.Recent work has unveiled important additional regulatory mechanisms: microRNAs regulate post-transcriptional processing of Rho GTPase-encoding mRNAs; palmitoylation and nuclear targeting affect intracellular distribution; post-translational phosphorylation, transglutamination and AMPylation modulate Rho GTPase signaling; and ubiquitination controls Rho GTPase protein stability and turnover. These modes of regulation add to the complexity of the Rho GTPase signaling network and allow precise spatiotemporal control of individual Rho GTPases.
Effectors
Each Rho protein affects numerous proteins downstream, all of which having roles in various cell processes. Over 60 targets of the three common Rho GTPases have been found. Two molecules that directly stimulate actin polymerization are the Arp2/3 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 | Sra1, IRSp53, PAK1, PAK2, PAK3 |
| Cdc42 | Wiskott-Aldrich syndrome protein, N-WASP, IRSp53, Dia2, Dia3, ROCK1, ROCK2, PAK4 |
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, it must have some manner of cell polarity.One example of Rho GTPases' role in cell polarity is seen in the much-studied yeast cell. Before the cell can bud, Cdc42 is used to locate the region of the cell's membrane that will begin to bulge into the new cell. When Cdc42 is removed from the cell, the outgrowths still form, but do so in an unorganized manner.
One of the most obvious changes to cell morphology controlled by Rho proteins is the formation of lamellipodia and filopodia, projecting processes that look like "fingers" or "feet" and often propel cells or growth cones across surfaces. Virtually all eukaryotic cells form such processes upon Rho activation. Fibroblasts such as Swiss 3T3 cells are often used to study these phenomena.
Study techniques
Much of what is known about cellular morphology changes and the effects of Rho proteins comes from the creation of a constitutively active mutated form of the protein. Mutation of a key amino acid can alter the conformation of the entire protein, causing it to permanently adopt a conformation that resembles the GTP-bound state. This protein cannot be inactivated normally, through GTP hydrolysis, and is thus "stuck on". When a Rho protein activated in this manner is expressed in 3T3 cells, morphological changes such as contractions and filopodia formation ensue.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. One method of maintaining the spatial zones of activation is through anchoring to the actin cytoskeleton, keeping the membrane-bound protein from diffusing away from the region where it is most needed. Another method of maintenance is through the formation of a large complex that is resistant to diffusion and more rigidly bound to the membrane than the Rho itself.