Cellular and Molecular Regulation

Much of the functional information on Rho family proteins has come from studies of the three classical members, Rac1, RhoA, and Cdc42. Hence, a majority of the information summarized in this article apply to the classical Rho GTPases and closely related isoforms. The GDP/GTP cycling of Rho family proteins is controlled by three distinct functional classes of regulatory proteins (Fig. 4). Guanine nucleotide exchange factors (GEFs) stimulate the weak intrinsic exchange activity of Rho family proteins to cause an exchange of the bound GDP for GTP to promote formation of active Rho-GTP. RhoGEFs are also called Dbl family proteins. Dbl family proteins (named after the founding member, a transforming protein identified from a human diffuse  B-cell lymphoma) share a tandem Dbl homology (DH) domain and pleckstrin homology domain. The DH domain is a catalytic domain that stimulates GDP/GTP exchange. The PH domain is believed to regulate DH domain catalytic activity and can also serve to promote Dbl protein association with the plasma membrane. A number of Dbl family proteins were initially identified in gene transfer screening searches for transforming (e.g., Dbl, Vav, Ect2, Lsc) or invasion-inducing ( Tiam1) genes. Others were identified as proteins with other catalytic functions, such as the breakpoint cluster region (BCR) protein. BCR is the translocation partner of the Abl tyrosine kinase present in Philadelphia chromosome-positive human leukemias, and this genetic rearrangement causes the formation of a chimeric BCR-ABL fusion oncoprotein. To date, 69 distinct human Dbl family proteins have been identified. Since they function as RhoGEFs, their transforming actions are due to chronic activation of Rho GTPases. Additionally, a second structurally distinct family of RhoGEFs, DOCK family proteins (15 human members), also serve as activators of Rho GTPases. Some atypical Rho GTPases (e.g., Rnd proteins) are GTPase deficient and constitutively GTP bound and active and, consequently, are not believed to be regulated by RhoGEFs.

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The second class of regulators of Rho family proteins is GTPase-activating proteins (GAPs) that stimulate the weak intrinsic GTP hydrolysis activity of Rho family proteins to promote formation of the inactive GDP-bound protein. Presently, at least 60 RhoGAPs have been identified (e.g., chimerins, BCR). The third class of regulators is the Rho guanine nucleotide dissociation inhibitory (GDI) factors. While RhoGDIs can inhibit GDP dissociation as well as GAP-stimulated GTP hydrolysis, their main cellular function involves regulation of the association of Rho family proteins with membranes. To date, three distinct Rho GDIs have been identified.

Structural analyses of Ras and Rho family proteins reveal that the GDP- and GTP-bound proteins differ in conformation in two regions, designated switch I and switch II (Fig. 2). The conformation of the GTP-bound protein results in increased binding affinity for downstream effector proteins. Each Rho family protein recognizes multiple effectors, and some effectors are recognized by multiple Rho family proteins. Rho family protein interaction and activation of effector function lead to the stimulation of effector-mediated cytoplasmic signaling pathways that regulate the diverse functions of Rho family proteins. The effectors for RhoA, Rac1, and Cdc42 have been the most intensively studied and characterized. The multitude of effectors identified for each Rho family protein reflects the complex and diverse functional properties of these proteins.

Genetically engineered structural mutants of Rho family proteins have provided very useful research reagents to evaluate the biochemical and biological functions of Rho family proteins. The first class of mutants is gain-of-function mutants. Single amino acid substitutions at residues analogous to those that convert normal Ras proteins into highly oncogenic, constitutively activated proteins (at Ras residues glycine 12 or glutamine 61) also create constitutively activated mutants of Rho family proteins. These mutations render Ras and Rho proteins insensitive to GAP stimulation, and thus, these proteins persist in the GTP-bound state. The second class of mutants is dominant-negative mutants that contain a serine to asparagine substitution at the residue analogous to amino acid 17 of Ras proteins. These mutants can prevent activation of specific Rho family proteins, presumably by forming inactive complexes with specific Dbl family proteins. The third class of mutants is effector domain mutants that possess single amino acid substitutions in the core effector domain. These impair interaction of Rho family proteins with downstream effectors, thus leading to impairment in biological activity. Since a particular effector domain mutation leads to differential impairment of effector interaction, such mutants have been very useful reagents in establishing the specific contribution of different effector targets to Rho family protein function.

Functions

A diverse spectrum of extracellular stimuli via interaction with  receptor tyrosine kinases (RTKs), heterotrimeric G protein-coupled receptors (GPCRs), or integrins cause activation of specific Rho family proteins, most commonly via activation of specific RhoGEFs (Fig. 5), for example, GPCRs that lead to activation of the heterotrimeric Gα13 subunit which then directly binds to the RGS (regulator of G protein signaling) box of the p115 RhoGEF, PDZ-RhoGEF, and Larg Dbl family proteins. These Dbl family proteins are specific GEFs for RhoA and related isoforms. Platelet-derived growth factor (PDGF) stimulation of the PDGF RTK causes activation of Rac1. This may be caused by activation of phosphatidylinositol 3-kinase and generation of phosphatidylinositol (3,4,5)-trisphosphate (PIP₃). PIP₃ can then bind the pleckstrin homology domain of the P-rex Rac-specific RhoGEF, leading to Rac activation. PDGF-stimulated activation of Ras can be another mechanism for Rac activation. Activated Ras binds to the Ras-binding domain (RBD) of the Tiam1 RhoGEF, promoting Rac activation. Bradykinin stimulation of a GPCR causes activation of Cdc42. These various extracellular signals cause a transient increase in the GTP-complexed protein that then rapidly cycles back to the GDP-complexed form to terminate the signal.

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Clinical Relevance

There is presently considerable experimental evidence linking Rho family proteins to cancer (Fig. 4). Unlike Ras, where mutational activation of Ras proteins is associated with 25% of human cancers, mutationally activated Rho GTPases are not found commonly in cancer. Rac1 activating mutations are found in melanoma, whereas apparent loss-of-function mutations in RhoA are found in gastric adenocarcoma. Instead, more commonly indirect mechanisms that include altered gene transcription or the altered function of Rho regulators are most commonly observed. For example, the Vav1 RhoGEF, normally hematopoietic cell restricted in expression, is ectopically overexpressed and activated in pancreatic cancers. Tiam1, a Rac-specific GEF, functions as an effector of Ras and is required for Ras-mediated oncogenesis. Ras binds to and activates Tiam1, leading to persistent Rac activation. The expression of DLC-1 and related RhoGAPs is lost in a wide variety of human cancers. Since DLC-1 (deleted in liver cancer 1) functions as a negative regulator of RhoA, the loss of DLC-1 may promote oncogenesis by causing persistent RhoA activation. Rac1b is a splice variant of Rac1 that is preferentially expressed in breast and colon cancers. Unlike Rac1, Rac1b is persistently activated and cannot bind RhoGDI and, consequently, is a transforming variant of Rac1.

Aberrant Rho GTPase function is also associated with other human diseases. Oligophrenin-1 is a RhoGAP whose expression is lost and involved in nonspecific X-linked mental retardation. FGD1 is a Cdc42-specific RhoGEF that is mutated and inactivated in Aarskog-Scott syndrome (also called faciogenital dysplasia), which is a rare, clinically and genetically heterogeneous condition characterized by facial dysmorphic features, short stature, brachydactyly, and genital anomalies. Salmonella typhimurium, Yersinia pestis, Shigella flexneri, and other virulent bacteria have evolved mechanisms that alter the Rho GTPase function of their human host cells that facilitate their ability to promote infection and disease. Salmonella typhimurium causes typhoid fever, Yersinia pestis was the causative agent for the Black Death plague which accounted for the death of approximately one-third of the European population in the fourteenth century, and Shigella flexneri causes dysentery. These bacteria use a type III secretion system to deliver bacterial effector proteins directly into the host cell. These function as activators or inactivators of human Rho GTPases and include proteins that act as RhoGEFs or RhoGAPs. Since bacteria do not possess Rho GTPases that are regulated by these effector proteins, these proteins exist solely for the purpose of deregulating the cellular functions of host cells.