The Rho GTPase family:a Racs to Wrchs storyMatthew Wherlock and HarryMellor*Department of Biochemistry, School of MedicalSciences, University of Bristol, Bristol BS8 1TD, UK*Author for correspondence (e-mail:h.mellor@bristol.ac.uk)

Journal of Cell Science 115, 239-240 (2002)© The Company of Biologists Ltd

The Rho family of small GTPasescomprises some 21 genes in humans,encoding at least 23 signalling proteins.Although these proteins control anamazingly diverse range of cellularfunctions, one general role is in theestablishment of polarity and of polarisedstructures through dynamic regulation ofthe actin cytoskeleton. This theme iscarried through all three eukaryotekingdoms - from bud formation in S.

cerevisiae, to pollen tube elongation inArabidopsis, to the formation of complexstructures such as cochlear stereocilia inmammals. Rho GTPases control thepolymerisation, branching and bundlingof actin, allowing them to regulate theremodelling of the actin cytoskeleton intodistinct architectural elements. Spatialand temporal control of these elementsallows Rho GTPases to direct complexmechanical processes such as cell motilityand phagocytosis (Bishop and Hall, 2000;Hall, 1998).

The best-characterised family membersare RhoA, Rac1 and Cdc42 (humannomenclature). Each controls theformation of a distinct cytoskeletalelement in mammalian cells; RhoAstimulates the bundling of actin filamentsinto stress fibres, Rac reorganises actin toproduce membrane sheets or lamellipodia,and Cdc42 is associated with the

formation of thin, actin-rich surfaceprojections called filopodia. These familymembers illustrate the high level ofconservation of both structure andfunction through eukaryote evolution. C.elegans and Drosophila have homologuesof all three small GTPases, anddownstream effector proteins are alsoconserved. Rac is absent from yeast, butboth the Cdc42 and RhoA (Rho1)homologues are present. The coupling ofthe yeast Rho1 to the Pck1 kinase is seenin worms, flies and humans in theinteraction of RhoA with the Pck1-relatedPRK/PKN kinases.

In humans these three archetypal familymembers form subgroups of relatedproteins. RhoA has two highly relatedhomologues: RhoB and C. Examination ofthe structures of the genes that encodethese proteins suggests that RhoC arosefrom duplication of the RhoA gene,

Cell Science at a Glance 239

Journal of Cell Science 2002 (115, pp. 239-240)

Cell ScienceJournal of

The Rho GTPase FamilyMatthew Wherlock and Harry Mellor

Dictyostelium discoideumDictyostelium disc

Drosophila mela Caenorhabdit

S h i i

Arabidopsis

S. cerevisiae

Dictyostelium

C. elegans

Drosophila

H. sapiens

-

Rho1

-

RhoA

Rho1

RhoA, RhoB, RhoC

?

-

Rac1a, Rac1b, Rac1cRac F1, RacF2

Rac1, Rac2

DRac1, DRac2

Rac1, Rac2, Rac3

-

Cdc42

-

Cdc42

Cdc42

Cdc42, TC10, TCL

RhoA Rac Cdc42

(See poster insert)

240

whereas the intronless RhoB gene appearsto represent a retrotransposition of RhoA.Similarly, distinct subfamilies of Cdc42-like (TC10 and TCL) and Rac1-likeisoforms (Rac2 and Rac3) are present. Inmany cases members of these subfamiliesshare subsets of downstream effectors.Thus, the value of these duplications to cellfunction may come from the differentialexpression and/or subcellular localisationof subfamily members.

The conservation of RhoA, Rac andCdc42 through evolution is striking butgives a somewhat misleading impressionof the development of this signallingfamily as a whole. Comparison of the RhoGTPase family structure between speciesshows that the overall picture is one ofgreat plasticity, individual species gainingand losing family members to give rise tounique sets of signalling proteins. Forexample, S. cerevisiae Rho3, Rho4 andRho5 have no apparent homologues in theother species shown; nor do their knowninteracting proteins suggest any obviousparallels. Perhaps the clearest example ofRho family plasticity comes from theexpansion of the Rac subfamily in plants.Arabidopsis thaliana has no apparentRhoA homologue, nor any proteinscontaining RhoA-binding motifs. Neitherdoes this species have any obvious Cdc42homologue or homologues of Cdc42 orRac effector proteins such as WASP andPAK. However, Arabidopsis has 11 Rhofamily GTPases that appear very distantlyrelated to the Rac GTPase; these aregenerally highly related (between 65-99%identical) to each other, but show onlyweak sequence similarity to Rho GTPasesin other species (see poster). The socialamoeba Dictyostelium discoideum has noCdc42 or RhoA homologue, but doeshave a group of Rac-like proteins (Rac1a,Rac1b, Rac1c, RacF1 and RacF2) andhomologues of WASP and PAK.However, Dictyostelium also has a host ofother more esoteric Rho family isoformsthat appear to lack equivalents in otherspecies.

One case that clearly supports loss of afamily member is that of the RhoBTBproteins. The majority of Rho familymembers are 21 kDa proteins terminatingin a C-terminal CAAX motif that becomesmodified by prenylation. In the highlyunusual RhoBTBs, this CAAX motif isreplaced by an approximately 400-residueextension that includes a BTB (for Broad-Complex, Tramtrack and Bric à Brac)

domain. Dictyostelium has a singleRhoBTB (RacA), Drosophila has one(RhoBTB) and humans have three.However, this highly distinctive RhoGTPase is clearly absent from C. elegans.Similarly, the Mig-2 GTPase, whichcontrols axon guidance in C. elegans andDrosophila (Mtl), has no apparenthomologue in the human genome.

It would seem that gene duplication anddivergence has allowed expansion of theRho GTPase family to continue late intoevolution. The overall picture is one wherefamily favourites may be retained andexpanded into closely related subfamilies,but these exist side by side with a host ofdivergent proteins that presumablyperform specialised functions. This placesimportant constraints on our interpretationof data from studies of different modelorganisms. While some pathways areclearly conserved, others are misleadinglynot. In yeast the Rho1 activation of Pck1signals to a well-defined MAP kinasepathway that responds to hyposmoticstress. In mammals, RhoA activates theanalogous PRK kinase, but a downstreamMAP kinase pathway is apparently absent.Similarly, Wilkins and Insall have pointedout that while Dictyostelium shares manyaspects of mammalian cell motility, it doesso without RhoA or Cdc42 (Wilkins andInsall, 2001). It seems that often the picturemay look highly familiar but thebrushstrokes differ.

Why so many different Rho GTPases?Perhaps this reflects the successful designof Ras-related small GTPases. The basicmolecular switch mechanism presents arich interaction surface to signallingpartners on activation of the protein. Whilethe switch mechanism itself is highlyconserved, the interacting surface caneasily be varied, with the consequentpotential to create new signallingjunctions. We also know from structuralstudies that different effectors can interactwith different regions of this surface,allowing several diverging pathways to bedriven by a single family member.

A harder question to explain is theclustering of function of Rho familymembers. With related Rho GTPasescarrying out seemingly highly relatedfunctions in cytoskeletal regulation, it iseasy to forget that the signallingintermediates are a diverse andstructurally unrelated set of proteins. Whyshould these downstream partners so

frequently end up signalling to the actincytoskeleton?

Finally, of the 21 human Rho GTPases,only RhoA, Rac and Cdc42 have beenstudied in detail. It seems unlikely thatany of the 19 uncharted isoforms couldpossibly have the amazing breadth offunction of the big three. However, it is aracing certainty that each will dosomething unique and interesting. This isan exciting time to be working on thisfascinating family of signalling proteins.

We thank Richard Sessions (University of Bristol)for help with the ribbon diagram of RhoAstructure. HM holds a Wellcome Trust ResearchCareer Development Fellowship. MW is supportedby a BBSRC Committee Studentship.

Poster: Sequences of previously identified RhoGTPases were used to search the genome databasesof the six species shown in the poster. The goal wasto produce a (near) complete, non-redundant list ineach case. Higher eukaryotes have many intronlessRho GTPase pseudogenes. Intronless genes withbroken coding sequences were excluded. In manycases, intronless genes were identified whosepredicted protein product was highly similar (oridentical) to known family members, but whosemRNA was not represented in the dbEST cDNAdatabase; these were also excluded. Finally,Drosophila sequences that mapped to identicalchromosomal locations in Canton and Oregonstrains, but showed 1-2 amino acid substitutions,were assumed to represent allelic variation and arepresentative sequence was used. Sequences werealigned using the ClustalW algorithm andsequence distances displayed as unrooteddendrograms in TreeView. Alternativenomenclature is shown in parenthesis. Splicevariants are indicated with a forward slash. Thereader should be aware that naming of Rho GTPaseisoforms has been largely by caprice, and thatRacX or RhoY does not necessarily implyrelatedness to archetypal Rac or Rho GTPases. Forthe BTB-containing Rho GTPases, the alignmentsconsidered only the Rho GTPase domain. Thehuman RhoBTB3 GTPase domain is highlydivergent and was excluded from the alignment.The human Chp sequence has been deposited withthe GenBank database (accession no. AY059636).

ReferencesBishop, A. L. and Hall, A. (2000). Rho GTPasesand their effector proteins. Biochem. J. 348, 241-255.Hall, A. (1998). Rho GTPases and the actincytoskeleton. Science 279, 509-514.Wilkins, A. and Insall, R. H. (2001). SmallGTPases in Dictyostelium: lessons from a socialamoeba. Trends Genet. 17, 41-48.

Journal of Cell Science 115 (2)

Cell Science at a Glance on the WebElectronic copies of the poster insert are availablein the online version of this article atjcs.biologists.org. JPEG and PDF files (seesupplemental material) can be downloaded forprinting or use as slides.