The Rheb family of GTP-binding proteins

Rheb proteins represent a novel and unique family of the Ras superfamily GTP-binding proteins that is conserved from yeast to human. Biochemical studies establish that they bind and hydrolyze GTP. Molecular modeling studies reveal a few structural differences between Rheb and Ras, which may suggest that residues involved in biochemical activities differ between the two G-proteins. The function of Rheb has been studied in a number of organisms that point to the involvement of Rheb in cell growth and cell cycle progression. In addition, studies in fungi suggest that Rheb is involved in arginine uptake. Further studies in Drosophila and mammalian cells have shown that the effects of Rheb on growth and cell cycle progression are mediated by the effect on the insulin/TOR/S6K signaling pathway. These studies have also shown that a complex consisting of the tuberous sclerosis gene products, Tsc1/Tsc2, serves as a GTPase activating protein (GAP) for Rheb, implying Rheb's role in this genetic disorder. Finally, Rheb proteins have been shown to be farnesylated and small molecule inhibitors of protein farnesyltransferase can block the ability of Rheb to activate the TOR/S6K signaling.     

Human ADP-ribosylation factors. A functionally conserved family of GTP-binding proteins

A new member, hARF4, of the ADP-ribosylation factor (ARF) family, a subset of the superfamily of regulatory GTP-binding proteins, has been cloned from a cDNA expression library. Two other human ARF cDNA sequences, designated human ARF1 and ARF3, have been reported previously and are 96% identical in amino acid sequence. A human ARF1 cDNA, significantly longer than previously described clones, was obtained, by cross-species hybridization using a bovine ARF1 cDNA probe. Bovine ARF1p and human ARF1p are 100% identical while each is only 80% identical to hARF4p. Thus, hARF4p is the most divergent of the mammalian ARF proteins identified. Northern blot analysis revealed the expression of at least three different ARF messages in human placenta and adrenal carcinoma cells. Both hARF1 and hARF4 encode GTP-binding proteins with predicted molecular masses of 20,000-21,000 Da. Biochemical analysis of the purified recombinant proteins revealed a high degree of conservation of nucleotide binding properties and in vitro ARF activities. ARF is an essential gene in the yeast, Saccharomyces cerevisiae, and is encoded by two genes. Expression of either hARF1p or hARF4p in yeast was found to rescue the lethal double mutant, arf1-arf2-, thus demonstrating the functional conservation of ARF functions between yeast and man. The combination of in vivo and in vitro assays for ARF function provides a specific and unambiguous means of determining bona fide ARF proteins from divergent species from among the rapidly increasing number of structurally related, small molecular weight GTP-binding proteins.     

RJLs: a new family of Ras-related GTP-binding proteins

The Ras superfamily of GTP binding proteins encompasses several gene families that regulate a plethora of events in the eukaryotic cell. Here we describe a novel branch of this superfamily which we have named RJLs. These are present in many unicellular organisms and also in deuterostomes but apparently missing in some intermediary phyla, suggesting an intriguing possibility of lateral gene transference between lower and higher eukaryotes. RJLs lack classical membrane targeting signals and the conserved glutamine residue that coordinates GTP hydrolysis in other proteins from the Ras superfamily. Interestingly, chordate orthologues are chimeras fused to "J" domains in their C-terminal, suggesting that these proteins recruit Hsc70 to specific sites in the cell. Expression analysis of RJLs from chordates suggests predominant expression in nervous tissues, possibly reflecting a role for RJLs in the development or maintenance of the sophisticated chordate nervous system.     

Effect of Rho family GTP-binding proteins on Amoeba proteus

Summary.  While there is a number of studies on the effects of Rho GTPases on the actin-based cytoskeleton in higher eukaryotes, studies in protozoans are rather limited. The problem seems to be intriguing since the structure of protozoan cytoskeletons is distinct from most vertebrate cells. By blocking endogenous Rho family proteins of highly motile Amoeba proteus with C3 transferase and antibodies against human RhoA and Rac1, we tried to assess the in vivo role of these proteins in amoebae. In migrating amoebae, both proteins are concentrated in the cortical layer and seem to colocalize with filamentous actin. Endogenous Rac1, but not RhoA, is accumulated in the perinuclear cytoskeleton. Blocking Rac- or Rho-like proteins caused distinct and irreversible changes in the locomotive shape of the examined amoebae and significant inhibition of their migration. Amoebae microinjected with anti-Rac1 antibodies were contracted, shortened, and developed only few wide pseudopodia. More pronounced changes were observed in cells treated with anti-RhoA antibodies. They exhibited an atypical locomotion not leading to their effective displacement. After treatment with 50 μg of C3 transferase per ml, cells rapidly contracted and almost completely rounded up, became refractile with the granules beaten into a dense mass, detached from the surface and died. Ten times lower concentration of the enzyme caused similar changes as the inhibition of endogenous RhoA-like protein. These results indicate that Rho family-based regulation plays a key role in amoebic migration. Received May 2, 2002; accepted August 2, 2002; published online November 29, 2002     

GTP-binding proteins in plants: new members of an old family

Regulatory guanine nucleotide-binding proteins (G proteins) have been studied extensively in animal and microbial organisms, and they are divided into the heterotrimeric and the small (monomeric) classes. Heterotrimeric G proteins are known to mediate signal responses in a variety of pathways in animals and simple eukaryotes, whiole small G proteins perform diverse functions including signal transduction, secretion, and regulation of cytoskeleton. In recent years, biochemical analyses have produced a large amount of information on the presence and possible functions of G proteins in plants. Further, molecular cloning has clearly demonstrated that plants have both heterotrimeric and small G proteins. Although the functions of the plant heterotrimeric G proteins are yet to be determined, expression analysis of an Arabidopsis G protein suggests that it may be involved in the regulation of cell division and differentiation. In contrast to the very few genes cloned thus far that encode heterotrimeric G proteins in plants, a large number of small G proteins have been identified by molecular cloning from various plants. In addition, several plant small G proteins have been shown to be functional homologues of their counterparts in animals and yeasts. Future studies using a number of approaches are likely to yield insights into the role plant G proteins play.     

Evolution of the Rab family of small GTP-binding proteins.

Rab proteins are small GTP-binding proteins that form the largest family within the Ras superfamily. Rab proteins regulate vesicular trafficking pathways, behaving as membrane-associated molecular switches. Here, we have identified the complete Rab families in the Caenorhabditis elegans (29 members), Drosophila melanogaster (29), Homo sapiens (60) and Arabidopsis thaliana (57), and we defined criteria for annotation of this protein family in each organism. We studied sequence conservation patterns and observed that the RabF motifs and the RabSF regions previously described in mammalian Rabs are conserved across species. This is consistent with conserved recognition mechanisms by general regulators and specific effectors. We used phylogenetic analysis and other approaches to reconstruct the multiplication of the Rab family and observed that this family shows a strict phylogeny of function as opposed to a phylogeny of species. Furthermore, we observed that Rabs co-segregating in phylogenetic trees show a pattern of similar cellular localisation and/or function. Therefore, animal and fungi Rab proteins can be grouped in "Rab functional groups" according to their segregating patterns in phylogenetic trees. These functional groups reflect similarity of sequence, localisation and/or function, and may also represent shared ancestry. Rab functional groups can help the understanding of the functional evolution of the Rab family in particular and vesicular transport in general, and may be used to predict general functions for novel Rab sequences.     

DRG represents a family of two closely related GTP-binding proteins

In a previous publication we identified a novel human GTP-binding protein that was related to DRG, a developmentally regulated GTP-binding protein from the central nervous system of mouse. Here we demonstrate that both the human and the mouse genome possess two closely related drg genes, termed drg1 and drg2. The two genes share 62% sequence identity at the nucleotide and 58% identity at the protein level. The corresponding proteins appear to constitute a separate family within the superfamily of the GTP-binding proteins. The DRG1 and the DRG2 mRNA are widely expressed in human and mouse tissues and show a very similar distribution pattern. The human drg1 gene is located on chromosome 22q12, the human drg2 gene on chromosome 17p12. Distantly related species including Caenorhabditis elegans, Schizosaccharomyces pombe and Saccharomyces cerevisiae also possess two drg genes. In contrast, the genomes of archaebacteria (Halobium, Methanococcus, Thermoplasma) harbor only one drg gene, while eubacteria do not seem to contain any. The high conservation of the polypeptide sequences between distantly related organisms indicates an important role for DRG1 and DRG2 in a fundamental pathway.     

The RGK family: a regulatory tail of small GTP-binding proteins

RGK proteins are small Ras-related GTP-binding proteins that function as potent inhibitors of voltage-dependent calcium channels, and two members of the family, Gem and Rad, modulate Rho-dependent remodeling of the cytoskeleton. Within the Ras superfamily, RGK proteins have distinct structural and regulatory characteristics. It is an open question as to whether RGK proteins catalyze GTP hydrolysis in vivo. Binding of calmodulin and the 14-3-3 protein to RGK proteins controls downstream pathways. Here, we discuss the structural and functional properties of RGK proteins and highlight recent work by Beguin and colleagues addressing the mechanism of Gem regulation by calmodulin and 14-3-3.     

A diverse family of GPCRs expressed in specific subsets of nociceptive sensory neurons

In vertebrates, peripheral chemosensory neurons express large families of G protein-coupled receptors (GPCRs), reflecting the diversity and specificity of stimuli they detect. However, somatosensory neurons, which respond to chemical, thermal, or mechanical stimuli, are more broadly tuned. Here we describe a family of approximately 50 GPCRs related to Mas1, called mrgs, a subset of which is expressed in specific subpopulations of sensory neurons that detect painful stimuli. The expression patterns of mrgs thus reveal an unexpected degree of molecular diversity among nociceptive neurons. Some of these receptors can be specifically activated in heterologous cells by RFamide neuropeptides such as NPFF and NPAF, which are analgesic in vivo. Thus, mrgs may regulate nociceptor function and/or development, including the sensation or modulation of pain.     

Evolution of the Rho family of ras-like GTPases in eukaryotes

GTPases of the Rho family are molecular switches that play important roles in converting and amplifying external signals into cellular effects. Originally demonstrated to control the dynamics of the F-actin cytoskeleton, Rho GTPases have been implicated in many basic cellular processes that influence cell proliferation, differentiation, motility, adhesion, survival, or secretion. To elucidate the evolutionary history of the Rho family, we have analyzed over 20 species covering major eukaryotic clades from unicellular organisms to mammals, including platypus and opossum, and have reconstructed the ontogeny and the chronology of emergence of the different subfamilies. Our data establish that the 20 mammalian Rho members are structured into 8 subfamilies, among which Rac is the founder of the whole family. Rho, Cdc42, RhoUV, and RhoBTB subfamilies appeared before Coelomates and RhoJQ, Cdc42 isoforms, RhoDF, and Rnd emerged in chordates. In vertebrates, gene duplications and retrotranspositions increased the size of each chordate Rho subfamily, whereas RhoH, the last subfamily, arose probably by horizontal gene transfer. Rac1b, a Rac1 isoform generated by alternative splicing, emerged in amniotes, and RhoD, only in therians. Analysis of Rho mRNA expression patterns in mouse tissues shows that recent subfamilies have tissue-specific and low-level expression that supports their implication only in narrow time windows or in differentiated metabolic functions. These findings give a comprehensive view of the evolutionary canvas of the Rho family and provide guides for future structure and evolution studies of other components of Rho signaling pathways, in particular regulators of the RhoGEF family.     

A novel member of the rho family of small GTP-binding proteins is specifically required for cytokinesis

Several members of the rho/rac family of small GTP-binding proteins are known to regulate the distribution of the actin cytoskeleton in various subcellular processes. We describe here a novel rac protein, racE, which is specifically required for cytokinesis, an actomyosin-mediated process. The racE gene was isolated in a molecular genetic screen devised to isolate genes required for cytokinesis in Dictyostelium. Phenotypic characterization of racE mutants revealed that racE is not essential for any other cell motility event, including phagocytosis, chemotaxis, capping, or development. Our data provide the first genetic evidence for the essential requirement of a rho-like protein, specifically in cytokinesis, and suggest a role for these proteins in coordinating cytokinesis with the mitotic events of the cell cycle.     

GTP-binding proteins in intracellular transport

One of the most exciting recent discoveries in the area of intracellular protein transport is the finding that many organelles involved in exocytic and endocytic membrane traffic have one or more Ras-like GTP-binding proteins on their cytoplasmic face that are specific for each membranous compartment. These proteins are attractive candidates for regulators of transport vesicle formation and the accurate delivery of transport vesicles to their correct targets.     

Human OLA1 defines an ATPase subfamily in the Obg family of GTP-binding proteins

Purine nucleotide-binding proteins build the large family of P-loop GTPases and related ATPases, which perform essential functions in all kingdoms of life. The Obg family comprises a group of ancient GTPases belonging to the TRAFAC (for translation factors) class and can be subdivided into several distinct protein subfamilies. The founding member of one of these subfamilies is the bacterial P-loop NTPase YchF, which had so far been assumed to act as GTPase. We have biochemically characterized the human homologue of YchF and found that it binds and hydrolyzes ATP more efficiently than GTP. For this reason, we have termed the protein hOLA1, for human Obg-like ATPase 1. Further biochemical characterization of YchF proteins from different species revealed that ATPase activity is a general but previously missed feature of the YchF subfamily of Obg-like GTPases. To explain ATP specificity of hOLA1, we have solved the x-ray structure of hOLA1 bound to the nonhydrolyzable ATP analogue AMPPCP. Our structural data help to explain the altered nucleotide specificity of YchF homologues and identify the Ola1/YchF subfamily of the Obg-related NTPases as an exceptional example of a single protein subfamily, which has evolved altered nucleotide specificity within a distinct protein family of GTPases.     

Relationships within the family of GTP-binding proteins isolated from bovine central nervous system

Four members of a family of GTP-binding proteins (G-proteins) which translate stimulation of extracellular receptors into regulation of intracellular enzymes were isolated from the bovine central nervous system. These proteins were examined for functional similarities and cross-reactivity with antibodies to the G-protein (transducin, Gt) from the photoreceptor system. Two proteins, Gs and Gi, can be distinguished by their respective abilities to stimulate or inhibit adenylate cyclase. The activated alpha subunits of Gt and a fourth member of the family, Go, did not affect this enzyme. Gt was shown to be unique in its ability to stimulate cGMP-dependent phosphodiesterase. While functionally diverse, the G-proteins were shown to have some common antigenic properties. Antibodies directed against the beta subunit of Gt recognize the beta 36 subunits of all preparations but not a putative second beta 35 subunit. Antibodies specific for the alpha subunit of Gt did not recognize other alpha subunits when immune blots from sodium dodecyl sulfate gels were examined. However, Go alpha, but not Gs alpha or Gi alpha, reacted strongly with the antibodies when the native subunit was spotted directly. This suggests that Go alpha and Gt alpha have homologous structural determinants. An antiserum that recognized Gt gamma did not recognize gamma subunits from other sources. These data support the proposed diversity of function and similarity of structure among the four G-proteins. The alpha and potentially gamma subunits appear to be responsible for the specificity of function.     

Structure/function relationship of proteins belonging to the family of receptors coupled to GTP-binding proteins.

The structural properties of a number of proteins belonging to the family of receptors coupled to GTP binding proteins are discussed in relation to their function. The structure of the ligand binding site and of the regions involved in coupling to the G proteins are analyzed mainly for the adrenergic and muscarinic cholinergic receptors, for which site-directed mutagenesis and chimaeric constructions have been studied. The structure of the genes are compared and the presence of various regulatory elements is discussed in relation to control of expression. Mechanism of desensitization and internalization, while mostly studied for the beta 2-adrenergic receptor, are proposed to be generally applicable to all G-protein-coupled receptors.