GEFs: structural basis for their activation of small GTP-binding proteins.

Small GTP-binding proteins of the Ras superfamily function as molecular switches in fundamental events such as signal transduction, cytoskeleton dynamics and intracellular trafficking. Guanine-nucleotide-exchange factors (GEFs) positively regulate these GTP-binding proteins in response to a variety of signals. GEFs catalyze the dissociation of GDP from the inactive GTP-binding proteins. GTP can then bind and induce structural changes that allow interaction with effectors. Representative structures of four main classes of exchange factors have been described recently and, in two cases, structures of the GTP-binding protein-GEF complex have been solved. These structures, together with biochemical studies, have allowed a deeper understanding of the mechanisms of activation of Ras-like GTP-binding proteins and suggested how they might represent targets for therapeutic intervention.     

The regulation of mitochondrial physiology by organelle-associated GTP-binding proteins

Recent studies have shown that GTP-binding proteins can modulate mitochondrial membrane fusion and fission. Furthermore, GTP-binding proteins can regulate the binding of ribosomes to the mitochondrial membrane and may facilitate the import of proteins through contact points between inner and outer mitochondrial membranes. Mitochondrial GTP-binding proteins therefore appear to have the potential to modulate physiological function of the organelle and may also be involved in cellular processes such as cellular transformation. A beginning has been made on the characterization of mitochondrial GTP-binding proteins and the DNA sequence of one protein has become newly available. Future studies are needed to determine whether GTP-binding proteins are interacting with cell signalling molecules such as protein kinases in the mitochondria.     

Identification of a conserved region common to cadherins and influenza strain A hemagglutinins

Cadherins are a family of integral membrane glycoproteins that mediate homophilic, calcium-dependent cell adhesion in vertebrate species. The primary structures of six members of the cadherin family have recently been determined. The extracellular portion of these proteins is composed of five domains, the first of which is the most highly conserved among cadherins. Previous searches of protein sequence databases have revealed little or no sequence homology between cadherins and other proteins. Here we report that the first extracellular domain of cadherins exhibits substantial sequence homology with the amino termini of influenza strain A hemagglutinins. These regions of sequence homology have been shown to be functionally important in both cadherins and hemagglutinins. Our observations suggest that a functional domain of cadherins is conserved among other proteins.     

Signal transducing GTP-binding proteins in olfaction

1. Several members of the family of heterotrimeric signal transducing GTP-binding proteins have been identified in the olfactory epithelium of vertebrates by biochemical and molecular cloning techniques. 2. Biochemical and electrophysiological evidence indicates that one or more GTP-binding proteins mediate many olfactory responses by coupling stimulus receptors to second messenger systems. 3. Although GTP-binding proteins may function in additional transduction events, a novel GTP-binding protein, expressed only in olfactory neurons, may mediate stimulus activation of adenylate cyclase in olfactory cilia.     

Small GTP-binding proteins on rat liver lysosomal membranes.

GTP-binding proteins have been identified on the membranes of highly purified dextran-filled lysosomes (dextranosomes) and Triton-filled lysosomes (tritosomes) obtained from rat liver. Autoradiography of blots of lysosomal membrane proteins incubated with [alpha-32P]GTP revealed the presence of several specific GTP-binding proteins with a relative molecular mass (M(r)) predominantly in the range of 26-30 kDa. These GTP-binding proteins migrated slower in polyacrylamide gels than purified c-Ha-ras protein expressed in E. coli, whose apparent M(r) was 23 kDa in the same blot. The relative contents of GTP-binding proteins in lysosomal membranes were comparable or greater than that of plasma membranes and of microsomes. Chemical extraction showed that lysosomal GTP-binding proteins were more tightly associated with the membranes than with microsomal GTP-binding proteins. The possible involvement of lysosomal GTP-binding proteins in cellular functions including vacuolar (lysosomal) acidification and organellar dynamics are discussed.     

Study of the Interaction of the Myelin Monomeric GTP-Binding Proteins with Other Brain Proteins

Abstract: Although several monomeric GTP-binding proteins have been found in myelin, the signaling pathways in which they operate are not known. To define these signaling pathways we searched for specific target proteins that interact with the myelin monomeric GTP-binding proteins. A blot overlay approach was used. Bovine white matter homogenate, myelin, and oligodendrocyte proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. The presence of proteins that interact with the myelin GTP-binding proteins was explored by incubating those blots with an enriched fraction of 22- and 25-kDa myelin GTP-binding proteins labeled with radioactive guanine nucleotides. When the GTP-binding proteins were in the inactive state (GDP-bound) they interacted with 28-, 47-, and 58-kDa oligodendrocyte polypeptides. Only the 28-kDa protein was present in myelin. In the active state (GTP-bound), they interacted only with a 47-kDa protein in myelin but with 31-, 38-, 47-, 58-, 60-, 68-, and 71-kDa proteins in oligodendrocytes and total homogenate. Under these experimental conditions the 28-kDa protein did not interact with the GTP-binding proteins. The fact that the myelin GTP-binding proteins in the active state formed complexes with a different set of proteins than when in the inactive state is a strong indication that these proteins are effector proteins. With the exception of the 31- and 38-kDa proteins that were detected only in the cytoplasmic fraction, these polypeptides were detected in the cytosolic fraction and total membrane fraction. The 25-kDa GTP-binding protein was present in all the complexes. Immunoblot analysis indicated that the 28-kDa polypeptide is RhoGDI, an effector protein that is known to regulate the activation and movement of several GTP-binding proteins between different cellular compartments. Thus, this study opens the way to identify the macromolecules participating in the myelin signaling pathway involving monomeric GTP-binding proteins.     

Characterization of small GTP-Binding proteins in plant cell

To identify and characterize small GTP-binding proteins in plant cells, GTP-binding studies were performed with electroblotted plant proteins following SDS-polyacrylamide gel electrophoresis using [α-³²P]GTP. Three species of small GTP-binding protein (21, 23, and 27 kD) which have a specific GTP-binding property were identified in the membrane and cytosolic fractions of both monocotyledons (Zea mays) and dicotyledons (Glycine max). Moreover, these three species of small GTP-binding protein were gradually decreased when membranes were treated with hydroxylamine. This result indicates that these small GTP-binding proteins in plant cells are fatty acylated to the membrane lipids. The 27 kDa component was partially purified from hypocotyl membranes of Glycinemax, following S-300 gel filtration, phenylsepharose CL-4B, hydroxyapatite, and Q-sepharose column chromatography. This 27 kD protein was found to have both GTP-binding and GTPase activities.     

Evidence for participation of GTP-binding proteins in elicitation of the rapid oxidative burst in cultured soybean cells.

GTP-binding proteins have been shown to serve as second messengers in the transduction of hormone signals across animal cell plasma membranes. We present here three lines of evidence to demonstrate that GTP-binding proteins are also involved in the elicitation of the defense response of cultured soybean cells. First, the antigen-binding fragment (Fab) of an antibody that specifically recognizes GTP-binding proteins in plants and animals was delivered into soybean cells using a non-destructive biotin-mediated delivery technique developed previously. Internalization of this Fab enhanced up to 10-fold the rapid oxidative burst induced by elicitor molecules, whereas internalization of its heat-denatured counterpart or unrelated proteins had no effect. Because the antibody recognizes a protein of molecular mass approximately 45 kDa in soybean cell membranes that is protected from ADP-ribosylation by GTP gamma S (guanosine 5'-O-(thiotriphosphate), we propose the 45-kDa GTP-binding protein is responsible for these effects. Second, mastoparan, a specific activator of GTP-binding proteins, was shown to induce the defense-related oxidative burst in the absence of elicitor stimulation, thus mimicking an activated receptor as it is thought to do in mammalian systems. Finally, but admittedly less convincing, the A subunit of cholera toxin, an activator of certain stimulatory GTP-binding proteins (Gs), was found to weakly enhance the conventional elicitor-induced oxidative burst. Taken together, these data argue for the involvement of GTP-binding proteins in elicitor signal transduction in soybean cells.     

Small GTP-binding proteins associated with secretory vesicles of Paramecium

GTP-binding proteins act as molecular switches in a variety of membrane-associated processes, including secretion. One group of GTP-binding proteins, 20-30 kDa, is related to the product of the ras proto-oncogene. In Saccharomyces cerevisiae, ras-like GTP-binding proteins regulate vesicular traffic in secretion. The ciliate protist Paramecium tetraurelia contains secretory vesicles (trichocysts) whose protein contents are released by regulated exocytosis. Using [alpha-32P]GTP and an on-blot assay for GTP-binding, we detected at least seven GTP-binding proteins of low molecular mass (22-31 kDa) in extracts of Paramecium tetraurelia. Subcellular fractions contained characteristic subsets of these seven; cilia were enriched for the smallest (22 kDa). The pattern of GTP-binding proteins was altered in two mutants defective in the formation or discharge of trichocysts. Trichocysts isolated with their surrounding membranes intact contained two minor GTP-binding proteins (23.5 and 29 kDa) and one major GTP-binding protein (23 kDa) that were absent from demembranated trichocysts. This differential localization of GTP-binding proteins suggests functional specialization of specific GTP-binding proteins in ciliary motility and exocytosis.     

Small GTP-binding Proteins Associated with Secretory Vesicles of Paramecium

GTP-binding proteins act as molecular switches in a variety of membrane-associated processes, including secretion. One group of GTP-binding proteins, 20–30 kDa, is related to the product of the ras proto-oncogene. In Saccharomyces cerevisiae, ras -like GTP-binding proteins regulate vesicular traffic in secretion. The ciliate protist Paramecium tetraurelia contains secretory vesicles (trichocysts) whose protein contents are released by regulated exocytosis. Using [α-³²P]GTP and an on-blot assay for GTP-binding, we detected at least seven GTP-binding proteins of low molecular mass (22–31 kDa) in extracts of Paramecium tetraurelia. Subcellular fractions contained characteristic subsets of these seven; cilia were enriched for the smallest (22 kDa). The pattern of GTP-binding proteins was altered in two mutants defective in the formation or discharge of trichocysts. Trichocysts isolated with their surrounding membranes intact contained two minor GTP-binding proteins (23.5 and 29 kDa) and one major GTP-binding protein (23 kDa) that were absent from demembranated trichocysts. This differential localization of GTP-binding proteins suggests functional specialization of specific GTP-binding proteins in ciliary motility and exocytosis.     

Structural and functional similarities between the nucleotide-binding domains of CFTR and GTP-binding proteins.

The opening and closing of the CFTR Cl- channel are regulated by ATP hydrolysis at its two nucleotide binding domains (NBDs). However, the mechanism and functional significance of ATP hydrolysis are unknown. Sequence similarity between the NBDs of CFTR and GTP-binding proteins suggested the NBDs might have a structure and perhaps a function like that of GTP-binding proteins. Based on this similarity, we predicted that the terminal residue of the LSGGQ motif in the NBDs of CFTR corresponds to a highly conserved glutamine residue in GTP-binding proteins that directly catalyzes the GTPase reaction. Mutations of this residue in NBD1 or NBD2, which were predicted to increase or decrease the rate of hydrolysis, altered the duration of channel closed and open times in a specific manner without altering ion conduction properties or ADP-dependent inhibition. These results suggest that the NBDs of CFTR, and consequently other ABC transporters, may have a structure and a function analogous to those of GTP-binding proteins. We conclude that the rates of ATP hydrolysis at NBD1 and at NBD2 determine the duration of the two states of the channel, closed and open, much as the rate of GTP hydrolysis by GTP-binding proteins determines the duration of their active state.     

The many faces of Ras: recognition of small GTP-binding proteins

The structures of over 30 complexes of Ras superfamily small GTP-binding proteins bound to diverse protein partners have been reported. Comparison of these complexes using the sequences of the small GTP-binding proteins to align the contact sites shows that virtually all surface positions make contacts with at least one partner protein. Rather than highlighting a single consensus binding site, these comparisons illustrate the remarkable diversity of contacts of Ras superfamily members. Here, a new analysis technique, the interface array, is introduced to quantify patterns of surface contacts. The interface array shows that small GTP-binding proteins are recognized in at least nine distinct ways. Remarkably, binding partners with similar functions, including those with distinct folds, recognize small GTP-binding proteins in similar ways. These classes of shared surface contacts support the occurrence of both divergent and convergent evolutionary processes and suggest that specific effector functions require particular protein–protein contacts.     

Identification and localization of low-molecular-mass GTP-binding proteins associated with synaptic vesicles and other membranes

GTP-binding proteins were studied in synaptic vesicles prepared from bovine brain by differential centrifugation and separated further from plasma membranes using gel permeation chromatography. Following separation by SDS-PAGE of proteins from the different fractions, and transfer to nitrocellulose sheets, the presence and localization of low-molecular-mass GTP-binding proteins were assessed by [alpha-32 P]GTP binding. The vesicle-membrane fraction (SV) was enriched in synaptophysin (p38, a synaptic vesicle marker) and contained low-molecular-mass GTP-binding proteins; these consisted of a major 27 kDa protein and minor components (Mr 26 and 24 kDa) which were trypsin-sensitive and immunologically distinguishable from ras p21 protein. GTP-binding proteins of low molecular mass, but displaying less sensitivity to trypsin, were also found in the plasma membrane fraction (PM; enriched in Na+/K(+)-ATPase). In addition, the PM fraction contained GTP-binding proteins with higher Mr (Gi alpha and G0 alpha), together with another GTP-binding protein, ras p21. Putative function(s) of these GTP-binding proteins with low mass are discussed.     

Regulatory GTP-binding proteins (ADP-ribosylation factor, Gt, and RAS) are not activated directly by nucleoside diphosphate kinase.

The expression of nucleoside diphosphate kinase (NDK) genes has been implicated as a negative regulator of murine and human tumor metastases and is critical to proper development in Drosophila melanogaster. Molecular mechanisms for the role(s) of NDK in these complex processes have not yet been elucidated, but several reports have suggested that these and many other signal transduction pathways may be activated by NDK acting directly on a regulatory GTP-binding protein(s). To test this hypothesis, we examined the ability of NDK to catalyze the phosphorylation of the GDP bound to the following three members of the superfamily of regulatory GTP-binding proteins: Gt, Ha-ras p21, and ARF. We have found no evidence to support the hypothesis that NDK can directly activate any GTP-binding protein. Rather, evidence is presented which clearly shows that all of the GTP formed upon incubation of GTP-binding proteins with NDK is the result of NDK utilizing free GDP as substrate. The GDP bound to the regulatory proteins is not a substrate for NDK under conditions in which free nucleotides are rapidly and efficiently phosphorylated. The importance of appropriate controls for dissociation of GDP from the regulatory proteins both during the NDK reaction and during the analysis of product is demonstrated. We believe there is currently no experimental evidence to support the hypothesis that NDK can directly activate a regulatory GTP-binding protein.     

Compartmentalization of low molecular mass GTP-binding proteins among neutrophil secretory granules

Neutrophils contain several distinct classes of secretory granules that may sequentially fuse with the phagosome after the ingestion of particulates, or that may be differentially exocytosed after cellular activation with soluble stimuli. The exocytosis of neutrophil secretory granules has been shown to be GTP-dependent at a step distal to activation of the transductional G proteins. Inasmuch as ras-related low molecular mass GTP-binding proteins have been shown to play regulatory roles in vesicle sorting in the secretory pathway in yeast, the differential mobilization of neutrophil granules might be regulated by distinct GTP-binding proteins. We therefore explored the distribution and identity of low molecular mass GTP-binding proteins in neutrophil secretory granules and other subcellular fractions. After lysis by nitrogen cavitation, four highly resolved fractions were harvested from discontinuous Percoll gradients: a microsomal fraction enriched for plasma membranes, specific granules, primary granules, and cytosol. At least seven bands of distinct Mr were detected by probing protein blots with [32P]GTP. Microsomes contained a prominent GTP-binding band at 26 kDa and weaker ones at 24 and 22.5 kDa; specific granules contained bands at 26, 24, 22, and 20 kDa; primary granules showed bands at 24 and 23 kDa; cytosol showed strong bands at 23.5 and 19 kDa and a weak band at 26 kDa. Antiserum against ADP-ribosylation factor reacted strongly with the 19-kDa band in cytosol but with none of the membrane fractions. None of these proteins was recognized by antibodies against ras or against Sec4p. Botulinum exoenzyme C3 labeled bands of molecular mass 20 and 21 kDa in cytosol and microsomes that have distinct mobilities from all the blotted [32P]GTP-binding proteins. The highly compartmentalized subcellular distribution of the blotted [32P]GTP-binding proteins in neutrophils is consistent with a regulatory role in the differential mobilization of granule compartments during cellular activation.     

Partial characterization of GTP-binding proteins in Neurospora

Six fractions of GTP-binding proteins separated by gel filtration of a mycelial extract containing membrane components of Neurospora crassa were partially characterized. [35S]GTP gamma S bound to GTP-binding protein was assayed by repeated treatments with a Norit solution and centrifugation. The binding of [35S]GTP gamma S to GTP-binding proteins was competitively prevented in the presence of 0.1 to 1 mM GTP but not in the presence of ATP. These GTP-binding proteins fractionated by the gel column had Km values of 20, 7, 4, 4, 80 and 2 nM. All six fractions of these GTP-binding proteins showed the capacity to be ADP-ribosylated by pertussis toxin.     

GEFs, GAPs, GDIs and effectors: taking a closer (3D) look at the regulation of Ras-related GTP-binding proteins

Cell biology depends on the interactions of macromolecules, such as protein—DNA, protein—protein or protein—nucleotide interactions. GTP-binding proteins are no exception to the rule. They regulate cellular processes as diverse as protein biosynthesis and intracellular membrane trafficking. Recently, a large number of genes encoding GTP-binding proteins and the proteins that interact witht these molecular switches have been cloned and expressed. The 3D structures of some of these have also been elucidated     

The P-loop--a common motif in ATP- and GTP-binding proteins

Many ATP- and GTP-binding proteins have a phosphate-binding loop (P-loop), the primary structure of which typically consists of a glycine-rich sequence followed by a conserved lysine and a serine or threonine. The three-dimensional structures of several ATP- and GTP-binding proteins containing P-loops have now been solved. In this review current knowledge of P-loops is discussed with the additional aim of illustrating the fascinating relationship between protein sequence, structure and function.     

Mannose-binding hemagglutinins in extracts of Pseudomonas aeruginosa.

Mannose-binding hemagglutinins were found in the extracts of a pyocyanin-forming Pseudomonas aeruginosa, which contain galactose-specific hemagglutinins. They were purified simultaneously with the latter proteins by heating to 70 degrees C, precipitating with ammonium sulfate, application to a Sepharose 4B column, and elution from it by 0.05 M mannose. The mannose-specific hemagglutinins were shown to be similar to the galactophilic ones in (a) being glycoproteins of very low molecular weight (about 11 000 by SDS gel electrophoresis), (b) their tendency to aggregate, and (c) their ability to effect stronger agglutination of erythrocytes treated with papain than of untreated ones. They were found to resemble them also in their reaction with simple sugars and interactions with divalent cations, which are essential for their activity. In these properties, as well as in their relative resistance to heat and to proteolytic enzymes, these two types of bacterial hemagglutinins are like most of the plant, contrasted with the animal, hemagglutinins. The reactions with mannose and mannose-bearing compounds (yeast mannan, horseradish peroxidase (EC 1.11.1.7), and serum globulins), which are not shared with the galactophilic Pseudomonas hemagglutinins, indicate a relationship of the mannose-binding protein of Pseudomonas to the plant lectin concanavalin A. The mannose-binding hemagglutinins do not exhibit identical cell-agglutinating spectra owing to difference in profiles of sugar specificity and relative affinity to mannose derivatives compared with free mannose.     

Characterization of G25K, a GTP-binding protein containing a novel putative nucleotide binding domain

Amino acid sequences were obtained for four peptides (p1, -2, -3 and 4) generated by chemical or proteolytic cleavage of a 25 kDa GTP-binding protein purified from human placental and platelet membranes. The peptides shared sequence similarities with those contained in several of the ras-related GTP-binding proteins. Peptide p2, a 12-mer, was homologous with a region of the GTP-binding proteins that contains a structural motif proposed to contribute to the nucleotide binding site. However, whereas nearly all GTP-binding proteins exhibit the residues NKXD as this motif, p2 contains TQID. Antisera (Ap1 and Ap3) raised against synthetic peptides corresponding to p1 and p3 specifically reacted on Western blots with the 25 kDa GTP-binding protein purified from human placenta, human platelet and bovine brain as well as with a 25 kDa polypeptide in various cell lines. These results demonstrate the widespread existence of an abundant 25 kDa GTP-binding protein which contains a putative nucleotide binding domain that is chemically distinct from that described for all GTP-binding proteins of known primary structure.