Conformations of the central transforming region (Ile 55-Met 67) of the p21 protein and their relationship to activation of the protein

The GTP-binding p21 protein, encoded by the ras-oncogene, becomes transforming if amino acid substitutions are made at critical positions in the polypeptide chain, e.g., at Gly 12, Gly 13, Ala 59, Gln 61 and Glu 63. Most of these substitutions occur in two phosphate-binding loop regions, Tyr 4-Thr 20, herein designated as segment 1, and Ile 55-Met 67, herein designated, as segment 2. These two segments are homologous to two corresponding regions in the two purine nucleotide binding proteins, bacterial elongation factor (EF-tu) (Val 12-Thr 28 corresponds to segment 1; His 78-Ile 92 corresponds to segment 2) and adenylate kinase (ADK) (Lys 9-Cys 25 corresponds to segment 1 and Tyr 95-Arg 107 corresponds to segment 2). We find that the conformations of the segment 1 region in the p21 protein, EF-tu and ADK are similar to one another and that the conformation of the segment 2 region of EF-tu is superimposable on that of segment 2 of ADK. Furthermore, the relative position of the two segments in EF-tu is strikingly similar to that of the two segments in ADK. In the originally proposed X-ray structure for the p21 protein, the conformation of segment 2 in the p21 protein is not similar to that found for the other two proteins, and its disposition relative to segment 1 and the remainder of the protein is also different from that observed for the other two proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prediction of the three-dimensional structure of the rap-1A protein from its homology to theras-gene-encoded p21 protein

rap-1A, an anti-oncogene-encoded protein, is aras-p21-like protein whose sequence is over 80% homologous to p21 and which interacts with the same intracellular target proteins and is activated by the same mechanisms as p21, e.g., by binding GTP in place of GDP. Both interact with effector proteins in the same region, involving residues 32–47. However, activated rap-1A blocks the mitogenic signal transducing effects of p21. Optimal sequence alignment of p21 and rap-1A shows two insertions of rap-1A atras positions 120 and 138. We have constructed the three-dimensional structure of rap-1A bound to GTP by using the energy-minimized three-dimensional structure ofras-p21 as the basis for the modeling using a stepwise procedure in which identical and homologous amino acid residues in rap-1A are assumed to adopt the same conformation as the corresponding residues in p21. Side-chain conformations for homologous and nonhomologous residues are generated in conformations that are as close as possible to those of the corresponding side chains in p21. The entire structure has been subjected to a nested series of energy minimizations. The final predicted structure has an overall backbone deviation of 0.7 å from that ofras-p21. The effector binding domains from residues 32–47 are identical in both proteins (except for different side chains of different residues at position 45). A major difference occurs in the insertion region at residue 120. This region is in the middle of another effector loop of the p21 protein involving residues 115–126. Differences in sequence and structure in this region may contribute to the differences in cellular functions of these two proteins.     

Prediction of the three-dimensional structure of the rap-1A protein from its homology to theras-gene-encoded p21 protein

rap-1A, an anti-oncogene-encoded protein, is aras-p21-like protein whose sequence is over 80% homologous to p21 and which interacts with the same intracellular target proteins and is activated by the same mechanisms as p21, e.g., by binding GTP in place of GDP. Both interact with effector proteins in the same region, involving residues 32–47. However, activated rap-1A blocks the mitogenic signal transducing effects of p21. Optimal sequence alignment of p21 and rap-1A shows two insertions of rap-1A atras positions 120 and 138. We have constructed the three-dimensional structure of rap-1A bound to GTP by using the energy-minimized three-dimensional structure ofras-p21 as the basis for the modeling using a stepwise procedure in which identical and homologous amino acid residues in rap-1A are assumed to adopt the same conformation as the corresponding residues in p21. Side-chain conformations for homologous and nonhomologous residues are generated in conformations that are as close as possible to those of the corresponding side chains in p21. The entire structure has been subjected to a nested series of energy minimizations. The final predicted structure has an overall backbone deviation of 0.7 å from that ofras-p21. The effector binding domains from residues 32–47 are identical in both proteins (except for different side chains of different residues at position 45). A major difference occurs in the insertion region at residue 120. This region is in the middle of another effector loop of the p21 protein involving residues 115–126. Differences in sequence and structure in this region may contribute to the differences in cellular functions of these two proteins.     

The posttranslational processing of ras p21 is critical for its stimulation of yeast adenylate cyclase.

Mammalian ras genes substitute for the yeast RAS gene, and their products activate adenylate cyclase in yeast cells, although the direct target protein of mammalian ras p21s remains to be identified. ras p21s undergo posttranslational processing, including prenylation, proteolysis, methylation, and palmitoylation, at their C-terminal regions. We have previously reported that the posttranslational processing of Ki-ras p21 is essential for its interaction with one of its GDP/GTP exchange proteins named smg GDS. In this investigation, we have studied whether the posttranslational processing of Ki- and Ha-ras p21s is critical for their stimulation of yeast adenylate cyclase in a cell-free system. We show that the posttranslationally fully processed Ki- and Ha-ras p21s activate yeast adenylate cyclase far more effectively than do the unprocessed proteins. The previous and present results suggest that the posttranslational processing of ras p21s is important for their interaction not only with smg GDS but also with the target protein.     

ralGDS family members interact with the effector loop of ras p21.

Using a yeast two-hybrid system, we identified a novel protein which interacts with ras p21. This protein shares 69% amino acid homology with ral guanine nucleotide dissociation stimulator (ralGDS), a GDP/GTP exchange protein for ral p24. We designated this protein RGL, for ralGDS-like. Using the yeast two-hybrid system, we found that an effector loop mutant of ras p21 was defective in interacting with the ras p21-interacting domain of RGL, suggesting that this domain binds to ras p21 through the effector loop of ras p21. Since ralGDS contained a region highly homologous with the ras p21-interacting domain of RGL, we examined whether ralGDS could interact with ras p21. In the yeast two-hybrid system, ralGDS failed to interact with an effector loop mutant of ras p21. In insect cells, ralGDS made a complex with v-ras p21 but not with a dominant negative mutant of ras p21. ralGDS interacted with the GTP-bound form of ras p21 but not with the GDP-bound form in vitro. ralGDS inhibited both the GTPase-activating activity of the neurofibromatosis gene product (NF1) for ras p21 and the interaction of Raf with ras p21 in vitro. These results demonstrate that ralGDS specifically interacts with the active form of ras p21 and that ralGDS can compete with NF1 and Raf for binding to the effector loop of ras p21. Therefore, ralGDS family members may be effector proteins of ras p21 or may inhibit interactions between ras p21 and its effectors.     

A Compact,Multifunctional Fusion Module Directs Cholesterol-Dependent Homomultimerization and Syncytiogenic Efficiency of Reovirus p10 FAST Proteins

The homologous p10 fusion-associated small transmembrane (FAST) proteins of the avian (ARV) and Nelson Bay (NBV) reoviruses are the smallest known viral membrane fusion proteins, and are virulence determinants of the fusogenic reoviruses. The small size of FAST proteins is incompatible with the paradigmatic membrane fusion pathway proposed for enveloped viral fusion proteins. Understanding how these diminutive viral fusogens mediate the complex process of membrane fusion is therefore of considerable interest, from both the pathogenesis and mechanism-of-action perspectives. Using chimeric ARV/NBV p10 constructs, the 36–40-residue ectodomain was identified as the major determinant of the differing fusion efficiencies of these homologous p10 proteins. Extensive mutagenic analysis determined the ectodomain comprises two distinct, essential functional motifs. Syncytiogenesis assays, thiol-specific surface biotinylation, and liposome lipid mixing assays identified an ∼25-residue, N-terminal motif that dictates formation of a cystine loop fusion peptide in both ARV and NBV p10. Surface immunofluorescence staining, FRET analysis and cholesterol depletion/repletion studies determined the cystine loop motif is connected through a two-residue linker to a 13-residue membrane-proximal ectodomain region (MPER). The MPER constitutes a second, independent motif governing reversible, cholesterol-dependent assembly of p10 multimers in the plasma membrane. Results further indicate that: (1) ARV and NBV homomultimers segregate to distinct, cholesterol-dependent microdomains in the plasma membrane; (2) p10 homomultimerization and cholesterol-dependent microdomain localization are co-dependent; and (3) the four juxtamembrane MPER residues present in the multimerization motif dictate species-specific microdomain association and homomultimerization. The p10 ectodomain therefore constitutes a remarkably compact, multifunctional fusion module that directs syncytiogenic efficiency and species-specific assembly of p10 homomultimers into cholesterol-dependent fusion platforms in the plasma membrane.     

Primary and tertiary structure of the principal human adenylate kinase.

1. Human adenylate kinase (isoenzyme AK-1-1) from skeletal muscle is a single polypeptide chain of 194 amino-acid residues with an acetylmethionine at the N-terminus and a lysine at the C-terminus. 2. The primary structure of the enzyme was determined: Ac-Met-Glu-Glu-Lys-Leu-Lys-Lys-Thr-Lys-Ile-Ile-Phe-Val-Val-Gly-Gly-Pro-Gly-Ser-Gly-Lys-Gly-Thr-Gln-Cys-Glu-Lys-Ile-Val-Gln-Lys-Tyr-Gly-Tyr-Thr-His-Leu-Ser-Thr-Gly-Asp-Leu-Leu-Arg-Ser-Glu-Val-Ser-Ser-Gly-Ser-Ala-Arg-Gly-Lys-Lys-Leu-Ser-Glu-Ile-Met-Glu-Lys-Gly-Gln-Leu-Val-Pro-Leu-Glu-Thr-Val-Leu-Asp-Met-Leu-Arg-Asp-Ala-Met-Val-Ala-Lys-Val-Asn-Thr-Ser-Lys-Gly-Phe-Leu-Ile-Asp-Gly-Tyr-Pro-Arg-Glu-Val-Gln-Gln-Gly-Glu-Glu-Phe-Glu-Arg-Arg-Ile-Gly-Gln-Pro-Thr-Leu-Leu-Leu-Tyr-Val-Asp-Ala-Gly-Pro-Glu-Thr-Met-Thr-Arg-Arg-Leu-Leu-Lys-Arg-Gly-Glu-Thr-Ser-Gly-Arg-Val-Asp-Asn-Glu-Glu-Thr-Ile-Lys-Lys-Arg-Leu-Glu-Thr-Tyr-Tyr-Lys-Ala-Thr-Glu-Pro-Val-Ile-Ala-Phe-Tyr-Glu-Lys-Arg-Gly-Ile-Val-Arg-Lys-Val-Asn-Ala-Glu-Gly-Ser-Val-Asp-Glu-Val-Phe-Ser-Gln-Val-Cys-Thr-His-Leu-Asp-Ala-Leu-Lys. 3. When the primary structure of the human enzyme was fitted to the electron density map of porcine adenylate kinase, all nine amino acids which are different in the homologous enzymes from pig and man were located on the surface of the molecule. 4. Precession photographs of crystalline human and of crystalline porcine adenylate kinase corroborated the result that the polypeptide chains of the two enzymes are folded in a closely related manner. 5. The structure of human adenylate kinase incorporates the so-called nucleotide-binding domain which is present in a wide variety of proteins in nature. Some implications of this phenomenom for the molecular biology and the molecular pharmacology of man are discussed.     

Comparison of the predicted structure for the activated form of the P21 protein with the X-ray crystal structure

The predicted conformation and position of the central transforming region (residues 55–67) of the p21 protein are compared with the conformation and position of this segment in a recently determined X-ray crystal structure of residues 1–166 of this protein in the activated state bound to a nonhydrolyzable GTP derivative. We previously predicted that this segment of the protein would adopt a roughly extended conformation from Ile 55-Thr 58, a reverse turn at Ala 59-Gln 61, followed by an -helix from Glu 62-Met 67. We further predicted that this region of the activated protein occupies a position that is virtually identical to corresponding regions in the homologous purine nucleotide-binding proteins, bacterial elongation factor (EF-tu), and adenylate kinase (ADK). We find that there is a close correspondence between the conformation and position of our predicted structure and those found in the X-ray crystal structure. A mechanism for activation of the protein is proposed and is corroborated by X-ray crystallographic data.     

Interaction of a synthetic fragment of the oncoprotein p21ras with cellular proteins

A decapeptide corresponding to residues 35-44(-Thr-Ile-Glu-Asp-Ser-Tyr-Arg-Lys-Gln-Val-) of p21ras was synthesized. It was found that peptide causes precipitation of some proteins from the Triton X-100 lysate of NIH 3T3 EJ cells. SDS-PAGE demonstrated the presence of many proteins in this precipitate. The peptide labeled with [125I]Bolton-Hunter reagent specifically recognized four proteins of M. W. 27, 35, 50 and 85 kDa. The order of charged amino acid residues in the fragment 35-44 of p21ras is "complementary" to that of the substrate sequence of tyrosine-specific protein kinases (-Arg-X-X-Glu-Asp-X-X-Tyr-). It is suggested that p21ras proteins directly regulate phosphorylation of the target proteins of these kinases. A model for functioning of p21ras proteins predicts the presence in their structure of certain sites homologous to sequences recognizable by tyrosine-specific kinases. Indeed two such sites are present in the sequences of all p21ras proteins, namely the residues 88-92 and 104-108.     

Different domains in the third intracellular loop of the GLP-1 receptor are responsible for Galpha(s) and Galpha(i)/Galpha(o) activation

It has previously been shown that the GLP-1 receptor is primarily coupled to the adenylate cyclase pathway via activation of Galpha(s) proteins. Recent studies have shown that the third intracellular loop of the receptor is important in the stimulation of cAMP production. We have studied the effect of three synthetic peptide sequences derived from the third intracellular loop of the GLP-1 receptor on signal transduction in Rin m5F cell membranes. The whole third intracellular loop strongly stimulates both pertussis toxin and cholera toxin-sensitive G proteins, while the N-terminal half exclusively stimulates cholera toxin-sensitive G proteins and the C-terminal half only stimulates pertussis toxin-sensitive G-proteins as demonstrated by measurements of GTPase activity. These data confirm that the principal stimulatory G-protein interaction site resides in the third intracellular loop, but also suggest that the GLP-1 receptor is not only coupled to the Galpha(s) but also to the Galpha(i)/Galpha(o) type of G proteins and that distinct domains within the third intracellular loop are responsible for the activation of the different G-protein subfamilies.     

GTP-binding proteins and adenylate cyclase activity in v-Ki-ras transformed NIH/3T3 fibroblast cells

To identify the role of ras oncogene and p21 in the coupling mechanism of GTP-binding proteins to adenylate cyclase, we used v-Ki-ras transformed NIH/3T3 fibroblast cells. In the previous study, we investigated that NaF, cholera toxin and forskolin remarkably enhanced the adenylate cyclase activity in transformed cells compared to normal NIH/3T3 cells. In the present study, adenylate cyclase was more enhanced by GTP gamma S in transformed cells than in normal cells. It was considered that p21 plays enhancing role in coupling of GTP-binding proteins to adenylate cyclase. Further, as measured by the degree of [32P] ADP-ribosylation of GTP-binding proteins by cholera toxin and pertussis toxin respectively, the amount of Gs (46 kDa) was almost equal in both cells, while the amount of Gi (41 kDa) in transformant was about one third of that in normal cells. This difference seems to be reflected in either the biological situations or the quantities of Gi. Our data suggest that v-Ki-ras transformation resulted in the decrease of Gi protein so that the inhibitory regulation on adenylate cyclase relatively becomes low and then stimulatory influence of Gs seems to be enhanced.     

ATP binding to hemoglobin response gene 1 protein is necessary for regulation of the mating type locus in Candida albicans

HBR1 (hemoglobin response gene 1) is an essential gene in Candida albicans that positively regulates mating type locus MTLα gene expression and thereby regulates cell type-specific developmental genes. Hbr1p contains a phosphate-binding loop (P-loop), a highly conserved motif characteristic of ATP- and GTP-binding proteins. Recombinant Hbr1p was isolated in an oligomeric state that specifically bound ATP with K(d) ～2 μM. ATP but not ADP, AMP, GTP, or dATP specifically protected Hbr1p from proteolysis by trypsin. Site-directed mutagenesis of the highly conserved P-loop lysine (K22Q) and the less conserved glycine (G19S) decreased the binding affinity for soluble ATP and ATP immobilized through its γ-phosphate. ATP bound somewhat more avidly than ATPγS to wild type and mutant Hbr1p. Although Hbr1p exhibits sequence motifs characteristic of adenylate kinases, and adenylate kinase and ATPase activities have been reported for the apparent human ortholog of Hbr1p, assays for adenylate kinase activity, autophosphorylation, and ATPase activity proved negative. Overexpression of wild type but not the mutant forms of Hbr1p restored MTlα2 expression in an HBR1/hbr1 mutant, indicating that ATP binding to the P-loop is necessary for this function of Hbr1p.     

Dissecting the localization and function of Atg18, Atg21 and Ygr223c

Atg18p and Atg21p are two highly homologous yeast autophagy proteins. Atg18p functions in both autophagy and the selective Cvt-pathway, while the function of Atg21p is restricted to the Cvt-pathway. The yeast genome encodes with Ygr223cp (Hsv2p), a third member of this protein family. So far no function has been assigned to Ygr223cp. By colocalization with the endosomal marker Snf7-RFP and an RFP-tagged FYVE domain, we here identify the localization of a pool of Atg18p, Atg21p and Ygr223cp at endosomes. Endosomal recruitment of all three proteins depends on PtdIns3P generated by the Vps34-complex II containing Vps38p, but not on the function of the Vps34-complex I. Since only the Vps34-complex I is essential for autophagy, we expect that at endosomes Atg18p, Atg21p and Ygr223cp have a function distinct from autophagy. Some Vps Class D mutants involved in Golgi-to-endosome transport are required for the endosomal recruitment of GFP-Atg18p, -Atg21p and -Ygr223cp. These include the Qa-SNARE Pep12p, its SM protein Vps45p, the Rab GTPase Vps21p and the Rab effector Vac1p. Deletion of ATG18, ATG21 and YGR223c, alone or simultaneously has no obvious function on the MVB-pathway and CPY-sorting. However, overexpression of ATG21 leads to CPY secretion. We further show, to our knowledge for the first time, that Ygr223cp affects an autophagic process, namely micronucleophagy.     

PGE2-induced desensitization of adenylate cyclase of a murine macrophage-like cell line (P388D1)

The mode of PGE2-induced desensitization of the adenylate cyclase of a murine macrophage-like cell line, P388D1 was investigated. The exposure of cells to PGE2 for 60 min induced PGE2-specific desensitization of the adenylate cyclase system which still responded normally to other specific ligand such as isoproterenol, 5'-guanylimidodiphosphate (Gpp(NH)p), or forskolin. The exposure of the cells to PGE2 for 6 hr induced heterologous desensitization, as the responses of adenylate cyclase to PGE2 as well as to isoproterenol or Gpp(NH)p were significantly reduced. The lowest concentration of PGE2 to induce both early homologous and late heterologous desensitization was found to be about two-fold over the KD of the low affinity PGE2-binding sites of P388D1 cells. The early homologous desensitization appeared to be due in part to the reduction in number of PGE2 receptors from the cell surface. The late heterologous desensitization may involve functional and/or structural alteration of Gs proteins, in addition to the reduction of PGE2 receptors from the cell surface.     

Mutations in the carboxy-terminus of the third intracellular loop of the human recombinant VPAC1 receptor impair VIP-stimulated [Ca2+]i increase but not adenylate cyclase stimulation

The vasoactive intestinal polypeptide (VIP) VPAC1 receptor is preferentially coupled to Galphas protein that stimulates adenylate cyclase activity and also to Galphaq and Galphai proteins that stimulate the inositol phosphate/calcium pathway. Previous studies indicated the importance of the third intracellular loop of the receptor for G protein coupling. By site-directed mutation of the human recombinant receptor expressed in Chinese hamster ovary cells, we identified two domains in this loop that contain clusters of basic residues conserved in most of the G-protein-coupled seven transmembrane domains receptors. We found that mutations in the proximal domain (K322) reduced the capability of VIP to increase adenylate cyclase activity without any change in the calcium response, whereas mutations in the distal part of the loop (R338, L339, R341) markedly reduced the calcium increase and Galphai coupling but only weakly the adenylate cyclase activity. Thus, the interaction of different G proteins with the VPAC1 receptor involves different receptor sub-domains.     

Molecular Dynamics on Complexes of ras-p21 and Its Inhibitor Protein, rap-1A, Bound to the ras-Binding Domain of the raf-p74 Protein: Identification of Effector Domains in the raf Protein

We have computed the average structures for the ras-p21 protein and its strongly homologous inhibitor protein, rap-1A, bound to the ras-binding domain (RBD) of the raf protein, using molecular dynamics. Our purpose is to determine the differences in structure between these complexes that would result in no mitogenic activity of rap-1A-RBD but full activity of p21-RBD. We find that despite the similarities of the starting structures for both complexes, the average structures differ considerably, indicating that these two proteins do not interact in the same way with this vital target protein. p21 does not undergo major changes in conformation when bound to the RBD, while rap-1 A undergoes significant changes in structure on binding to the RBD, especially in the critical region around residue 61. The p21 and rap-1A make substantially different contacts with the RBD. For example, the loop region from residues 55–71 of rap-la makes extensive hydrogen-bond contacts with the RBD, while the same residues of p21 do not. Comparison of the structures of the RBD in both complexes reveals that it undergoes considerable changes in structure when its structure bound to p21 is compared with that bound to rap-1A. These changes in structure are due to displacements of regular structure (e.g., α-helices and β-sheets) rather than to changes in the specific conformations of the segments themselves. Three regions of the RBD have been found to differ significantly from one another in the two complexes: the binding interface between the two proteins at residues 60 and 70, the region around residues 105–106, and 118–120. These regions may constitute effector domains of the RBD whose conformations determine whether or not mitogenic signal transduction will occur.     

Increased adenylate cyclase activity in rat thyroid epithelial cells expressing viral ras genes

The activity of the adenylate cyclase catalytic subunit is higher in Harvey and Kirsten Murine Sarcoma Viruses-infected thyroid epithelial cells than in uninfected control cells either in the presence of Mg2+ alone or following stimulation by Mn2+ or forskolin. The higher activity is associated with an increased cAMP cellular content. The Gpp(NH)p and F- anion are more effective positive modulators in the control than in the virus infected cells: these results exclude therefore that the ras p21 proteins can act as the G-protein alpha-subunit and suggest that they negatively interfere with the G-protein modulation of the adenylate cyclase system.     

Exchange of Guanine Nucleotides Between Tubulin and GTP-Binding Proteins That Regulate Adenylate Cyclase: Cytoskeletal Modification of Neuronal Signal Transduction

Tubulin, the primary constituent of microtubules, is a GTP-binding proteins with structural similarities to other GTP-binding proteins. Whereas microtubules have been implicated as modulators of the adenylate cyclase system, the mechanism of this regulation has been elusive. Tubulin, polymerized with the hydrolysis-resistant GTP analog, 5'-guanylylimidodiphosphate [Gpp(NH)p], can promote inhibition of synaptic membrane adenylate cyclase which persists subsequent to washing. Tubulin with Gpp(NH)p bound was slightly less potent than free Gpp(NH)p in the inhibition of adenylate cyclase, but tubulin without nucleotide bound had no effect on the enzyme. A GTP-binding protein from the rod outer segment (transducin), with Gpp(NH)p bound, was also without effect on adenylate cyclase. Tubulin (regardless of the nucleotide bound to it) did not alter the activity of the adenylate cyclase catalytic unit directly. When tubulin was polymerized with the hydrolysis-resistant photoaffinity GTP analog, [32P]P3(4-azidoanilido)-P1-5'-GTP ([32P]AAGTP), and this protein was added to synaptic membranes, AAGTP was transferred from tubulin to the inhibitory GTP-binding protein, Gi. This transfer was blocked by prior incubation of the membranes with Gpp(NH)p or covalent binding of AAGTP to tubulin prior to exposure of that tubulin to membranes. Incubation of membranes with Gpp(NH)p subsequent to incubation with tubulin-AAGTP results in a decrease in AAGTP bound to Gi and a compensatory increase in AAGTP bound to the stimulatory GTP-binding protein, Gs. Likewise, persistent inhibition of adenylate cyclase by tubulin-Gpp(NH)p could be overridden by the inclusion of 100 microM Gpp(NH)p in the assay inhibition. Whereas Gpp(NH)p promotes persistent inhibition of synaptic membrane adenylate cyclase without incubation at elevated temperatures, tubulin [with AAGTP or Gpp(NH)p bound] requires 30 s incubation at 23 degrees C to effect adenylate cyclase inhibition. Photoaffinity experiments yield parallel results. These data are consistent with synaptic membrane tubulin regulating neuronal adenylate cyclase by transferring GTP to Gi and, subsequently, to Gs.     

The ral gene: a new ras related gene isolated by the use of a synthetic probe.

We synthesized a set of 20-mer oligonucleotides corresponding to a sequence of seven amino acids strictly conserved in all the different ras proteins, from yeast to man, as well as in rho and YPT, two proteins distantly related to p21 ras (approximately 30% amino acid homology). This oligonucleotide probe was used to search for new members of the ras family. We describe here a new ras related gene named ral, isolated from a cDNA library of immortalized simian B-lymphocytes. The ral gene codes for a 206 amino acid protein of expected mol. wt 23.5 kd that shares greater than 50% homology with H-ras, K-ras or N-ras. The GTP binding regions of p21 ras and a C-terminal cysteine involved in membrane anchoring are also present in ral; this strongly suggests that ral is a GTP binding protein with membrane localization. Furthermore, several external regions of p21 ras presumably involved in the interaction with effector, receptor and/or regulatory proteins are highly homologous to the corresponding regions in ral. Therefore some of the proteins that interact with ral might be identical or closely related to those interacting with p21 ras.