Rem Is a New Member of the Rad- and Gem/Kir Ras-related GTP-binding Protein Family Repressed by Lipopolysaccharide Stimulation
1997; Elsevier BV; Volume: 272; Issue: 35 Linguagem: Inglês
10.1074/jbc.272.35.21982
ISSN1083-351X
AutoresBrian S. Finlin, Douglas Andres,
Tópico(s)Ion channel regulation and function
ResumoWe report the cDNA cloning and characterization of a novel GTP-binding protein, termed Rem (for Rad and Gem-related), that was identified as a product of polymerase chain reaction amplification using oligonucleotide primers derived from conserved regions of the Rad, Gem, and Kir Ras subfamily. Alignment of the full-length open reading frame of mouse Rem revealed the encoded protein to be 47% identical to the Rad, Gem, and Kir proteins. The distinct structural features of the Rad, Gem, and Kir subfamily are maintained including a series of nonconservative amino acid substitutions at positions important for GTPase activity and a unique sequence motif thought to direct membrane association. Recombinant Rem binds GTP in a specific and saturable manner. Ribonuclease protection analysis found Rem to be expressed at comparatively high levels in cardiac muscle and at moderate levels in lung, skeletal muscle, and kidney. The administration of lipopolysaccharide to mice, a potent activator of the inflammatory and immune systems, results in the general repression of Rem mRNA levels in a dose- and time-dependent manner. Thus, Rem is the first Ras-related gene whose mRNA levels have been shown to be regulated by repression. We report the cDNA cloning and characterization of a novel GTP-binding protein, termed Rem (for Rad and Gem-related), that was identified as a product of polymerase chain reaction amplification using oligonucleotide primers derived from conserved regions of the Rad, Gem, and Kir Ras subfamily. Alignment of the full-length open reading frame of mouse Rem revealed the encoded protein to be 47% identical to the Rad, Gem, and Kir proteins. The distinct structural features of the Rad, Gem, and Kir subfamily are maintained including a series of nonconservative amino acid substitutions at positions important for GTPase activity and a unique sequence motif thought to direct membrane association. Recombinant Rem binds GTP in a specific and saturable manner. Ribonuclease protection analysis found Rem to be expressed at comparatively high levels in cardiac muscle and at moderate levels in lung, skeletal muscle, and kidney. The administration of lipopolysaccharide to mice, a potent activator of the inflammatory and immune systems, results in the general repression of Rem mRNA levels in a dose- and time-dependent manner. Thus, Rem is the first Ras-related gene whose mRNA levels have been shown to be regulated by repression. The Ras family of low molecular weight GTP-binding proteins has been implicated in a wide range of cellular processes, including cell growth and differentiation, intracellular vesicular trafficking, nucleocytoplasmic transport, and cytoskeletonal reorganization. To date, six subfamilies have been identified: Ras, Rho, Rab, Ran, ARF, and a newly described family composed of the Rad, Gem, and Kir proteins (1Bourne H.R. Sanders D.A. McCormick F. Nature. 1990; 348: 125-132Crossref PubMed Scopus (1837) Google Scholar, 2Zerial M. Huber L.A. Guidebook to the Small GTPases. Oxford University Press, New York1995Google Scholar). These subfamilies are defined largely by primary sequence relationships but also by their regulation of common cellular functions. All GTPases of the Ras superfamily contain five well conserved amino acid motifs involved in guanine nucleotide binding and hydrolysis (1Bourne H.R. Sanders D.A. McCormick F. Nature. 1990; 348: 125-132Crossref PubMed Scopus (1837) Google Scholar, 3Valencia A. Chardin P. Wittinghofer A. Sander C. Biochemistry. 1991; 30: 4637-4648Crossref PubMed Scopus (56) Google Scholar). These primary sequence motifs have been evolutionarily conserved and define a conserved structure whose importance has been confirmed through extensive mutational analysis (4Bourne H.R. Sanders D.A. McCormick F. Nature. 1991; 349: 117-127Crossref PubMed Scopus (2690) Google Scholar). Therefore, the sequence of all GTPases share approximately 20–30% amino acid identity, whereas the sequence identity is considerably higher within subfamilies (5Kahn R.A. Der C.J. Bokoch G.M. FASEB J. 1992; 6: 2512-2513Crossref PubMed Scopus (97) Google Scholar). In addition, most family members share conserved COOH-terminal cysteine rich motifs needed for covalent modification by isoprenoid lipids (6Glomset J.A. Farnesworth C.C. Annu. Rev. Cell Biol. 1994; 10: 181-205Crossref PubMed Scopus (278) Google Scholar). Prenylation is the initial step in the attachment of these proteins to the cytoplasmic leaflets of a variety of cellular organelles (7Casey P.J. Seabra M.C. J. Biol. Chem. 1996; 271: 5289-5292Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar) and has been shown to be required for normal membrane localization and biological activity (8Hancock J.F. Magee A.I. Childs J.E. Marshall C.J. Cell. 1989; 57: 1167-1177Abstract Full Text PDF PubMed Scopus (1460) Google Scholar). The Ras-related GTPases are thought to act as binary switching molecules, alternating between an active GTP-bound and an inactive GDP-bound structural state (4Bourne H.R. Sanders D.A. McCormick F. Nature. 1991; 349: 117-127Crossref PubMed Scopus (2690) Google Scholar). They respond to external signals by exchanging GTP for constitutively bound GDP, thereby triggering intracellular signaling cascades. The signal is terminated when the protein hydrolyzes its bound GTP to GDP in a reaction that is stimulated by guanosine triphosphatase (GTPase)-activating proteins (GAPs) 1The abbreviations used are: GAP, GTPase-activating protein; LPS, lipopolysaccharide; RGK, Rad, Gem, and Kir Ras-related GTP-binding proteins; GST, glutathioneS-transferase; PCR, polymerase chain reaction; bp, base pair(s); TNF, tumor necrosis factor-α; Pipes, 1,4-piperazinediethanesulfonic acid; GTPγS, guanosine 5′-3-O-(thio)triphosphate. (9Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1760) Google Scholar). Progression through this GTPase cycle is regulated by additional regulatory proteins, including factors that stimulate guanine nucleotide exchange. These regulatory proteins may themselves possess intrinsic effector activity (9Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1760) Google Scholar) and associate with specific GTPases largely through interactions with their G2 effector domains. A variety of G2 regions allow the Ras-related GTPases to interact with a wide range of cellular effector molecules (4Bourne H.R. Sanders D.A. McCormick F. Nature. 1991; 349: 117-127Crossref PubMed Scopus (2690) Google Scholar). Thus, small GTPases are extremely versatile and found to regulate an array of cellular processes (2Zerial M. Huber L.A. Guidebook to the Small GTPases. Oxford University Press, New York1995Google Scholar, 9Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1760) Google Scholar). The Rad, Gem, and Kir proteins are the first members of a new class of Ras-like GTPases (10Kahn C.R. Reynet C. Science. 1993; 262: 1441-1444Crossref PubMed Scopus (0) Google Scholar, 11Maguire J. Santoro P.J. Siebenlist J.Y. Kelly K. Science. 1994; 265: 241-244Crossref PubMed Scopus (163) Google Scholar, 12Cohen L. Mohr R. Chen Y. Huang M. Kato R. Dorin D. Tamanoi F. Goga A. Afar D. Rosenberg N. Witte O. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12448-12452Crossref PubMed Scopus (82) Google Scholar). Rad shares 61% identity at the amino acid level with Gem (11Maguire J. Santoro P.J. Siebenlist J.Y. Kelly K. Science. 1994; 265: 241-244Crossref PubMed Scopus (163) Google Scholar) and Kir (12Cohen L. Mohr R. Chen Y. Huang M. Kato R. Dorin D. Tamanoi F. Goga A. Afar D. Rosenberg N. Witte O. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12448-12452Crossref PubMed Scopus (82) Google Scholar), whereas the coding sequences of the Gem and Kir genes are 98% identical, differing significantly only in the 5′-untranslated sequences (12Cohen L. Mohr R. Chen Y. Huang M. Kato R. Dorin D. Tamanoi F. Goga A. Afar D. Rosenberg N. Witte O. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12448-12452Crossref PubMed Scopus (82) Google Scholar). The RGK proteins share structural features that are distinct from other Ras-related proteins. These include several nonconservative amino acid substitutions within regions known to be involved in guanine nucleotide binding and hydrolysis including unique G2 effector and G3 domains, extended NH2and COOH termini and a conserved COOH-terminal motif thought to mediate membrane association but lacking classical CAAX motifs needed to direct protein isoprenylation. In addition, the members of this Ras subfamily are subject to transcriptional regulation. Rad was found to be overexpressed in muscle of type II diabetes patients (10Kahn C.R. Reynet C. Science. 1993; 262: 1441-1444Crossref PubMed Scopus (0) Google Scholar), and Gem expression is induced in mitogen-stimulated T-cells (11Maguire J. Santoro P.J. Siebenlist J.Y. Kelly K. Science. 1994; 265: 241-244Crossref PubMed Scopus (163) Google Scholar), whereas Kir expression is induced by oncogenic kinases (12Cohen L. Mohr R. Chen Y. Huang M. Kato R. Dorin D. Tamanoi F. Goga A. Afar D. Rosenberg N. Witte O. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12448-12452Crossref PubMed Scopus (82) Google Scholar). Although the cellular function of these proteins remains to be established, Rad has been shown to associate with skeletal muscle β-tropomyosin and the cytoskeleton of muscle cells (13Zhu J. Bilan P.J. Moyers J.S. Antonetti D.A. Kahn C.R. J. Biol. Chem. 1996; 271: 768-773Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) and to inhibit insulin-stimulated glucose uptake in a variety of cultured cell lines (14Moyers J.S. Bilan P.J. Reynet C. Kahn C.R. J. Biol. Chem. 1996; 271: 23111-23116Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). This suggests a role for Rad in skeletal muscle function and cytoskeletal organization. The deregulated expression of Gem prevents proliferation of normal and transformed 3T3 cells, suggesting that Gem is involved in regulating signaling pathways that influence cell growth (11Maguire J. Santoro P.J. Siebenlist J.Y. Kelly K. Science. 1994; 265: 241-244Crossref PubMed Scopus (163) Google Scholar). Finally, the cellular levels of Kir are dramatically increased in pre-B cells transformed by a select set of abl tyrosine kinase oncogenes (12Cohen L. Mohr R. Chen Y. Huang M. Kato R. Dorin D. Tamanoi F. Goga A. Afar D. Rosenberg N. Witte O. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12448-12452Crossref PubMed Scopus (82) Google Scholar). The correlation between Kir expression and the tumorigenic and metastatic potential of BCL/ABL and v-ABL transformed cells suggests that Kir may participate in the processes of invasion or metastasis. When expressed in yeast, Kir leads to the formation of pseudohyphae, a developmental transition normally induced by nitrogen starvation (15Dorin D. Cohen L. Del Villar K. Poullet P. Mohr R. Whiteway M. Witte O. Tamanoi F. Oncogene. 1995; 11: 2267-2271PubMed Google Scholar). Genetic analysis suggests that Kir acts upstream of theSTE20 kinase and results in the activation of a mitogen-activated protein kinase cascade (15Dorin D. Cohen L. Del Villar K. Poullet P. Mohr R. Whiteway M. Witte O. Tamanoi F. Oncogene. 1995; 11: 2267-2271PubMed Google Scholar). These results are consistent with a model in which Kir, and perhaps other members of the RGK family, may regulate cellular signaling cascades by controlling the activity of mitogen-activated protein kinases that have yet to be determined. In this report, we describe the initial characterization of a novel Ras-related GTP-binding protein, Rem, first identified using a degenerate PCR strategy. On the basis of structural criteria, Rem is a new member of the Rad, Gem, and Kir subfamily of Ras-related proteins. Rem mRNA is expressed predominantly in skeletal and cardiac muscle, lung, and kidney, and the bacterially expressed protein is shown to bind GTP in a specific and saturable manner. Because other members of the RGK family are transcriptionally regulated, we examined whether a similar method of regulation controlled the expression of Rem. Surprisingly, we find that in mice treated with lipopolysaccharide, a potent activator of cells of the immune and inflammatory systems, the levels of Rem mRNA are repressed in a dose- and time-dependent manner. Thus, Rem is the first Ras-related GTP-binding protein whose mRNA levels are regulated by repression. Standard molecular biology techniques were used (16Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). cDNA clones were subcloned to plasmid pBluescript II vectors (Stratagene) and sequenced by the dideoxy chain termination method (16Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) using the M13 universal primer or specific internal primers. Nick-translated probes were synthesized using a labeling kit (Life Technologies, Inc.). The strategy used to generate the PCR-derived fragment of Rem from conserved amino acid sequences found within the Rad and Gem/Kir small GTP-binding proteins is outlined in Fig. 1. First strand cDNA from mouse testis and brain total RNA was a kind gift from Dr. Kevin Sarge of this department. The first strand cDNA was used for PCR with the degenerate PCR primers AT(A/C/T)AT(A/C/T)(C/T)TNGTNGGNAA(C/T)AA and GT(T/C)TC(A/T/G) AT(A/G)AA(C/T)TT(G/A)CA(A/G)TC(AG)AA. The resultant 105-base PCR product was blunt ended with T4 polymerase, and 5′-phosphate was added with polynucleotide kinase (17Wang K. Koop B.F. Hood L. BioTechniques. 1994; 17: 236-238PubMed Google Scholar). The PCR product was then subcloned into the EcoRV site of pBluescript KS+(Stratagene, La Jolla, CA). Sixty individual clones were sequenced using a Sequenase™ kit (U. S. Biochemical Corp.). Two PCR products, when sequenced, were found to contain a unique cDNA fragment that was used to design a Rem-specific oligonucleotide (Rem-3; see Fig. 1). The Rem-3 oligonucleotide was then used to screen an oligo(dT)-primed Uni-ZAP XR adult mouse testis cDNA library (the kind gift of Dr. Debra J. Wolgemuth, Center for Reproductive Sciences, Columbia University College of Physicians and Surgeons, New York, NY). Approximately 30,000 plaques were transferred in duplicate to filters that were then probed with 1 × 106cpm/ml of the end labeled oligonucleotide, TTCTACCGAGACTTCCCGGC. Filters were hybridized at 42 °C in hybridization solution containing 6 × SSC, 1 × Denhardt's solution, 100 μg/ml yeast tRNA, and 0.05% sodium pyrophosphate (18Chen W.-J. Andres D.A. Goldstein J.L. Russell D.W. Brown M.S. Cell. 1991; 66: 327-334Abstract Full Text PDF PubMed Scopus (153) Google Scholar). The filters were washed for 2 h with 6 × SSC and 0.05% sodium pyrophosphate at room temperature with three changes of wash buffer. Three positives were identified, and the largest (Rem λ10-1, 1.1-kilobase insert) was plaque purified, rescued using phage mediated in vivo excision (according to manufacturer's protocol, Stratagene), and sequenced. To obtain a larger cDNA clone, the mouse testis library was rescreened with 1 × 106 cpm/ml of the nick-translatedEcoRI/XhoI fragment of pRem 10-1. Duplicate filters were hybridized at 42 °C in a 5 × SSPE solution (18Chen W.-J. Andres D.A. Goldstein J.L. Russell D.W. Brown M.S. Cell. 1991; 66: 327-334Abstract Full Text PDF PubMed Scopus (153) Google Scholar) containing 50% formamide. The filters were washed in 0.2 × SSC and 0.1% SDS for 1 h at 60 °C with three changes of wash buffer. Of five positives, the clone Rem λ6-2 was plaque purified and rescued as a pBS plasmid because of its ability to hybridize to 5′-directed Rem-specific oligonucleotides. To obtain a full-length cDNA clone, a bacteriophage cDNA library was constructed from mouse kidney. Poly(A)+ RNA was isolated using the Straight A's™ mRNA isolation system (Novagen, Madison, WI) and used to construct a random primed cDNA library (Directional RH Random Primer cDNA Library Construction System, Novagen). Approximately 30,000 plaques were transferred in duplicate to filters that were probed under high stringency with 1 × 106 cpm/ml of the nick-translatedEcoRI/XhoI fragment of pRem λ6-2. Of the four positive clones identified, pRem λ5, which contained the largest and most 5′-extended insert (0.9 kilobase), was rescued and characterized by DNA sequencing. To construct a plasmid that contained the full Rem coding region, nucleotides 1–502 of pRem 5 were ligated to nucleotides 56–1050 of pRem 6-2 using a unique NheI site. The resulting plasmid, pRemWT, was characterized by restriction mapping and sequencing. Mouse total RNA was isolated using a STAT-60 kit (Tel-Test B, Friendswood, TX) according to the manufacturer's protocol. The PstI fragment of pRem 6-2 was subcloned into pBluescript KS+ (Stratagene, La Jolla, CA) to create the plasmid pRem PstI. Plasmids containing an 89-bp fragment of the ribosomal protein L32 (19Hobbs M.V. Weigle W.O. Noonan D.J. Torbett B.E. McEvilly R.J. Koch R.J. Cardenas G.J. Ernst D.N. J. Immunol. 1993; 150: 3602-3614PubMed Google Scholar) or a 303-bp fragment of mouse TNF-α cDNA subcloned in the vector pGEM-4 were a gift of Dr. Daniel Noonan of this department. Antisense radiolabeled riboprobes were prepared using linearized templates and a Maxiscript™ (Ambion, Austin, TX) kit according to the manufacturer's protocol. RNase protection assays were performed according to the method of Hobbs (19Hobbs M.V. Weigle W.O. Noonan D.J. Torbett B.E. McEvilly R.J. Koch R.J. Cardenas G.J. Ernst D.N. J. Immunol. 1993; 150: 3602-3614PubMed Google Scholar) with minor modifications. Briefly 20 μg of total RNA was dissolved in 8 μl of hybridization buffer (80% formamide, 0.3 m NaCl, 1 mm EDTA, 40 mm Pipes, pH 6.7). For LPS experiments, two protection assays were performed for each tissue. The first contained a mixture of Rem and L32 riboprobes, whereas the second contained the TNF-α and L32 riboprobe mix. 1 μl (500,000 cpm) of each probe (in hybridization buffer) was added to each sample. Samples were overlaid with mineral oil, incubated for 1 min at 90 °C, and subsequently incubated for 12–16 h at 56 °C. Single-stranded RNA was digested as described by Hobbs (19Hobbs M.V. Weigle W.O. Noonan D.J. Torbett B.E. McEvilly R.J. Koch R.J. Cardenas G.J. Ernst D.N. J. Immunol. 1993; 150: 3602-3614PubMed Google Scholar). The reaction was stopped by the addition of an equivalent volume (110 μl) of 4 mguanidine thiocyanate, 0.5% sodium N-lauryl-sarcosine, 25 mm sodium citrate, pH 7.0, 0.1 mβ-mercaptethanol. Yeast tRNA (25 μg) was added as carrier, and total RNA was precipitated by addition of isopropanol (225 μl) followed by centrifugation (20Hod Y. BioTechniques. 1992; 13: 852-854PubMed Google Scholar). The pellet was dissolved in 10 μl of loading buffer (80% formamide, 10 mm EDTA, 1 mg/ml bromphenol blue, 1 mg/ml xylene cyanol) and electrophoresed in a 5% acrylamide/8 m urea sequencing gel. The gel was dried and exposed to X-OMAT AR film (Eastman Kodak Co.) for the indicated time. The gel was quantitated using a Molecular Dynamics PhosphorImager SF (model 455A). Simultaneous measurement of the rpL32 transcripts, which encode the L32 ribosomal protein (19Hobbs M.V. Weigle W.O. Noonan D.J. Torbett B.E. McEvilly R.J. Koch R.J. Cardenas G.J. Ernst D.N. J. Immunol. 1993; 150: 3602-3614PubMed Google Scholar), served as an internal control for housekeeping gene levels. The tissues of adult female C57BL6/C3H mice were kindly provided by Dr. Mark Kindy of this department. Lipopolysaccharide stimulation was achieved by intraperitoneal injection (10 μg/animal) to paired animals for each time point. Lipopolysaccharide was from Escherichia coli 0111:B4 (DIFCO). Control animals were not injected. At the indicated times the animals were treated with metafane and euthanized by cervical dislocation, and tissues were harvested immediately and quick frozen on dry ice. RNA was isolated and used for ribonuclease protection analysis as described above. These studies were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Recombinant Rem was expressed as a glutathione S-transferase (GST)-fusion protein. A Rem PCR product containing a 5′ BamHI restriction site was generated using oligonucleotides 5′-CGCGGATCCATGACTCTTAACACGCA and 3′-TTCTACCGAGACTTCCCGGC, sequenced to verify the cDNA, and subcloned to BamHI/NheI-digested pRemWT to create pRem express. The BamHI/XhoI fragment of pRem express was subsequently cloned in-frame to pGEX-KG (21Hakes D.J. Dixon J.E. Anal. Biochem. 1992; 202: 293-298Crossref PubMed Scopus (222) Google Scholar) to create pRem GEX. GST-Rem was produced in BL21DE3 cells upon isopropyl-β-d-thiogalactopyranoside addition, purified on glutathione agarose beads (Sigma), and removed from GST by thrombin cleavage as described (22Cicchetti P. Ridley A.J. Zheng Y. Cerione R.A. Baltimore D. EMBO J. 1995; 14: 3127-3135Crossref PubMed Scopus (64) Google Scholar). Thrombin cleavage was deemed to be >95% efficient as judged by Coomassie Blue staining after SDS-polyacrylamide gel electrophoresis analysis and Western blotting with anti-GST antibodies. A bacteria protein co-purified with Rem and was resistant to extensive high salt washes of the glutathione-agarose beads (see Fig. 4 C). Thrombin was removed by incubation with benzamidene-Sepharose 6B (Pharmacia Biotech Inc.) for 1 h at 4 °C with mixing. The protein was then dialyzed against 50 mm Tris, pH 7.5, 150 mm NaCl, 1 mmdithiothreitol, and 5% glycerol and stored in multiple aliquots at −70 °C. GTP binding to Rem was determined with the rapid filtration assay (23Albright C.F. Giddings B.W. Liu J. Vito R. Weinberg A. EMBO J. 1993; 12: 339-347Crossref PubMed Scopus (159) Google Scholar). Rem (1 μg) or bovine serum albumin (1 μg) was incubated in binding buffer (20 mm Tris, pH 7.5, 50 mm NaCl, 0.1% Triton X-100, 1 mmdithiothreitol, 40 μg/ml bovine serum albumin, and 1 μmGTPγS (0.45 μCi/sample)) with the indicated concentration of Mg2+ or EDTA at 22 °C. At the indicated times, aliquots of 100 μl were withdrawn in duplicate, and the reaction was stopped by addition of 400 μl of ice-cold wash buffer (20 mmTris, pH 7.5, 50 mm NaCl, and the indicated Mg2+ concentration) and immediately filtered through BA 85 nitrocellulose filters (Schleicher & Schuell) followed by washing with 12 ml of ice-cold wash buffer. The radioactivity remaining on the filters was determined by scintillation counting. Nonspecific background was determined by performing the binding assay in the absence of added protein. To assess the specificity of GTP binding, cold ribonucleotides were added to a final concentration of 20 μm, and the binding reaction was allowed to proceed for 1 h prior to rapid filtration analysis. We sought a comprehensive method to identify additional members of the RGK family of low molecular weight GTP-binding proteins. A PCR-based strategy using degenerate oligonucleotide primers designed to exploit amino acid differences between the guanine nucleotide-binding domains of the RGK family and other Ras-related GTP-binding proteins was used to isolate novel RGK-related cDNA fragments from mouse testis cDNA (Fig. 1 and see "Experimental Procedures"). The 5′ oligonucleotide was designed to partially overlap the G4 guanine nucleotide-binding site motif IILVGNK, whereas the 3′ oligonucleotide recognized a portion of the G5 guanine nucleotide-binding region and a flanking region of amino acids highly conserved within the RGK family FDCKFIET. PCR generated a pool of cDNAs of the approximate predicted size (105 bp), which were subcloned and sequenced. Sequencing of 60 individual clones revealed that approximately 90% of the products were identical to the previously characterized Rad, Gem, or Kir genes (10Kahn C.R. Reynet C. Science. 1993; 262: 1441-1444Crossref PubMed Scopus (0) Google Scholar, 11Maguire J. Santoro P.J. Siebenlist J.Y. Kelly K. Science. 1994; 265: 241-244Crossref PubMed Scopus (163) Google Scholar, 12Cohen L. Mohr R. Chen Y. Huang M. Kato R. Dorin D. Tamanoi F. Goga A. Afar D. Rosenberg N. Witte O. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12448-12452Crossref PubMed Scopus (82) Google Scholar). Two of the remaining clones encoded a previously unknown sequence that was homologous to but clearly different from RGK genes (Fig. 1). To emphasize this relationship we designated this clone Rem, for Rad and Gem-related. A Rem-specific oligonucleotide probe was designed (Rem-3) and used to screen a mouse testis cDNA library, from which several clones were isolated (Fig. 1; see "Experimental Procedures"). The largest of these cDNA clones was λRem 6-2, which extended from a polyadenylation tract to nucleotide 441 (nucleotide positions refer to the final sequence of the cDNA that can be retrieved from GenBank™ U91601). Preliminary ribonuclease protection assays using a portion of the λRem 6-2 cDNA indicated that Rem was abundantly expressed in kidney (data not shown). Based on the high levels of expression in kidney, a random-primed mouse kidney cDNA library was constructed and screened with a probe generated from the λRem 6-2 clone to obtain the 5′ end of the Rem cDNA (see "Experimental Procedures"). Four additional cDNA clones were isolated, one of which (λRem 5) extended from nucleotides 1 to 881. These overlapping cDNA clones (λRem 6-2 and λRem 5) were spliced together to form a full-length cDNA. The 1.5-kilobase cDNA sequence included a portion of the 5′-untranslated region, an 891-nucleotide (219–1109) open reading frame with a putative initiator methionine in a region that matched the Kozak sequence motif (24Kozak M. Nucleic Acids Res. 1984; 12: 3873-3893Crossref PubMed Scopus (231) Google Scholar), and a large 3′-untranslated region followed by a polyadenylate tail. No other methionines were observed 5′ to the putative start codon in any other reading frame. In addition, 5′ to the Kozak sequence and 3′ to the in-frame stop codon, stop codons were observed in all three reading frames. Analysis of the open reading frame revealed significant identity at the nucleotide level with members of the Ras superfamily. The deduced amino acid sequence of Rem is shown in Fig. 2. This cDNA predicts a protein of 297 amino acids with a calculated molecular size of 32,893 Da. A data base search was performed to determine the degree of homology of Rem with the RGK family and to identify additional G-proteins with structural similarities. The Rem protein contains a core sequence (amino acids 84–246) that is highly related to members of the Ras superfamily of small GTP-binding proteins. The highest degree of similarity was with mouse and human Gem, Kir, and Rad (46.7–47.2% sequence identity), but there was also a high degree of homology with additional Ras-like GTPases, including 22.8, 25.9, and 22.3% identity to dictyostelium RasA (25Reymond C.D. Gomer R.H. Medhy M.C. Firtel R.A. Cell. 1984; 39: 141-148Abstract Full Text PDF PubMed Scopus (149) Google Scholar), human Rap-2A (26Farrell F.X. Ohmstede C.A. Reep B.R. Lapetina E.G. Nucleic Acids Res. 1990; 18: 4281Crossref PubMed Scopus (34) Google Scholar), and Rap-2B (27Pizon V. Chardin P. Lerosey I. Olofsson B. Tavitian A. Oncogene. 1988; 3: 201-204PubMed Google Scholar). To assure ourselves that Rem was a new member of the RGK Ras subfamily, we performed a general comparative protein analysis among the different Ras-like subfamilies (data not shown). On the basis of this comparison, it is evident that Rem is a novel member of the RGK Ras-related subfamily. Fig. 2 depicts the alignment of Rem with a select subset of these proteins. The greatest similarity exists in regions that correspond to the guanine nucleotide-binding domains conserved in all Ras family members. The Rem protein exhibits all five of the domains (G1–G5) that have been shown to take part in both guanine nucleotide binding and the catalytic functions of the Ras protein superfamily (4Bourne H.R. Sanders D.A. McCormick F. Nature. 1991; 349: 117-127Crossref PubMed Scopus (2690) Google Scholar). Although both the NH2- and COOH-terminal extensions past the Ras core region are divergent, the COOH-terminal 10 amino acids of Rem are highly conserved in Rad, Gem, and Kir proteins (Fig. 2). This region does not contain a typical CAAX, XXCC, or CXC prenylation site (where A is an aliphatic amino acid and X is any amino acid) present in almost all Ras family members, although it does contain a conserved cysteine residue at position 7 from the COOH terminus. This conserved COOH-terminal motif may therefore represent a novel lipid modification site or direct the association of these proteins with membranes by interaction with an anchoring protein. Ribonuclease protection analysis revealed that Rem was expressed at detectable levels in every mouse organ examined (Fig. 3). The highest basal levels were detected in cardiac and skeletal muscle with slightly lower levels of Rem mRNA in lung and kidney. Low levels of mRNA were identified in spleen and brain with barely detectable levels in several additional tissues. The significance of the abundance and distribution of Rem message in these tissues is unclear. The tissue distribution of Rem contrasts with that of both Rad and Gem; Gem mRNA is most abundant in kidney, lung, and spleen, whereas Rad is expressed in cardiac and skeletal muscle and lung (10Kahn C.R. Reynet C. Science. 1993; 262: 1441-1444Crossref PubMed Scopus (0) Google Scholar, 11Maguire J. Santoro P.J. Siebenlist J.Y. Kelly K. Science. 1994; 265: 241-244Crossref PubMed Scopus (163) Google Scholar). The close homology between Rem and Ras in the regions associated with the binding of GTP suggested that Rem was a GTP-binding
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