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Molecular Cloning and Characterization of a Novel Retinoic Acid-inducible Gene That Encodes a Putative G Protein-coupled Receptor

1998; Elsevier BV; Volume: 273; Issue: 52 Linguagem: Inglês

10.1074/jbc.273.52.35008

ISSN

1083-351X

Autores

Yijun Cheng, Reuben Lotan,

Tópico(s)

interferon and immune responses

Resumo

The effects of retinoids such as all-trans-retinoic acid (ATRA) on cell growth, differentiation, and apoptosis are thought to be mediated by nuclear retinoid receptors, which are involved in ligand-dependent transcriptional activation of target genes. Using differential display, we identified the cDNA of a novel gene, designated retinoic acid-inducible gene 1 (RAIG1), which was induced by ATRA in the squamous carcinoma cell line UMSCC-22B. Two RAIG1 transcripts of 2.4 and 6.8 kilobase pairs, respectively, have the same ORF that encodes a 357-amino acid polypeptide. RAIG1 mRNA is expressed at high level in fetal and adult lung tissues. Induction of RAIG1 expression by ATRA is rapid (within 2 h) and dose-dependent in the range between 1 nm to 1 μm. The constitutive RAIG1 mRNA levels, which were low in three of five head and neck and four of six lung cancer cell lines, increased after ATRA treatment in most cell lines. The deduced RAIG1 protein sequence contains seven transmembrane domains, characteristic of G protein-coupled receptors. A fusion protein of RAIG1 and the green fluorescent protein was localized in the cell surface membrane and perinuclear vesicles in transiently transfected cells. RAIG1 was mapped to chromosome 12p12.3-p13. Our results provide novel evidence for a possible interaction between retinoid and G protein signaling pathways. The effects of retinoids such as all-trans-retinoic acid (ATRA) on cell growth, differentiation, and apoptosis are thought to be mediated by nuclear retinoid receptors, which are involved in ligand-dependent transcriptional activation of target genes. Using differential display, we identified the cDNA of a novel gene, designated retinoic acid-inducible gene 1 (RAIG1), which was induced by ATRA in the squamous carcinoma cell line UMSCC-22B. Two RAIG1 transcripts of 2.4 and 6.8 kilobase pairs, respectively, have the same ORF that encodes a 357-amino acid polypeptide. RAIG1 mRNA is expressed at high level in fetal and adult lung tissues. Induction of RAIG1 expression by ATRA is rapid (within 2 h) and dose-dependent in the range between 1 nm to 1 μm. The constitutive RAIG1 mRNA levels, which were low in three of five head and neck and four of six lung cancer cell lines, increased after ATRA treatment in most cell lines. The deduced RAIG1 protein sequence contains seven transmembrane domains, characteristic of G protein-coupled receptors. A fusion protein of RAIG1 and the green fluorescent protein was localized in the cell surface membrane and perinuclear vesicles in transiently transfected cells. RAIG1 was mapped to chromosome 12p12.3-p13. Our results provide novel evidence for a possible interaction between retinoid and G protein signaling pathways. Malignant transformation is often associated with abrogation of signaling pathways that are essential for maintaining normal development, differentiation, and homeostasis. One of the approaches to cancer prevention and therapy is to restore aberrant pathways to normalcy. In this regard, retinoids have been shown to affect many fundamental cellular processes including embryogenesis, differentiation, and tumorigenesis (1Love J.M. Gudas L.J. Curr. Opin. Cell Biol. 1994; 6: 825-831Crossref PubMed Scopus (139) Google Scholar, 2Morriss-Kay G.M. Sokolova N. FASEB J. 1996; 10: 961-968Crossref PubMed Scopus (169) Google Scholar, 3Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry and Medicine. 2nd Ed. Raven Press, New York, NY1994Google Scholar). Furthermore, these compounds have been found to exert significant preventive and therapeutic effects against cancer in human patients (4Lotan R. FASEB J. 1996; 10: 1031-1039Crossref PubMed Scopus (398) Google Scholar, 5Hong W.K. Sporn M.B. Science. 1997; 278: 1073-1077Crossref PubMed Scopus (791) Google Scholar). The ability of retinoids to reverse malignant transformation is best demonstrated in several successful chemoprevention trials. It has been shown that retinoids suppress oral premalignant leukoplakia lesions (6Hong W.K. Endicott J. Itri L.M. Doos W. Batsakis J.G. Bell R. Fofonff S. Byers R. Atkinson E.N. Vaughan C. Toth B.B. Kramer A. Dimery I.W. Skipper P. Strong S. N. Engl. J. Med. 1986; 315: 1501-1505Crossref PubMed Scopus (738) Google Scholar) and decrease the incidence of second primary tumors in head and neck cancer patients (7Hong W.K. Lippman S.M. Itri L.M. Karp D.D. Lee J.S. Byers R.M. Schultz S.P. Kramer A.M. Lotan R. Peters L.J. Dimery I.W. Brown B.W. Goepfert H. N. Eng. J. Med. 1990; 323: 795-801Crossref PubMed Scopus (1193) Google Scholar). Most of the effects of retinoids are thought to be mediated by retinoid receptors through regulating gene expression in the cell. Retinoids interact with two classes of nuclear receptors, the retinoic acid receptors (RARs) 1The abbreviations used are: RAR, retinoic acid receptor; RARE, retinoic acid receptor response element; HNSCC, head and neck squamous carcinoma; RAIG1, retinoic acid-induced gene 1; G protein, guanine nucleotide-binding protein; NSCLC, non-small cell lung cancer; ATRA, all-trans-retinoic acid; 9-cis-RA, 9-cis-retinoic acid; 13-cis-RA, 13-cis-retinoic acid; PCR, polymerase chain reaction; DD-PCR, differential display PCR; bp, base pair(s); kbp, kilobase pair(s); SSC, saline sodium citrate; GFP, green fluorescent protein; STS, sequence-tagged site; YAC, yeast artificial chromosome; GPCR, G protein-coupled receptor; mGluR, metabotropic glutamate receptor; ORF, open reading frame; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 1The abbreviations used are: RAR, retinoic acid receptor; RARE, retinoic acid receptor response element; HNSCC, head and neck squamous carcinoma; RAIG1, retinoic acid-induced gene 1; G protein, guanine nucleotide-binding protein; NSCLC, non-small cell lung cancer; ATRA, all-trans-retinoic acid; 9-cis-RA, 9-cis-retinoic acid; 13-cis-RA, 13-cis-retinoic acid; PCR, polymerase chain reaction; DD-PCR, differential display PCR; bp, base pair(s); kbp, kilobase pair(s); SSC, saline sodium citrate; GFP, green fluorescent protein; STS, sequence-tagged site; YAC, yeast artificial chromosome; GPCR, G protein-coupled receptor; mGluR, metabotropic glutamate receptor; ORF, open reading frame; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.and retinoid X receptors, which belong to a superfamily of ligand-inducible transcription factors that include the steroid and thyroid hormone receptors, vitamin D receptor, and a number of orphan receptors with unknown ligands (8Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2589) Google Scholar, 9Minucci S. Ozato K. Curr. Opin. Genet. Dev. 1996; 6: 567-574Crossref PubMed Scopus (59) Google Scholar, 10Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2818) Google Scholar). Retinoid receptors can form homodimers and heterodimers that recognize retinoic acid receptor response element (RARE) consensus sequence, usually a direct repeat (purineG(G/T)TCA) separated by 1–5 nucleotides. Upon ligand binding, nuclear receptors recognize and bind to RARE, thereby activating or repressing gene transcription (8Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2589) Google Scholar, 9Minucci S. Ozato K. Curr. Opin. Genet. Dev. 1996; 6: 567-574Crossref PubMed Scopus (59) Google Scholar, 10Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2818) Google Scholar). A number of co-factors have been found to interact with nuclear receptors and modulate their transcriptional activity. Co-activators are thought to serve as bridging molecules between the receptors and components of the basic transcriptional machinery, thus mediating activation of transcription (11Onate S.A. Tsai S.Y. Tsai M.J. O'Malley B. Science. 1995; 270: 1354-1357Crossref PubMed Scopus (2042) Google Scholar, 12Voegel J.J. Heine M.J.S. Zechel C. Chambon P. Gronemeyer H. EMBO J. 1996; 15: 3667-3675Crossref PubMed Scopus (947) Google Scholar, 13Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S-C. Heyman R.A. Rose D.W. Glass C.K. Rosenfeld M.G. Cell. 1996; 85: 403-414Abstract Full Text Full Text PDF PubMed Scopus (1916) Google Scholar). Co-repressors may associate with ligand-free receptors and squelch their activity. Ligand-dependent activation of nuclear receptors may involve dissociation of co-repressor and recruitment of co-activator by the receptor (14Horlein A.J. Naar A.M. Heinzel T. Torchia J. Gloss B. Kurokawa R. Ryan A. Kamei Y. Soderstrom M. Glass C.K. Rosenfeld M.G. Nature. 1995; 377: 397-404Crossref PubMed Scopus (1695) Google Scholar, 15Chen J.D. Evans R.M. Nature. 1995; 377: 454-457Crossref PubMed Scopus (1699) Google Scholar, 16Le Douarin B. Nielsen A.L. Chambon P. Losson R. Biochem. Soc. Trans. 1997; 25: 605-615Crossref PubMed Scopus (20) Google Scholar). Various genes, which are induced directly by ATRA, were found to contain RARE in their promoter region (1Love J.M. Gudas L.J. Curr. Opin. Cell Biol. 1994; 6: 825-831Crossref PubMed Scopus (139) Google Scholar, 3Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry and Medicine. 2nd Ed. Raven Press, New York, NY1994Google Scholar, 8Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2589) Google Scholar, 9Minucci S. Ozato K. Curr. Opin. Genet. Dev. 1996; 6: 567-574Crossref PubMed Scopus (59) Google Scholar, 10Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2818) Google Scholar). Only a few of these genes can potentially mediate retinoid signaling for modulation of cell growth (1Love J.M. Gudas L.J. Curr. Opin. Cell Biol. 1994; 6: 825-831Crossref PubMed Scopus (139) Google Scholar, 3Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry and Medicine. 2nd Ed. Raven Press, New York, NY1994Google Scholar).The mechanisms through which retinoids suppress carcinogenesis are not well understood. However, it is thought that nuclear receptors play an important role in this mechanism (1Love J.M. Gudas L.J. Curr. Opin. Cell Biol. 1994; 6: 825-831Crossref PubMed Scopus (139) Google Scholar, 4Lotan R. FASEB J. 1996; 10: 1031-1039Crossref PubMed Scopus (398) Google Scholar, 17Lotan R. Environ. Health Perspect. 1997; 105: 985-988Crossref PubMed Scopus (27) Google Scholar). In vitrostudies have shown that retinoids inhibit cell proliferation and suppress squamous cell differentiation in head and neck squamous cell carcinoma (HNSCC) cell lines (18Zou C-P. Clifford J.L. Xu X-C. Sacks P.G. Chambon P. Hong W.K. Lotan R. Cancer Res. 1994; 54: 5479-5487PubMed Google Scholar). Human oral squamous carcinoma cell UMSCC-22B is sensitive to ATRA in a clonogenic assay (19Jetten A.M. Kim J.S. Sacks P.G. Rearick J.I. Lotan D. Hong W.K. Lotan R. Int. J. Cancer. 1990; 45: 195-202Crossref PubMed Scopus (88) Google Scholar) and exhibits diminished growth as xeno-transplants in nude mice treated with RAR-selective retinoid ALRT1150 (20Shalinsky D.R. Bischoff E.D. Lamph W.W. Zhang L. Boehm M.F. Davies P.J.A. Nadzan A.M. Heyman R.A. Cancer Res. 1997; 57: 162-168PubMed Google Scholar). To better understand molecular events in retinoid-induced cell growth inhibition and differentiation, we use the differential display technique (21Liang P. Pardee A.B. Science. 1992; 257: 967-971Crossref PubMed Scopus (4688) Google Scholar) to identify retinoic acid-regulated genes in UMSCC-22B cells. Here, we present the cDNA of a novel retinoic acid-responsive gene, retinoic acid-induced gene 1 (RAIG1), which encodes a putative guanine nucleotide-binding protein (G protein)-coupled receptor. Our results suggest that retinoids may exert biological function through interaction with a yet to be defined G-protein signaling pathway.DISCUSSIONIn this study, we cloned and characterized the cDNA of a novel retinoic acid-responsive gene, RAIG1, from UMSCC-22B cells. A unique feature of RAIG1 is the presence of two mRNA species, which presumably results from alternative utilization of different polyadenylation signals. There are four potential poly(A) signals in the primary transcript unit of RAIG1, of which only the first and the last are used in 3′ end RNA polyadenylation/cleavage reaction. In many occasions, RNA processing involves a selection among multiple polyadenylation sites (33Wahle E. Kuhn U. Prog. Nucleic Acids Res. Mol. Biol. 1997; 57: 41-71Crossref PubMed Scopus (79) Google Scholar). Polyadenylation signals can be strong or weak relative to one another, depending on their surrounding sequence context (34Edwalds-Gilbert G. Veraldi K.L. Milcarek C. Nucleic Acids Res. 1997; 25: 2547-2561Crossref PubMed Scopus (451) Google Scholar). The relative stable ratios between the two RAIG1 mRNA species in different cell and tissue types suggested that the competition between the upstream and downstream polyadenylation sites is well balanced regardless of the cell type and the level of transcription. Because both RAIG1 transcripts encode the same protein, it would be interesting to analyze the effects of 3′-end untranslated region on translation and stability of RAIG1 mRNA.The deduced RAIG1 amino acid sequence contains a characteristic secondary structure of seven transmembrane α-helical domains. The presence of this characteristic motif and the plasma membrane localization of the RAIG1-GFP chimeric protein support the conclusion that RAIG1 is a member of GPCRs superfamily. GPCRs represent an increasingly large and functionally diverse superfamily of receptors that mediate their intracellular actions by signaling pathways involving G proteins and downstream secondary messengers (27Dohlman H.G. Thorner J. Caron M.G. Lefkowitz R.J. Annu. Rev. Biochem. 1991; 60: 653-688Crossref PubMed Scopus (1130) Google Scholar, 35Baldwin J.M. Curr. Opin. Cell Biol. 1994; 6: 180-190Crossref PubMed Scopus (340) Google Scholar, 36Wess J. FASEB J. 1997; 11: 346-354Crossref PubMed Scopus (507) Google Scholar). Receptors of this class respond to a variety of extracellular signals including light, lipid-derived messengers, neurotransmitters, and peptide hormones. Because the seven transmembrane domains are common to all members of GPCRs, most GPCRs bear sequence similarity with one another, primarily in the transmembrane regions (37Probst W.C. Snyder L.A. Schuster D.I. Brosius J. Sealfon S.C. DNA Cell Biol. 1992; 11: 1-20Crossref PubMed Scopus (677) Google Scholar). Among known members of GPCRs, RAIG1 protein shares a low sequence homology (25%) in the transmembrane domains only with mGluR2 and 3, which indicates that RAIG1 protein may be a novel GPCR rather than a member of the mGluR subfamily. Because GPCRs are targets for many types of therapeutics, studies on orphan receptors have become a seminal point for drug discovery using reverse molecular pharmacology strategy (38Stadel J.M. Wilson S. Bergsma D.J. Trends Pharmacol. Sci. 1997; 18: 430-437Abstract Full Text PDF PubMed Scopus (141) Google Scholar). Therefore, it will be of interest to elucidate the function of RAIG1 protein and determine whether it too is a potential target for therapeutics.The lack of a sequence homology between RAIG1 protein and known GPCRs makes it difficult to predict the specific ligand for RAIG1 protein and the identity of its coupled G protein and secondary messengers. However, RAIG1's expression pattern provides some hints about its physiological functions. In normal tissues, RAIG1 mRNA was detected in various epithelial tissues but not in mesoderm-derived tissues. The expression of RAIG1 in epithelial tissues, notably the earodigestive tract, indicates its possible function in modulating differentiation and maintaining homeostasis of epithelial cells. The comparable messenger level of RAIG1 in fetal lung and kidney with adult tissues suggests a possible role in embryonic development and maturation of these organs. Retinoids are required for the proper embryonic development and epithelial cell differentiation (2Morriss-Kay G.M. Sokolova N. FASEB J. 1996; 10: 961-968Crossref PubMed Scopus (169) Google Scholar, 39Morriss-Kay G. BioEssays. 1993; 15: 9-15Crossref PubMed Scopus (145) Google Scholar, 40Lotan R. Cancer Res. 1994; 54 (suppl.): 1987-1990Google Scholar). Such actions are carried out by regulating the expression of downstream effectors, and RAIG1 could be one such target gene.RAIG1 mRNA level increased 3-fold within 2 h of ATRA (1 μm) treatment and almost reached a plateau by 4 h of treatment. The rapid response to ATRA suggests that RAIG1 is a primary target gene. Indeed, preliminary analysis of the upstream regulatory sequence of RAIG1 demonstrated that it confers transcriptional activation by ATRA in a promoter-luciferase construct (41Wang Y. Cheng Y. Lotan R. Proc. Am. Assoc. Cancer Res. 1998; 39 (Abstr. 1881): 275Google Scholar). 9-cis-RA, a ligand for both RARs and retinoid X receptors, can up-regulate RAIG1 expression, whereas another natural retinoic acid, 13-cis-RA shows little effect. 13-cis-RA does not bind to retinoic acid receptors and is thought to act through its metabolites including ATRA (3Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry and Medicine. 2nd Ed. Raven Press, New York, NY1994Google Scholar). Therefore, treatment with 13-cis-RA may not result in sufficiently high levels of biological active retinoids to stimulate RAIG1 expression.Several previous reports linked effects of ATRA on cell differentiation to G-protein signaling pathways. ATRA induces F9 mouse teratocarcinoma cells to differentiate into primitive endoderm-like cells and decreases the steady-state level of the Gαi2 subunit (42Galvin-Parton P.A. Watkins D.C. Malbon C.C. J. Biol. Chem. 1990; 265: 17771-17779Abstract Full Text PDF PubMed Google Scholar). The reduction of the Gαi2 protein by antisense RNA has ATRA-like effects on F9 cell differentiation. Overexpression of constitutively active mutant Gαi2 blocks the ATRA-induced F9 cell differentiation (43Watkins D.C. Johnson G.L. Malbon C.C. Science. 1992; 258: 1373-1375Crossref PubMed Scopus (91) Google Scholar). These results indicate that suppression of Gαi2 expression is required for ATRA-induced F9 differentiation. In contrast, ATRA induces P19 mouse embryonic carcinoma cells to differentiate to endoderm by up-regulating expression of Gα12 and Gα13, which in turn activate c-Jun amino-terminal kinase (44Jho E.H. Davis R.J. Malbon C.C. J. Biol. Chem. 1997; 272: 24468-24474Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In ATRA-induced human TC teratocarcinoma cells differentiation, protein kinase A-associated Gαs and Gαi2 expression level increased (45Kurie J.M. Allopena J. Dmitrovsky E. Biochem. Biophys. Acta. 1994; 1222: 88-94Crossref PubMed Scopus (7) Google Scholar). Exposure to ATRA induced HL-60 human leukemia cells to differentiate into granulocyte-like cells with increased expression of receptors for the chemoattractants interleukin-8, C5a (46Zahn S. Zwirner J. Spengler H.P. Gotze O. Eur. J. Immunol. 1997; 27: 935-940Crossref PubMed Scopus (28) Google Scholar), and leukotriene B4 (47Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (847) Google Scholar). Those receptors are members of the GPCR superfamily and are required for normal leukocyte function in inflammatory response and host defense against infection. The ability of ATRA to modulate the expression of G proteins and GPCRs suggests that at least some aspects of ATRA-induced cell differentiation are mediated by G protein signaling pathways. Therefore, modulation of RAIG1 expression could contribute to the effects of retinoids on epithelial cell differentiation.RAIG1 expression in HNSCC and NSCLC cells varies from high abundance to undetectable levels. Because of the high level of RAIG1 mRNA in normal lung tissue, it is notable that RAIG1 was expressed at a low level in three of five HNSCC and four of six NSCLC cell lines tested. This finding raises the hypothesis that the decrease in RAIG1 expression is associated with the malignant transformation of some airway epithelial tissues. The observation that ATRA treatment can increase RAIG1 expression in cancer cells with low constitutive expression level indicates that retinoids can restore aberrant target gene expression in cancer cells and suggests that this may play a role in suppression of carcinogenesis. Further, RAIG1 induction may serve as a marker for ATRA-induced epithelial cell differentiation. Malignant transformation is often associated with abrogation of signaling pathways that are essential for maintaining normal development, differentiation, and homeostasis. One of the approaches to cancer prevention and therapy is to restore aberrant pathways to normalcy. In this regard, retinoids have been shown to affect many fundamental cellular processes including embryogenesis, differentiation, and tumorigenesis (1Love J.M. Gudas L.J. Curr. Opin. Cell Biol. 1994; 6: 825-831Crossref PubMed Scopus (139) Google Scholar, 2Morriss-Kay G.M. Sokolova N. FASEB J. 1996; 10: 961-968Crossref PubMed Scopus (169) Google Scholar, 3Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry and Medicine. 2nd Ed. Raven Press, New York, NY1994Google Scholar). Furthermore, these compounds have been found to exert significant preventive and therapeutic effects against cancer in human patients (4Lotan R. FASEB J. 1996; 10: 1031-1039Crossref PubMed Scopus (398) Google Scholar, 5Hong W.K. Sporn M.B. Science. 1997; 278: 1073-1077Crossref PubMed Scopus (791) Google Scholar). The ability of retinoids to reverse malignant transformation is best demonstrated in several successful chemoprevention trials. It has been shown that retinoids suppress oral premalignant leukoplakia lesions (6Hong W.K. Endicott J. Itri L.M. Doos W. Batsakis J.G. Bell R. Fofonff S. Byers R. Atkinson E.N. Vaughan C. Toth B.B. Kramer A. Dimery I.W. Skipper P. Strong S. N. Engl. J. Med. 1986; 315: 1501-1505Crossref PubMed Scopus (738) Google Scholar) and decrease the incidence of second primary tumors in head and neck cancer patients (7Hong W.K. Lippman S.M. Itri L.M. Karp D.D. Lee J.S. Byers R.M. Schultz S.P. Kramer A.M. Lotan R. Peters L.J. Dimery I.W. Brown B.W. Goepfert H. N. Eng. J. Med. 1990; 323: 795-801Crossref PubMed Scopus (1193) Google Scholar). Most of the effects of retinoids are thought to be mediated by retinoid receptors through regulating gene expression in the cell. Retinoids interact with two classes of nuclear receptors, the retinoic acid receptors (RARs) 1The abbreviations used are: RAR, retinoic acid receptor; RARE, retinoic acid receptor response element; HNSCC, head and neck squamous carcinoma; RAIG1, retinoic acid-induced gene 1; G protein, guanine nucleotide-binding protein; NSCLC, non-small cell lung cancer; ATRA, all-trans-retinoic acid; 9-cis-RA, 9-cis-retinoic acid; 13-cis-RA, 13-cis-retinoic acid; PCR, polymerase chain reaction; DD-PCR, differential display PCR; bp, base pair(s); kbp, kilobase pair(s); SSC, saline sodium citrate; GFP, green fluorescent protein; STS, sequence-tagged site; YAC, yeast artificial chromosome; GPCR, G protein-coupled receptor; mGluR, metabotropic glutamate receptor; ORF, open reading frame; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 1The abbreviations used are: RAR, retinoic acid receptor; RARE, retinoic acid receptor response element; HNSCC, head and neck squamous carcinoma; RAIG1, retinoic acid-induced gene 1; G protein, guanine nucleotide-binding protein; NSCLC, non-small cell lung cancer; ATRA, all-trans-retinoic acid; 9-cis-RA, 9-cis-retinoic acid; 13-cis-RA, 13-cis-retinoic acid; PCR, polymerase chain reaction; DD-PCR, differential display PCR; bp, base pair(s); kbp, kilobase pair(s); SSC, saline sodium citrate; GFP, green fluorescent protein; STS, sequence-tagged site; YAC, yeast artificial chromosome; GPCR, G protein-coupled receptor; mGluR, metabotropic glutamate receptor; ORF, open reading frame; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.and retinoid X receptors, which belong to a superfamily of ligand-inducible transcription factors that include the steroid and thyroid hormone receptors, vitamin D receptor, and a number of orphan receptors with unknown ligands (8Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2589) Google Scholar, 9Minucci S. Ozato K. Curr. Opin. Genet. Dev. 1996; 6: 567-574Crossref PubMed Scopus (59) Google Scholar, 10Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2818) Google Scholar). Retinoid receptors can form homodimers and heterodimers that recognize retinoic acid receptor response element (RARE) consensus sequence, usually a direct repeat (purineG(G/T)TCA) separated by 1–5 nucleotides. Upon ligand binding, nuclear receptors recognize and bind to RARE, thereby activating or repressing gene transcription (8Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2589) Google Scholar, 9Minucci S. Ozato K. Curr. Opin. Genet. Dev. 1996; 6: 567-574Crossref PubMed Scopus (59) Google Scholar, 10Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2818) Google Scholar). A number of co-factors have been found to interact with nuclear receptors and modulate their transcriptional activity. Co-activators are thought to serve as bridging molecules between the receptors and components of the basic transcriptional machinery, thus mediating activation of transcription (11Onate S.A. Tsai S.Y. Tsai M.J. O'Malley B. Science. 1995; 270: 1354-1357Crossref PubMed Scopus (2042) Google Scholar, 12Voegel J.J. Heine M.J.S. Zechel C. Chambon P. Gronemeyer H. EMBO J. 1996; 15: 3667-3675Crossref PubMed Scopus (947) Google Scholar, 13Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S-C. Heyman R.A. Rose D.W. Glass C.K. Rosenfeld M.G. Cell. 1996; 85: 403-414Abstract Full Text Full Text PDF PubMed Scopus (1916) Google Scholar). Co-repressors may associate with ligand-free receptors and squelch their activity. Ligand-dependent activation of nuclear receptors may involve dissociation of co-repressor and recruitment of co-activator by the receptor (14Horlein A.J. Naar A.M. Heinzel T. Torchia J. Gloss B. Kurokawa R. Ryan A. Kamei Y. Soderstrom M. Glass C.K. Rosenfeld M.G. Nature. 1995; 377: 397-404Crossref PubMed Scopus (1695) Google Scholar, 15Chen J.D. Evans R.M. Nature. 1995; 377: 454-457Crossref PubMed Scopus (1699) Google Scholar, 16Le Douarin B. Nielsen A.L. Chambon P. Losson R. Biochem. Soc. Trans. 1997; 25: 605-615Crossref PubMed Scopus (20) Google Scholar). Various genes, which are induced directly by ATRA, were found to contain RARE in their promoter region (1Love J.M. Gudas L.J. Curr. Opin. Cell Biol. 1994; 6: 825-831Crossref PubMed Scopus (139) Google Scholar, 3Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry and Medicine. 2nd Ed. Raven Press, New York, NY1994Google Scholar, 8Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2589) Google Scholar, 9Minucci S. Ozato K. Curr. Opin. Genet. Dev. 1996; 6: 567-574Crossref PubMed Scopus (59) Google Scholar, 10Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2818) Google Scholar). Only a few of these genes can potentially mediate retinoid signaling for modulation of cell growth (1Love J.M. Gudas L.J. Curr. Opin. Cell Biol. 1994; 6: 825-831Crossref PubMed Scopus (139) Google Scholar, 3Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry and Medicine. 2nd Ed. Raven Press, New York, NY1994Google Scholar). The mechanisms through which retinoids suppress carcinogenesis are not well understood. However, it is thought that nuclear receptors play an important role in this mechanism (1Love J.M. Gudas L.J. Curr. Opin. Cell Biol. 1994; 6: 825-831Crossref PubMed Scopus (139) Google Scholar, 4Lotan R. FASEB J. 1996; 10: 1031-1039Crossref PubMed Scopus (398) Google Scholar, 17Lotan R. Environ. Health Perspect. 1997; 105: 985-988Crossref PubMed Scopus (27) Google Scholar). In vitrostudies have shown that retinoids inhibit cell proliferation and suppress squamous cell differentiation in head and neck squamous cell carcinoma (HNSCC) cell lines (18Zou C-P. Clifford J.L. Xu X-C. Sacks P.G. Chambon P. Hong W.K. Lotan R. Cancer Res. 1994; 54: 5479-5487PubMed Google Scholar). Human oral squamous carcinoma cell UMSCC-22B is sensitive to ATRA in a clonogenic assay (19Jetten A.M. Kim J.S. Sacks P.G. Rearick J.I. Lotan D. Hong W.K. Lotan R. Int. J. Cancer. 1990; 45: 195-202Crossref PubMed Scopus (88) Google Scholar) and exhibits diminished growth as xeno-transplants in nude mice treated with RAR-selective retinoid ALRT1150 (20Shalinsky D.R. Bischoff E.D. Lamph W.W. Zhang L. Boehm M.F. Davies P.J.A. Nadzan A.M. Heyman R.A. Cancer Res. 1997; 57: 162-168PubMed Google Scholar). To better understand molecular events in retinoid-induced cell growth inhibition and differentiation, we use the differential display technique (21Liang P. Pardee A.B. Science. 1992; 257: 967-971Crossref PubMed Scopus (4688) Google Scholar) to identify retinoic acid-regulated genes in UMSCC-22B cells. Here, we present the cDNA of a novel retinoic acid-responsive gene, retinoic acid-induced gene 1 (RAIG1), which encodes a putative guanine nucleotide-binding protein (G protein)-coupled receptor. Our results suggest that retinoids may exert biological function through interaction with a yet to be defined G-protein signaling pathway. DISCUSSIONIn this study, we cloned and characterized the cDNA of a novel retinoic acid-responsive gene, RAIG1, from UMSCC-22B cells. A unique feature of RAIG1 is the presence of two mRNA species, which presumably results from alternative utilization of different polyadenylation signals. There are four potential poly(A) signals in the primary transcript unit of RAIG1, of which only the first and the last are used in 3′ end RNA polyadenylation/cleavage reaction. In many occasions, RNA processing involves a selection among multiple polyadenylation sites (33Wahle E. Kuhn U. Prog. Nucleic Acids Res. Mol. Biol. 1997; 57: 41-71Crossref PubMed Scopus (79) Google Scholar). Polyadenylation signals can be strong or weak relative to one another, depending on their surrounding sequence context (34Edwalds-Gilbert G. Veraldi K.L. Milcarek C. Nucleic Acids Res. 1997; 25: 2547-2561Crossref PubMed Scopus (451) Google Scholar). The relative stable ratios between the two RAIG1 mRNA species in different cell and tissue types suggested that the competition between the upstream and downstream polyadenylation sites is well balanced regardless of the cell type and the level of transcription. Because both RAIG1 transcripts encode the same protein, it would be interesting to analyze the effects of 3′-end untranslated region on translation and stability of RAIG1 mRNA.The deduced RAIG1 amino acid sequence contains a characteristic secondary structure of seven transmembrane α-helical domains. The presence of this characteristic motif and the plasma membrane localization of the RAIG1-GFP chimeric protein support the conclusion that RAIG1 is a member of GPCRs superfamily. GPCRs represent an increasingly large and functionally diverse superfamily of receptors that mediate their intracellular actions by signaling pathways involving G proteins and downstream secondary messengers (27Dohlman H.G. Thorner J. Caron M.G. Lefkowitz R.J. Annu. Rev. Biochem. 1991; 60: 653-688Crossref PubMed Scopus (1130) Google Scholar, 35Baldwin J.M. Curr. Opin. Cell Biol. 1994; 6: 180-190Crossref PubMed Scopus (340) Google Scholar, 36Wess J. FASEB J. 1997; 11: 346-354Crossref PubMed Scopus (507) Google Scholar). Receptors of this class respond to a variety of extracellular signals including light, lipid-derived messengers, neurotransmitters, and peptide hormones. Because the seven transmembrane domains are common to all members of GPCRs, most GPCRs bear sequence similarity with one another, primarily in the transmembrane regions (37Probst W.C. Snyder L.A. Schuster D.I. Brosius J. Sealfon S.C. DNA Cell Biol. 1992; 11: 1-20Crossref PubMed Scopus (677) Google Scholar). Among known members of GPCRs, RAIG1 protein shares a low sequence homology (25%) in the transmembrane domains only with mGluR2 and 3, which indicates that RAIG1 protein may be a novel GPCR rather than a member of the mGluR subfamily. Because GPCRs are targets for many types of therapeutics, studies on orphan receptors have become a seminal point for drug discovery using reverse molecular pharmacology strategy (38Stadel J.M. Wilson S. Bergsma D.J. Trends Pharmacol. Sci. 1997; 18: 430-437Abstract Full Text PDF PubMed Scopus (141) Google Scholar). Therefore, it will be of interest to elucidate the function of RAIG1 protein and determine whether it too is a potential target for therapeutics.The lack of a sequence homology between RAIG1 protein and known GPCRs makes it difficult to predict the specific ligand for RAIG1 protein and the identity of its coupled G protein and secondary messengers. However, RAIG1's expression pattern provides some hints about its physiological functions. In normal tissues, RAIG1 mRNA was detected in various epithelial tissues but not in mesoderm-derived tissues. The expression of RAIG1 in epithelial tissues, notably the earodigestive tract, indicates its possible function in modulating differentiation and maintaining homeostasis of epithelial cells. The comparable messenger level of RAIG1 in fetal lung and kidney with adult tissues suggests a possible role in embryonic development and maturation of these organs. Retinoids are required for the proper embryonic development and epithelial cell differentiation (2Morriss-Kay G.M. Sokolova N. FASEB J. 1996; 10: 961-968Crossref PubMed Scopus (169) Google Scholar, 39Morriss-Kay G. BioEssays. 1993; 15: 9-15Crossref PubMed Scopus (145) Google Scholar, 40Lotan R. Cancer Res. 1994; 54 (suppl.): 1987-1990Google Scholar). Such actions are carried out by regulating the expression of downstream effectors, and RAIG1 could be one such target gene.RAIG1 mRNA level increased 3-fold within 2 h of ATRA (1 μm) treatment and almost reached a plateau by 4 h of treatment. The rapid response to ATRA suggests that RAIG1 is a primary target gene. Indeed, preliminary analysis of the upstream regulatory sequence of RAIG1 demonstrated that it confers transcriptional activation by ATRA in a promoter-luciferase construct (41Wang Y. Cheng Y. Lotan R. Proc. Am. Assoc. Cancer Res. 1998; 39 (Abstr. 1881): 275Google Scholar). 9-cis-RA, a ligand for both RARs and retinoid X receptors, can up-regulate RAIG1 expression, whereas another natural retinoic acid, 13-cis-RA shows little effect. 13-cis-RA does not bind to retinoic acid receptors and is thought to act through its metabolites including ATRA (3Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry and Medicine. 2nd Ed. Raven Press, New York, NY1994Google Scholar). Therefore, treatment with 13-cis-RA may not result in sufficiently high levels of biological active retinoids to stimulate RAIG1 expression.Several previous reports linked effects of ATRA on cell differentiation to G-protein signaling pathways. ATRA induces F9 mouse teratocarcinoma cells to differentiate into primitive endoderm-like cells and decreases the steady-state level of the Gαi2 subunit (42Galvin-Parton P.A. Watkins D.C. Malbon C.C. J. Biol. Chem. 1990; 265: 17771-17779Abstract Full Text PDF PubMed Google Scholar). The reduction of the Gαi2 protein by antisense RNA has ATRA-like effects on F9 cell differentiation. Overexpression of constitutively active mutant Gαi2 blocks the ATRA-induced F9 cell differentiation (43Watkins D.C. Johnson G.L. Malbon C.C. Science. 1992; 258: 1373-1375Crossref PubMed Scopus (91) Google Scholar). These results indicate that suppression of Gαi2 expression is required for ATRA-induced F9 differentiation. In contrast, ATRA induces P19 mouse embryonic carcinoma cells to differentiate to endoderm by up-regulating expression of Gα12 and Gα13, which in turn activate c-Jun amino-terminal kinase (44Jho E.H. Davis R.J. Malbon C.C. J. Biol. Chem. 1997; 272: 24468-24474Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In ATRA-induced human TC teratocarcinoma cells differentiation, protein kinase A-associated Gαs and Gαi2 expression level increased (45Kurie J.M. Allopena J. Dmitrovsky E. Biochem. Biophys. Acta. 1994; 1222: 88-94Crossref PubMed Scopus (7) Google Scholar). Exposure to ATRA induced HL-60 human leukemia cells to differentiate into granulocyte-like cells with increased expression of receptors for the chemoattractants interleukin-8, C5a (46Zahn S. Zwirner J. Spengler H.P. Gotze O. Eur. J. Immunol. 1997; 27: 935-940Crossref PubMed Scopus (28) Google Scholar), and leukotriene B4 (47Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (847) Google Scholar). Those receptors are members of the GPCR superfamily and are required for normal leukocyte function in inflammatory response and host defense against infection. The ability of ATRA to modulate the expression of G proteins and GPCRs suggests that at least some aspects of ATRA-induced cell differentiation are mediated by G protein signaling pathways. Therefore, modulation of RAIG1 expression could contribute to the effects of retinoids on epithelial cell differentiation.RAIG1 expression in HNSCC and NSCLC cells varies from high abundance to undetectable levels. Because of the high level of RAIG1 mRNA in normal lung tissue, it is notable that RAIG1 was expressed at a low level in three of five HNSCC and four of six NSCLC cell lines tested. This finding raises the hypothesis that the decrease in RAIG1 expression is associated with the malignant transformation of some airway epithelial tissues. The observation that ATRA treatment can increase RAIG1 expression in cancer cells with low constitutive expression level indicates that retinoids can restore aberrant target gene expression in cancer cells and suggests that this may play a role in suppression of carcinogenesis. Further, RAIG1 induction may serve as a marker for ATRA-induced epithelial cell differentiation. In this study, we cloned and characterized the cDNA of a novel retinoic acid-responsive gene, RAIG1, from UMSCC-22B cells. A unique feature of RAIG1 is the presence of two mRNA species, which presumably results from alternative utilization of different polyadenylation signals. There are four potential poly(A) signals in the primary transcript unit of RAIG1, of which only the first and the last are used in 3′ end RNA polyadenylation/cleavage reaction. In many occasions, RNA processing involves a selection among multiple polyadenylation sites (33Wahle E. Kuhn U. Prog. Nucleic Acids Res. Mol. Biol. 1997; 57: 41-71Crossref PubMed Scopus (79) Google Scholar). Polyadenylation signals can be strong or weak relative to one another, depending on their surrounding sequence context (34Edwalds-Gilbert G. Veraldi K.L. Milcarek C. Nucleic Acids Res. 1997; 25: 2547-2561Crossref PubMed Scopus (451) Google Scholar). The relative stable ratios between the two RAIG1 mRNA species in different cell and tissue types suggested that the competition between the upstream and downstream polyadenylation sites is well balanced regardless of the cell type and the level of transcription. Because both RAIG1 transcripts encode the same protein, it would be interesting to analyze the effects of 3′-end untranslated region on translation and stability of RAIG1 mRNA. The deduced RAIG1 amino acid sequence contains a characteristic secondary structure of seven transmembrane α-helical domains. The presence of this characteristic motif and the plasma membrane localization of the RAIG1-GFP chimeric protein support the conclusion that RAIG1 is a member of GPCRs superfamily. GPCRs represent an increasingly large and functionally diverse superfamily of receptors that mediate their intracellular actions by signaling pathways involving G proteins and downstream secondary messengers (27Dohlman H.G. Thorner J. Caron M.G. Lefkowitz R.J. Annu. Rev. Biochem. 1991; 60: 653-688Crossref PubMed Scopus (1130) Google Scholar, 35Baldwin J.M. Curr. Opin. Cell Biol. 1994; 6: 180-190Crossref PubMed Scopus (340) Google Scholar, 36Wess J. FASEB J. 1997; 11: 346-354Crossref PubMed Scopus (507) Google Scholar). Receptors of this class respond to a variety of extracellular signals including light, lipid-derived messengers, neurotransmitters, and peptide hormones. Because the seven transmembrane domains are common to all members of GPCRs, most GPCRs bear sequence similarity with one another, primarily in the transmembrane regions (37Probst W.C. Snyder L.A. Schuster D.I. Brosius J. Sealfon S.C. DNA Cell Biol. 1992; 11: 1-20Crossref PubMed Scopus (677) Google Scholar). Among known members of GPCRs, RAIG1 protein shares a low sequence homology (25%) in the transmembrane domains only with mGluR2 and 3, which indicates that RAIG1 protein may be a novel GPCR rather than a member of the mGluR subfamily. Because GPCRs are targets for many types of therapeutics, studies on orphan receptors have become a seminal point for drug discovery using reverse molecular pharmacology strategy (38Stadel J.M. Wilson S. Bergsma D.J. Trends Pharmacol. Sci. 1997; 18: 430-437Abstract Full Text PDF PubMed Scopus (141) Google Scholar). Therefore, it will be of interest to elucidate the function of RAIG1 protein and determine whether it too is a potential target for therapeutics. The lack of a sequence homology between RAIG1 protein and known GPCRs makes it difficult to predict the specific ligand for RAIG1 protein and the identity of its coupled G protein and secondary messengers. However, RAIG1's expression pattern provides some hints about its physiological functions. In normal tissues, RAIG1 mRNA was detected in various epithelial tissues but not in mesoderm-derived tissues. The expression of RAIG1 in epithelial tissues, notably the earodigestive tract, indicates its possible function in modulating differentiation and maintaining homeostasis of epithelial cells. The comparable messenger level of RAIG1 in fetal lung and kidney with adult tissues suggests a possible role in embryonic development and maturation of these organs. Retinoids are required for the proper embryonic development and epithelial cell differentiation (2Morriss-Kay G.M. Sokolova N. FASEB J. 1996; 10: 961-968Crossref PubMed Scopus (169) Google Scholar, 39Morriss-Kay G. BioEssays. 1993; 15: 9-15Crossref PubMed Scopus (145) Google Scholar, 40Lotan R. Cancer Res. 1994; 54 (suppl.): 1987-1990Google Scholar). Such actions are carried out by regulating the expression of downstream effectors, and RAIG1 could be one such target gene. RAIG1 mRNA level increased 3-fold within 2 h of ATRA (1 μm) treatment and almost reached a plateau by 4 h of treatment. The rapid response to ATRA suggests that RAIG1 is a primary target gene. Indeed, preliminary analysis of the upstream regulatory sequence of RAIG1 demonstrated that it confers transcriptional activation by ATRA in a promoter-luciferase construct (41Wang Y. Cheng Y. Lotan R. Proc. Am. Assoc. Cancer Res. 1998; 39 (Abstr. 1881): 275Google Scholar). 9-cis-RA, a ligand for both RARs and retinoid X receptors, can up-regulate RAIG1 expression, whereas another natural retinoic acid, 13-cis-RA shows little effect. 13-cis-RA does not bind to retinoic acid receptors and is thought to act through its metabolites including ATRA (3Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry and Medicine. 2nd Ed. Raven Press, New York, NY1994Google Scholar). Therefore, treatment with 13-cis-RA may not result in sufficiently high levels of biological active retinoids to stimulate RAIG1 expression. Several previous reports linked effects of ATRA on cell differentiation to G-protein signaling pathways. ATRA induces F9 mouse teratocarcinoma cells to differentiate into primitive endoderm-like cells and decreases the steady-state level of the Gαi2 subunit (42Galvin-Parton P.A. Watkins D.C. Malbon C.C. J. Biol. Chem. 1990; 265: 17771-17779Abstract Full Text PDF PubMed Google Scholar). The reduction of the Gαi2 protein by antisense RNA has ATRA-like effects on F9 cell differentiation. Overexpression of constitutively active mutant Gαi2 blocks the ATRA-induced F9 cell differentiation (43Watkins D.C. Johnson G.L. Malbon C.C. Science. 1992; 258: 1373-1375Crossref PubMed Scopus (91) Google Scholar). These results indicate that suppression of Gαi2 expression is required for ATRA-induced F9 differentiation. In contrast, ATRA induces P19 mouse embryonic carcinoma cells to differentiate to endoderm by up-regulating expression of Gα12 and Gα13, which in turn activate c-Jun amino-terminal kinase (44Jho E.H. Davis R.J. Malbon C.C. J. Biol. Chem. 1997; 272: 24468-24474Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In ATRA-induced human TC teratocarcinoma cells differentiation, protein kinase A-associated Gαs and Gαi2 expression level increased (45Kurie J.M. Allopena J. Dmitrovsky E. Biochem. Biophys. Acta. 1994; 1222: 88-94Crossref PubMed Scopus (7) Google Scholar). Exposure to ATRA induced HL-60 human leukemia cells to differentiate into granulocyte-like cells with increased expression of receptors for the chemoattractants interleukin-8, C5a (46Zahn S. Zwirner J. Spengler H.P. Gotze O. Eur. J. Immunol. 1997; 27: 935-940Crossref PubMed Scopus (28) Google Scholar), and leukotriene B4 (47Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (847) Google Scholar). Those receptors are members of the GPCR superfamily and are required for normal leukocyte function in inflammatory response and host defense against infection. The ability of ATRA to modulate the expression of G proteins and GPCRs suggests that at least some aspects of ATRA-induced cell differentiation are mediated by G protein signaling pathways. Therefore, modulation of RAIG1 expression could contribute to the effects of retinoids on epithelial cell differentiation. RAIG1 expression in HNSCC and NSCLC cells varies from high abundance to undetectable levels. Because of the high level of RAIG1 mRNA in normal lung tissue, it is notable that RAIG1 was expressed at a low level in three of five HNSCC and four of six NSCLC cell lines tested. This finding raises the hypothesis that the decrease in RAIG1 expression is associated with the malignant transformation of some airway epithelial tissues. The observation that ATRA treatment can increase RAIG1 expression in cancer cells with low constitutive expression level indicates that retinoids can restore aberrant target gene expression in cancer cells and suggests that this may play a role in suppression of carcinogenesis. Further, RAIG1 induction may serve as a marker for ATRA-induced epithelial cell differentiation. We thank Dafna Lotan for excellent technical assistance.

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