Heterogeneous RNA-binding Protein M4 Is a Receptor for Carcinoembryonic Antigen in Kupffer Cells

Abstract

Here we report the isolation of the recombinant cDNA clone from rat macrophages, Kupffer cells (KC) that encodes a protein interacting with carcinoembryonic antigen (CEA). To isolate and identify the CEA receptor gene we used two approaches: screening of a KC cDNA library with a specific antibody and the yeast two-hybrid system for protein interaction using as a bait the N-terminal part of the CEA encoding the binding site. Both techniques resulted in the identification of the rat heterogeneous RNA-binding protein(hnRNP) M4 gene. The rat ortholog cDNA sequence has not been previously described. The open reading frame for this gene contains a 2351-base pair sequence with the polyadenylation signal AATAAA and a termination poly(A) tail. The mRNA shows ubiquitous tissue expression as a 2.4-kilobase transcript. The deduced amino acid sequence comprised a 78-kDa membrane protein with 3 putative RNA-binding domains, arginine/methionine/glutamine-rich C terminus and 3 potential membrane spanning regions. When hnRNP M4 protein is expressed in pGEX4T-3 vector system in Escherichia coli it binds125I-labeled CEA in a Ca2+-dependent fashion. Transfection of rathnRNP M4 cDNA into a non-CEA binding mouse macrophage cell line p388D1 resulted in CEA binding. These data provide evidence for a new function of hnRNP M4 protein as a CEA-binding protein in Kupffer cells. Here we report the isolation of the recombinant cDNA clone from rat macrophages, Kupffer cells (KC) that encodes a protein interacting with carcinoembryonic antigen (CEA). To isolate and identify the CEA receptor gene we used two approaches: screening of a KC cDNA library with a specific antibody and the yeast two-hybrid system for protein interaction using as a bait the N-terminal part of the CEA encoding the binding site. Both techniques resulted in the identification of the rat heterogeneous RNA-binding protein(hnRNP) M4 gene. The rat ortholog cDNA sequence has not been previously described. The open reading frame for this gene contains a 2351-base pair sequence with the polyadenylation signal AATAAA and a termination poly(A) tail. The mRNA shows ubiquitous tissue expression as a 2.4-kilobase transcript. The deduced amino acid sequence comprised a 78-kDa membrane protein with 3 putative RNA-binding domains, arginine/methionine/glutamine-rich C terminus and 3 potential membrane spanning regions. When hnRNP M4 protein is expressed in pGEX4T-3 vector system in Escherichia coli it binds125I-labeled CEA in a Ca2+-dependent fashion. Transfection of rathnRNP M4 cDNA into a non-CEA binding mouse macrophage cell line p388D1 resulted in CEA binding. These data provide evidence for a new function of hnRNP M4 protein as a CEA-binding protein in Kupffer cells. carcinoembryonic antigen Kupffer cell heterogeneous RNA-binding protein RNA-binding domain amino acid interleukin polyacrylamide gel electrophoresis base pair(s) reverse transcriptase-polymerase chain reaction open reading frame myelin gene expression factor 2 glutathione S-transferase The liver is a common site for metastasis from various forms of primary malignancies. Both experimental and clinical results reveal that the presence of carcinoembryonic antigen (CEA)1 enhances liver metastasis from colorectal carcinoma cells (1Kim J.C. Gong G. Roh S.A. Park K.C. Mol. Cells. 1999; 9: 133-137PubMed Google Scholar, 2Jessup J.M. Thomas P. Stanners C.P. Cell Adhesion and Communication Mediated by the CEA Family: Basic and Clinical Perspectives. Harwood Academic Publishers, Amsterdam, Netherlands1998: 195-222Google Scholar). CEA is a highly characterized, cell surface glycoprotein overexpressed by various tumor cells and provides a tool for tumor tissue-specific targeting. Increasing amounts of CEA in the serum correlates with the development of metastatic recurrence after surgical removal of the primary tumor (3Keesee S.K. Briggman J.V. Thill G. Wu Y.-J. Crit. Rev. Eukaryotic Gene Exp. 1996; 6: 189-214Crossref PubMed Scopus (124) Google Scholar). Earlier we have shown that CEA production of human colorectal cancer cell lines directly correlates with the metastatic potential (4Wagner H.E. Toth C.A. Steele G.D. Thomas P. Clin. Exp. Metastasis. 1992; 10: 25-31Crossref PubMed Scopus (84) Google Scholar). Poorly metastatic colon cancer cell lines become highly metastatic when transfected with the cDNA coding for CEA (5Hashino J. Fukada Y. Oikawa S. Nakazato H. Nakanishi T. Clin. Exp. Metastasis. 1994; 12: 324-328Crossref PubMed Scopus (77) Google Scholar, 6Thomas P. Gangopadhyay A. Steele Jr., G. Andrews C. Nakazato H. Oikawa S. Jessup J.M. Cancer Lett. 1995; 25: 59-66Crossref Scopus (96) Google Scholar). As a member of the immunoglobulin supergene family, CEA is involved in intercellular recognition and may facilitate attachment of colorectal carcinoma cells to sites of metastasis. In an experimental metastasis model of colorectal carcinoma in athymic nude mice, systemic injection of CEA enhanced experimental liver metastasis and implantation in liver by weakly metastatic tumor cells (7Leconte A. Garambois V. Ychou M. Robert B. Pourquier D. Terskikh A. Mach J.P. Pelegrin A. Br. J. Cancer. 1999; 9: 1373-1379Crossref Scopus (17) Google Scholar, 8Hostetter R.B. Augustus L.B. Mankarious R. Chi K. Fan D. Toth C.A. Thomas P. Jessup J.M. J. Natl. Cancer Inst. 1990; 82: 380-385Crossref PubMed Scopus (181) Google Scholar). To successfully treat cancers, it is necessary to prevent the development of metastasis after the treatment or removal of the primary malignancy. Therefore, it is important to elucidate the mechanism by which CEA enhances metastatic potential. The molecular basis by which CEA can influence metastasis is only partly understood. We have shown that CEA is rapidly cleared from the circulation of experimental animals, accumulates in the liver, and is endocytosed in vitro by Kupffer cells. This initiates a series of signaling events that leads to tyrosine phosphorylation on at least two intracellular proteins (9Gangopadhyay A. Lazure D.A. Thomas P. Cancer Lett. 1997; 118: 1-6Crossref PubMed Scopus (24) Google Scholar) and is followed by induction of IL-1α, IL-6, IL-10, and tumor necrosis factor-α cytokines (10Gangopadhyay A. Bajenova O. Kelly T.M. Thomas P. Cancer Res. 1996; 56: 4805-4810PubMed Google Scholar). CEA uptake by Kupffer cells is independent of its carbohydrate composition and is mediated by an 80-kDa binding protein (11Toth C.A. Thomas P. Broitman S.A. Zamcheck N. Biochem. J. 1982; 204: 377-381Crossref PubMed Scopus (45) Google Scholar). CEA is recognized by this binding protein through a 5-amino acid sequence, Pro-Glu-Leu-Pro-Lys (PELPK), located at the hinge region (amino acids 108–112) between the N-terminal and the first immunoglobulin loop domain (12Gangopadhyay A. Thomas P. Arch. Biochem. Biophys. 1996; 334: 151-157Crossref PubMed Scopus (25) Google Scholar). Molecular modeling studies have suggested that this region is exposed on the surface of the molecule. 2P. A. Bates, personal communication. We have recently shown in a subset of colorectal cancer patients that mutations in the PELPK region of CEA results in the accumulation of large amounts of CEA in the serum (13Zimmer R. Thomas P. Cancer Res. 2001; 61: 2822-2826PubMed Google Scholar). To further study the interaction between the peptide sequence, PELPK, and rat Kupffer cells, the CEA-binding protein was purified using a combination of gel filtration, preparative polyacrylamide gel electrophoresis, and affinity chromatography on CEA-Sepharose (14Gangopadhyay A. Lazure D.A. Kelly T.M. Thomas P. Arch. Biochem. Biophys. 1996; 328: 151-157Crossref PubMed Scopus (15) Google Scholar). A polyclonal antibody to the rat 80-kDa protein was produced in mice that blocks uptake by isolated rat Kupffer cells of both CEA and PELPK peptide conjugated to albumin. This antibody shows a high degree of specificity for the rat 80-kDa protein by both fluorescein isothiocyanate analysis and Western blotting (14Gangopadhyay A. Lazure D.A. Kelly T.M. Thomas P. Arch. Biochem. Biophys. 1996; 328: 151-157Crossref PubMed Scopus (15) Google Scholar). In this study we used this antibody to clone the rat CEA-binding protein. We have isolated the rat ortholog of the human hnRNP M4 protein and present the data on the analysis and tissue distribution of this protein. It was also shown that transfection of rat hnRNP M4cDNA into a mouse P388D1 macrophages resulted in CEA binding. Kupffer cells were isolated according to our standard laboratory protocol (15Toth C.A. Thomas P. Broitman S.A. Zamcheck N. Cancer Res. 1985; 45: 392-397PubMed Google Scholar) from the livers of male Harlan Sprague-Dawley rats. The rodents were starved overnight and the livers perfused through the portal vein with collagenase. Suspended cells were separated into parenchymal and non-parenchymal fractions by differential centrifugation. Kupffer cells were purified from the non-parenchymal cell fraction using a gradient of 17.5% metrizamide in Gey's balanced salt solution. The interface layer containing Kupffer cells was isolated and washed in phosphate-buffered saline. Further purification of the cells is achieved by attachment to plastic plates for 2 h. Generally 3–5 × 108 cells are routinely isolated from one liver. 1 × 108 Kupffer cells remain after the adhesion step. Kupffer cells are identified by their ability to phagocytose 1-µm latex particles, and by staining for endogenous peroxidase activity. The cells were >90% viable by trypan blue staining and 85% phagocytic. Rat KC library was constructed on the basis of the λZAP Express vector system according to the manufacturers instructions (Stratagene). Total RNA from various tissues, KC, and cell lines was isolated using the RNAzol method (Biotex Laboratories, Inc.) and mRNA using the mRNA isolation kit (Qiagen Inc.). To screen the cDNA library was with recombinant 1 × of the library were on plates containing and at for h. were in in the and at for h. The were and in to to the antibody (14Gangopadhyay A. Lazure D.A. Kelly T.M. Thomas P. Arch. Biochem. Biophys. 1996; 328: 151-157Crossref PubMed Scopus (15) Google Scholar). The enhanced was used to cDNA screening one clone was and after the was in vector for We also used the system to identify CEA-binding cDNA from rat Kupffer cells were using the Express cDNA kit according to the manufacturers using of mRNA which was purified as the vector we rat cDNA into the The domain vector was as a the region was isolated from a CEA from T. for and to used as of the was by and This bait not in to binding of CEA with yeast cells were by with the and bait at for in a containing 1 × interaction were in plates and interaction between and bait was by a are to in and for to of interaction was by from yeast cells. was purified by the method and to the The was using method with the were purified by and separated on the gel The sequence was and using the The were by of the production of CEA-binding protein in a to hnRNP M4 cDNA from the recombinant was into sites of vector The was from the of to the reading The was into coli To the cells were in to a after which was and were at for 2 h. Total proteins were and proteins were purified using affinity according to the manufacturers instructions Kupffer cells × were washed in phosphate-buffered and in with 2 and cells were by with Cell were at for at were and protein were using the method amounts of protein were on electrophoresis were into membrane To CEA binding the membrane was with CEA in containing and exposed to a antibody of CEA was using the was using the Molecular to CEA-binding protein a reaction was containing of and of of and of 1 × and 1 of containing reverse and The was 1 of for 2 of for for for of for for for with of for and a of for The were the region encoding amino acids and of the rat hnRNP M4 protein. This two of and the hnRNP M4 and hnRNP M4 with the As for that cDNA reverse were in independent were for transfection of hnRNP M4 was in P388D1 mouse mouse and rat macrophage cell lines of cells lines were to CEA standard We used rat Kupffer cells as a The P388D1 cells were in with The cells were in with serum The cells were in with cells were at in tissue were from for transfection were at × cells in tissue were to for and the was transfected in using the Gene system The cells were washed with phosphate-buffered and at a of in of M4 expression vector was transfected of the cell was used in The cells were at to and after the cells were into at a of the cells were and in a of for of CEA binding activity. These were as previously (15Toth C.A. Thomas P. Broitman S.A. Zamcheck N. Cancer Res. 1985; 45: 392-397PubMed Google Scholar). CEA was in with the cells at were at and These were through an of at using an The cells through the and can separated from the for by the of the plastic The rat Kupffer cell cDNA library was with the mouse polyclonal antibody (14Gangopadhyay A. Lazure D.A. Kelly T.M. Thomas P. Arch. Biochem. Biophys. 1996; 328: 151-157Crossref PubMed Scopus (15) Google Scholar). of from the λZAP library resulted in the identification of one cDNA clone with an of and a of human data for that this cDNA was a gene with of cDNA to the M4 gene The sequence are of and a of in a region between two putative N-terminal RNA-binding and A is also to M4 protein cDNA The cDNA sequence of the rat gene was to the with the used to the rat KC library is polyclonal (14Gangopadhyay A. Lazure D.A. Kelly T.M. Thomas P. Arch. Biochem. Biophys. 1996; 328: 151-157Crossref PubMed Scopus (15) Google is potential to protein To that hnRNP M4 was a CEA-binding protein we for CEA-binding proteins using the yeast two-hybrid system The bait was an of the CEA containing a acid the site. that to the M4 gene. Both screening antibody and the yeast two-hybrid resulted in the identification of the gene sequence a new for the hnRNP M4 gene encoding the 80-kDa CEA-binding protein in Kupffer cells. of the rat cDNA an open reading frame from the sequence and containing a region of followed by with the polyadenylation signal AATAAA and a termination poly(A) tail. shows this The cDNA has 3 putative RNA-binding that are present in and hnRNP and polyadenylation Biochem. PubMed Scopus Google Scholar). The first and and are in to the terminus and is located the C terminus The N-terminal and have and with the H. myelin gene expression factor cDNA S. A. S. K. J. 1995; PubMed Scopus Google Scholar). These are on The protein contains two RNA-binding and previously shown to for sequence specific binding to both RNA and S. A. S. K. J. 1995; PubMed Scopus Google Scholar). is a factor that was isolated from the mouse to mouse and of the myelin protein gene. The hnRNP M4 and a degree of to in a of RNA and proteins M. G. Mol. PubMed Scopus Google Scholar). analysis M4 contains recognized binding as or to the RNA binding can for binding and RNA binding of hnRNP with was by The rat M4 cDNA sequence has to a H. clone The human gene was previously to of The of the hnRNP M4 in the rat is not sequence analysis on the cDNA that rat hnRNP M4 encodes a protein of amino It is with its molecular of on gel electrophoresis and is with the molecular previously for the Kupffer protein (14Gangopadhyay A. Lazure D.A. Kelly T.M. Thomas P. Arch. Biochem. Biophys. 1996; 328: 151-157Crossref PubMed Scopus (15) Google Scholar). on cDNA hnRNP M4 is a signaling protein that has the potential to by of analysis of the protein using data base that in to 3 putative RNA-binding and this protein has 3 potential membrane spanning and 2 It also potential protein C phosphorylation phosphorylation and 2 The intracellular domain contains to a tyrosine phosphorylation and two attachment sites and The C terminus of hnRNP M4 is to this and contains of and is the only amino acid in that the of proteins is to have an important on their G. Mol. 1995; PubMed Scopus Google Scholar). is part of the mechanism by which are recognized for 12: PubMed Scopus Google Scholar). The amino acid sequence of rat hnRNP M4 has been recently P. M. A. Biochem. J. PubMed Scopus Google Scholar) and it is in of our protein This sequence is the N-terminal part of the protein and has the sequence It also the the in the region between and The ability of the hnRNP M4 protein to with CEA was by membrane from λZAP M4 plates and CEA or PELPK (11Toth C.A. Thomas P. Broitman S.A. Zamcheck N. Biochem. J. 1982; 204: 377-381Crossref PubMed Scopus (45) Google Scholar) in the presence of or Both CEA and PELPK to the in the presence of was binding when was These data on the of the CEA-binding protein using isolated Kupffer cells (13Zimmer R. Thomas P. Cancer Res. 2001; 61: 2822-2826PubMed Google Scholar) and further evidence that hnRNP M4 is the Kupffer cell CEA-binding protein. To further this and to the molecular of the the cDNA to rat hnRNP M4 was into a expression production of a M4 protein. A series of were for high intracellular expression of the cDNA as a protein protein The hnRNP M4 cDNA was into vector to the reading The protein was as The Kupffer cell and M4 proteins were to and to a The membrane was exposed to 1 CEA followed by an KC was used as a and the vector as a A 80-kDa was shown in Kupffer cells and in the proteins 2 and The was for the vector This data that rat hnRNP M4 protein by the cDNA is to CEA to the 80-kDa KC The molecular of the hnRNP M4 protein and an 80-kDa receptor from KC were Both rat and human were for the presence of the binding protein mRNA using A with to hnRNP M4 cDNA was The results from rat and human were analysis that the mRNA encoding the hnRNP M4 is expressed as a of in the liver, and The RNA to have a ubiquitous distribution the analysis was to between the and To the with the in a region between the and is expressed in Kupffer cells we that the region as part of their results in a for the and a for the The results that the encoding is rat The is also present in to Both mRNA forms and the were of the rat the with the was in human KC These data that are of hnRNP M4 mRNA that may by and may in To elucidate macrophage cell lines can vitro with CEA were We 3 macrophage cell and CEA uptake by isolated rat KC was used as a were with for and shows that of the macrophage cell lines was to the CEA in Earlier it was also that isolated macrophages the 80-kDa protein and rapidly CEA in a to Kupffer cells C.A. A. Zamcheck N. Steele G. Thomas P. J. 45: PubMed Scopus Google Scholar). These data that the ability of CEA uptake is cell specific and is tissue to Kupffer cells and To further the of hnRNP M4 as a CEA-binding protein and to to elucidate the mechanism the intracellular signaling with Kupffer cell the hnRNP M4cDNA in the expression vector was transfected into macrophage cell The mouse P388D1 and mouse macrophages were transfected with of hnRNP expression shows that after binding in P388D1 cells with in the transfected as with the cell A containing a of the vector not This data shows that transfection of hnRNP M4 cDNA results in CEA binding by P388D1 cells and expression of the hnRNP M4 protein on the cell only not cells were to the for of by cells are not is experimental and clinical data that an important for CEA in of metastasis from colorectal cancer J.M. Thomas P. Stanners C.P. Cell Adhesion and Communication Mediated by the CEA Family: Basic and Clinical Perspectives. Harwood Academic Publishers, Amsterdam, Netherlands1998: 195-222Google Scholar). A mechanism for this function the identification of the proteins involved in metastatic signaling through In this we two independent to identify the gene for CEA binding to Kupffer cells. The expression using an specific to the 80-kDa CEA-binding protein and the yeast two-hybrid system to identify and isolate the cDNA encoding the CEA Both techniques resulted in the isolation of a cDNA with high to the hnRNP To further study the interaction we an in vitro model that the signaling events that in the of between KC and macrophage cell which hnRNP M4 function in cell lines to the involved in the CEA receptor expression and binding. To elucidate the hnRNP M4 cDNA can CEA the macrophage cell and were transfected with the expression M4 In to our it was shown that the ability to CEA on the tissue and is to Kupffer cells and macrophages P. M. A. Biochem. J. 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This report on isolation of CEA-binding protein provides both a for further analysis of the mechanism of KC and and the clinical to expression of hnRNP M4 is involved in metastatic vitro model using of hnRNP M4 in macrophage cell lines may used to the signaling events that in in It also to signaling events and the protein to and to the in macrophages by These studies have a potential to not only our of to into new We for and reading of this We also Thomas for and with the of the