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• W2034961094 abstract "We have sought to develop methodologies to identify genes that are preferentially expressed during the differentiation of mast cells from their hematopoietic stem cell precursors. By using a modified differential display protocol, we compared a subset of transcripts expressed in bone marrow cells differentiated into immature mast cells with the exogenous addition of stem cell factor (SCF) or interleukin 3. One gene was identified that was preferentially expressed in the SCF-derived cells and encodes a novel murine integrin β subunit-like molecule, dubbed Pactolus-1 (Pactolus). Two distinct forms of Pactolus mRNA were detected which, via alternative splicing, are predicted to encode a membrane-bound form and truncated version of the protein. The full-length Pactolus gene product is very similar to a number of β subunit integrin chains, particularly β2, with the notable exceptions of the apparent deletion of the metal-binding site within the putative metal ion-dependent adhesion site-like domain of the Pactolus gene product and a cytoplasmic domain that shares no obvious homology to similar domains of the other β subunit integrin proteins. Although the Pactolus sequence was first identified in immature mast cell samples, screening of murine tissues indicated the highest level of Pactolus expression was found in the bone marrow, suggesting that the expression of Pactolus is confined to immature and maturing bone marrow-derived cells, and that the SCF-derived mast cells are more representative of this state than are the interleukin 3-derived mast cells. Immunoprecipitation of Pactolus revealed a cell-surface protein with an apparent molecular mass of about 95 kDa. Surprisingly, no associating α integrin subunit could be identified suggesting that either Pactolus does not associate with another integrin subunit or the association is too weak to be identified. These data suggest that Pactolus represents a gene and gene product related to those of the integrin β subunits but whose function(s) may be quite distinct from those of the integrin β subunits. We have sought to develop methodologies to identify genes that are preferentially expressed during the differentiation of mast cells from their hematopoietic stem cell precursors. By using a modified differential display protocol, we compared a subset of transcripts expressed in bone marrow cells differentiated into immature mast cells with the exogenous addition of stem cell factor (SCF) or interleukin 3. One gene was identified that was preferentially expressed in the SCF-derived cells and encodes a novel murine integrin β subunit-like molecule, dubbed Pactolus-1 (Pactolus). Two distinct forms of Pactolus mRNA were detected which, via alternative splicing, are predicted to encode a membrane-bound form and truncated version of the protein. The full-length Pactolus gene product is very similar to a number of β subunit integrin chains, particularly β2, with the notable exceptions of the apparent deletion of the metal-binding site within the putative metal ion-dependent adhesion site-like domain of the Pactolus gene product and a cytoplasmic domain that shares no obvious homology to similar domains of the other β subunit integrin proteins. Although the Pactolus sequence was first identified in immature mast cell samples, screening of murine tissues indicated the highest level of Pactolus expression was found in the bone marrow, suggesting that the expression of Pactolus is confined to immature and maturing bone marrow-derived cells, and that the SCF-derived mast cells are more representative of this state than are the interleukin 3-derived mast cells. Immunoprecipitation of Pactolus revealed a cell-surface protein with an apparent molecular mass of about 95 kDa. Surprisingly, no associating α integrin subunit could be identified suggesting that either Pactolus does not associate with another integrin subunit or the association is too weak to be identified. These data suggest that Pactolus represents a gene and gene product related to those of the integrin β subunits but whose function(s) may be quite distinct from those of the integrin β subunits. It has been the goal of many investigations to isolate those genes whose expression is intimately tied to hematopoietic cell differentiation and maturation (reviewed in Refs. 1Clevers H.C. Grosschedl R. Immunol. Today. 1996; 17: 336-343Abstract Full Text PDF PubMed Scopus (90) Google Scholar and 2Shivdasani R. Orkin S.H. Blood. 1996; 87: 4025-4028Crossref PubMed Google Scholar). This goal has been approached using a variety of different experimental approaches. One of the most successful techniques used to isolate cell-specific gene products has been cDNA subtraction (3Swaroop A. Xu J.Z. Agarwal N. Weissman S.M. Nucleic Acids Res. 1991; 19: 1954-1962Crossref PubMed Scopus (72) Google Scholar). This protocol has been used to identify and isolate a number of significant gene products, notably the T cell receptor genes (4Hedrick S.M. Cohen D.I. Nielson E.A. Davis M.M. Nature. 1984; 308: 149-153Crossref PubMed Scopus (877) Google Scholar, 5Yanagi Y. Yoshikai Y. Leggett K. Clark S.P. Aleksander I. Mak T.W. Nature. 1984; 308: 145-149Crossref PubMed Scopus (876) Google Scholar). There are, however, several drawbacks to the subtraction technique when it is applied to study hematopoiesis. Most conspicuously, it requires relatively large amounts of mRNA and is labor intensive. A successful subtraction normally requires several rounds of hybridization and physical dissociation. This protocol also leads to the preferential enrichment of abundant mRNA species over the rarer species.More recently, an alternative approach to subtraction was introduced in which mRNA species are randomly amplified from total RNA preparations. This technique, known as differential display (DD) 1The abbreviations used are: DD, differential display; CTMC, connective tissue mast cells; MMC, mucosal mast cells; RT-RPCR, reverse transcriptase-rapid polymerase chain reaction; PCR, polymerase chain reaction; SCF, stem cell factor; IL-3, interleukin 3; n, nucleotides; bp, base pairs; aa, amino acids; MIDAS, metal ion-dependent adhesion site. 1The abbreviations used are: DD, differential display; CTMC, connective tissue mast cells; MMC, mucosal mast cells; RT-RPCR, reverse transcriptase-rapid polymerase chain reaction; PCR, polymerase chain reaction; SCF, stem cell factor; IL-3, interleukin 3; n, nucleotides; bp, base pairs; aa, amino acids; MIDAS, metal ion-dependent adhesion site. (6Liang P. Pardee A. Science. 1992; 257: 967-971Crossref PubMed Scopus (4687) Google Scholar, 7Liang P. Averboukh L. Pardee A. Nucleic Acids Res. 1993; 21: 3269-3275Crossref PubMed Scopus (883) Google Scholar), is based on the theory that every mRNA in the cell can be amplified, via a cDNA intermediate, with a specific combination of a poly(T) containing anchoring primer and a random decamer oligonucleotide. This protocol allows for the rapid comparison of transcript species between two closely related cell types such as normal and transformed or quiescent and activated cells. By using this protocol the total complexity of transcripts within a cell can be displayed, thus achieving transcript saturation. Any transcript product that is specific for the cell type in question can, with this protocol, be identified, isolated, and sequenced. The advantages of this protocol are many and include the requirement of much less RNA, the simplicity of PCR amplification, and the ease of product resolution (6Liang P. Pardee A. Science. 1992; 257: 967-971Crossref PubMed Scopus (4687) Google Scholar, 7Liang P. Averboukh L. Pardee A. Nucleic Acids Res. 1993; 21: 3269-3275Crossref PubMed Scopus (883) Google Scholar, 8Bauer D. Muller H. Reich J. Riedel H. Ahrenkiel V. Warthoe P. Strauss M. Nucleic Acids Res. 1993; 21: 4272-4276Crossref PubMed Scopus (501) Google Scholar). The primary difficulties with DD have been the large number of false positives generated and the requirement of closely matched cell types with which to compare.Mast cells arise from the multipotent bone marrow stem cells. There are two types of tissue mast cells in the mouse (mucosal mast cells and connective tissue mast cells) that are believed to be derived from the same precursor cells. A variety of researchers have demonstrated that immature mucosal-like and connective tissue-like mast cells can be derived in vitro by culturing mouse bone marrow with either IL-3 or SCF, respectively (9Mekori Y.A. Oh C.K. Metcalfe D.D. J. Immunol. 1993; 151: 3775-3783PubMed Google Scholar, 10Levi-Schaffer Austen K.F. Gravellese P.M. Stevens R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6485-6492Crossref PubMed Scopus (247) Google Scholar, 11Tsai M. Takeishi T. Thompson H. Langley K.E. Zsebo K.M. Metcalfe D.D. Geissler E.N. Galli S.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6382Crossref PubMed Scopus (375) Google Scholar, 12Gurish M.F. Ghildyal N. McNeil H.P. Austen K.F. Gillis S. Stevens R.L. J. Exp. Med. 1992; 175: 1003-1012Crossref PubMed Scopus (167) Google Scholar). The molecular events that drive the differentiation of these two cell types have remained an enigma despite the recent characterization of mast cell progenitor cells (13Rodewald H.R. Dessing M. Dvorak A.M. Galli S.J. Science. 1996; 271: 818-821Crossref PubMed Scopus (300) Google Scholar). Since these two cell types are very similar to one another and can, given the correct circumstances, shift their phenotype back and forth, they are ideal candidates for the utilization of DD to identify those gene products specific for each cell phenotype.In this work, we describe a modification of the original differential display protocol designed to identify transcripts implicated in bone marrow maturation. This protocol not only allows for a more rapid progression of the protocol but also appears to increase the specificity of the products such that the generation of false positives is greatly diminished. By using this protocol we identified a transcript preferentially expressed in SCF-derived mast cells but absent in those derived with IL-3. The gene fragment identified in this protocol was used to screen a mast cell cDNA library from which an apparent full-length transcript was obtained. This cDNA predicts a novel murine integrin β subunit-like molecule, Pactolus, that, via alternative splicing, would be expected to produce both a membrane-bound and truncated form. The expression of this gene is most pronounced in the murine bone marrow. The expression of Pactolus in cells derived in SCF but not the IL-3 cells suggests that the SCF-derived cells may represent a more immature mast cell type than those derived in IL-3 culture.DISCUSSIONIn this report, we describe the isolation of a novel murine gene (Pactolus) that shares a high degree of homology with the family of adhesion molecules known as the integrin β subunits. The Pactolus sequence was obtained from a differential display (DD) analysis of murine bone marrow-derived mast cells in which transcripts derived from cells cultured in IL-3 were compared with those derived in SCF. Based upon our data and data from others (11Tsai M. Takeishi T. Thompson H. Langley K.E. Zsebo K.M. Metcalfe D.D. Geissler E.N. Galli S.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6382Crossref PubMed Scopus (375) Google Scholar, 14Marietta E.V. Chen Y. Weis J.H. Eur. J. Immunol. 1996; 26: 49-56Crossref PubMed Scopus (40) Google Scholar, 32Ducharme L. Weis J.H. Eur. J. Immunol. 1992; 22: 2603-2608Crossref PubMed Scopus (37) Google Scholar), those cells derived solely in IL-3 possess a phenotype associated with mast cells found in the intestinal mucosa, whereas those derived in SCF possess characteristics of mast cells found in the skin and peritoneal cavity of the animal. Following this cell culture rationale and utilizing a modified DD protocol, we isolated a novel sequence from bone marrow cells cultured in SCF which was lacking, as an apparent transcript, from those cells cultured in IL-3. Subsequent cloning and sequence analysis indicated this novel gene transcript was very similar to the members of the integrin β subunit gene family.The protein predicted by the Pactolus sequence is related to the integrin β subunits but is clearly divergent in two important domains. The first of these is within the proposed I (inserted) domain-like structure (also termed A for activation domain) which is also present within the majority of the α integrin subunits (reviewed in Ref. 33Humpries M.J. Curr. Opin. Cell Biol. 1996; 8: 632-640Crossref PubMed Scopus (203) Google Scholar). This region resides at the amino terminus of the protein (thus extracellular) and is implicated in ligand binding, heteroduplex formation, and metal ion binding. Single amino acid mutations in this site abrogate stable expression of the integrin heterodimers and block ligand binding. Structural analysis of the I domain of the CD11b chain of the CR3 integrin complex suggested the presence of a MIDAS motif (metal ion-dependent adhesion site) that was formed in a three-dimensional fold utilizing a conserved DXSXS-(65 amino acids)-T-(25 amino acids)-D sequence, where the X residues and spacer amino acids are not conserved (26Lee J.-O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (798) Google Scholar). The analagous sequence present in Pactolus differs from that of the α and β integrin subunits. Not only does Pactolus lack the amino-terminal Asp residue, but the spacing between the Ser and Thr residues, which presumably is critical for the ternary folds of the protein, is 21 amino acids shorter than the consensus sequence. Of the more than 40 gene sequences that possess the proposed MIDAS motif, the shortest distance between the analogous Ser and Thr residues is 61 amino acids, compared with 44 for Pactolus. This deletion would, with respect to the proposed MIDAS motif structure, apparently delete the α2,α3 helices, thus placing the Thr residue in opposite orientation to the Mg2+ ion within the MIDAS structure. These two alterations in this region of Pactolus suggest that this site may not be functionally similar to those of the integrin β subunits.The other site of high homology within the integrin β subunit family is the cytoplasmic domain. Sequences have been defined in the cytoplasmic tail of these subunits that are implicated in the binding of the β chain to cytosolic proteins including α-actinin, talin, paxillin, and others (reviewed in Ref. 34Dedhar S. Hannigan G.E. Curr. Opin. Cell Biol. 1996; 8: 657-669Crossref PubMed Scopus (344) Google Scholar). Pactolus is lacking these conserved residues in its proposed cytoplasmic domain suggesting it may bind to a different set of cytoplasmic proteins than those described for the integrin β subunits.The Pactolus gene transcripts also differ from members of the integrin β subunit family in predicting two distinct forms of the protein, plus or minus the transmembrane and cytoplasmic domains. Attempts to generate an antisera specific for the truncated form of the Pactulus protein have so far been unsuccessful; thus we cannot conclude whether the protein produced by the truncated transcript is stably expressed in mammalian cells. Some integrin β subunits utilize alternative splicing to produce variant isoforms of the proteins. In particular, at least four distinct alternative cytoplasmic domains have been described for integrin subunit β1 which alter the functional characteristics of the protein (35Zhidkova N.I. Blkin A.M. Mayne R. Biochem. Biophys. Res. Commun. 1995; 214: 279-285Crossref PubMed Scopus (80) Google Scholar, 36Atruda F. Cervella P. Tarone G. Botta C. Balzac F. Stefunato G. Silengo L. Gene (Amst .). 1990; 95: 261-266Crossref PubMed Scopus (84) Google Scholar, 37Languino L.R. Ruoslahiti E. J. Biol. Chem. 1992; 267: 7116-7120Abstract Full Text PDF PubMed Google Scholar, 38Meridith Jr., J. Takada Y. Fornaro M. Languino L.R. Schwartz M.A. Science. 1995; 269: 1570-1572Crossref PubMed Scopus (117) Google Scholar). However, we do not know of any β integrin subunit that would, like Pactolus, predict a secreted form of the protein.The expression of the Pactolus gene appears to be limited to immature cells of bone marrow derivation. The murine tissue demonstrating the highest level of expression is the mouse marrow. Based upon β-actin transcript levels, the quantity of Pactolus transcripts in the splenic sample was 10% or less than that of the bone marrow. Since the spleen is primarily populated by mature cells of bone marrow origin (B cells, T cells, and macrophages), the absence of appreciable Pactolus transcripts in the spleen may suggest the down-regulation of this gene during cellular maturation.Two key findings were provided in the analysis of the Pactolus protein. First, the protein is expressed on the surface of the bone marrow cells with an apparent molecule mass of 95 kDa. Second, we cannot detect any significant association of Pactolus with any other cell-surface protein, as might be expected if Pactolus was to function as a β integrin subunit. Based upon the sequence of the Pactolus gene product and the lack of an apparent heterodimer complex, it is likely that Pactolus does not function as the typical β integrin subunit despite its sequence homology with β2. Therefore placing Pactolus within the integrin gene/protein family would imply a functionality of the protein that it may not possess.The major question left open by this study is the function of Pactolus. Its expression pattern suggests it is expressed by immature and maturing cells of bone marrow derivation. Its similarity in structure to the integrin β subunit gene family suggests it may be a receptor mediating adhesion of such cells within the marrow stroma. Previously the α4 integrin has been shown to be critical in marrow maintenance (39Arroyo A.G. Yang J.T. Rayburn H. Hynes R.O. Cell. 1996; 85: 997-1008Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 40Papayannopoulou T. Nakamoto B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9374-9378Crossref PubMed Scopus (415) Google Scholar). Alternatively, Pactolus may be playing a signaling role for cells within the marrow. For example, one of the alternative cytoplasmic domains for the integrin β1 subunit (β1c) acts to directly inhibit cell cycle progression (38Meridith Jr., J. Takada Y. Fornaro M. Languino L.R. Schwartz M.A. Science. 1995; 269: 1570-1572Crossref PubMed Scopus (117) Google Scholar). The marrow consists of many cell types, some of which are held in a state of low replicative activity. The two different forms of the Pactolus protein may, upon ligation with ligand, send two quite distinct signals into the cell. This model might be especially appropriate if a modulation of splicing between the two forms is evident during bone marrow cell maturation and if we can detect the stable expression of the truncated form of the Pactolus protein. It has been the goal of many investigations to isolate those genes whose expression is intimately tied to hematopoietic cell differentiation and maturation (reviewed in Refs. 1Clevers H.C. Grosschedl R. Immunol. Today. 1996; 17: 336-343Abstract Full Text PDF PubMed Scopus (90) Google Scholar and 2Shivdasani R. Orkin S.H. Blood. 1996; 87: 4025-4028Crossref PubMed Google Scholar). This goal has been approached using a variety of different experimental approaches. One of the most successful techniques used to isolate cell-specific gene products has been cDNA subtraction (3Swaroop A. Xu J.Z. Agarwal N. Weissman S.M. Nucleic Acids Res. 1991; 19: 1954-1962Crossref PubMed Scopus (72) Google Scholar). This protocol has been used to identify and isolate a number of significant gene products, notably the T cell receptor genes (4Hedrick S.M. Cohen D.I. Nielson E.A. Davis M.M. Nature. 1984; 308: 149-153Crossref PubMed Scopus (877) Google Scholar, 5Yanagi Y. Yoshikai Y. Leggett K. Clark S.P. Aleksander I. Mak T.W. Nature. 1984; 308: 145-149Crossref PubMed Scopus (876) Google Scholar). There are, however, several drawbacks to the subtraction technique when it is applied to study hematopoiesis. Most conspicuously, it requires relatively large amounts of mRNA and is labor intensive. A successful subtraction normally requires several rounds of hybridization and physical dissociation. This protocol also leads to the preferential enrichment of abundant mRNA species over the rarer species. More recently, an alternative approach to subtraction was introduced in which mRNA species are randomly amplified from total RNA preparations. This technique, known as differential display (DD) 1The abbreviations used are: DD, differential display; CTMC, connective tissue mast cells; MMC, mucosal mast cells; RT-RPCR, reverse transcriptase-rapid polymerase chain reaction; PCR, polymerase chain reaction; SCF, stem cell factor; IL-3, interleukin 3; n, nucleotides; bp, base pairs; aa, amino acids; MIDAS, metal ion-dependent adhesion site. 1The abbreviations used are: DD, differential display; CTMC, connective tissue mast cells; MMC, mucosal mast cells; RT-RPCR, reverse transcriptase-rapid polymerase chain reaction; PCR, polymerase chain reaction; SCF, stem cell factor; IL-3, interleukin 3; n, nucleotides; bp, base pairs; aa, amino acids; MIDAS, metal ion-dependent adhesion site. (6Liang P. Pardee A. Science. 1992; 257: 967-971Crossref PubMed Scopus (4687) Google Scholar, 7Liang P. Averboukh L. Pardee A. Nucleic Acids Res. 1993; 21: 3269-3275Crossref PubMed Scopus (883) Google Scholar), is based on the theory that every mRNA in the cell can be amplified, via a cDNA intermediate, with a specific combination of a poly(T) containing anchoring primer and a random decamer oligonucleotide. This protocol allows for the rapid comparison of transcript species between two closely related cell types such as normal and transformed or quiescent and activated cells. By using this protocol the total complexity of transcripts within a cell can be displayed, thus achieving transcript saturation. Any transcript product that is specific for the cell type in question can, with this protocol, be identified, isolated, and sequenced. The advantages of this protocol are many and include the requirement of much less RNA, the simplicity of PCR amplification, and the ease of product resolution (6Liang P. Pardee A. Science. 1992; 257: 967-971Crossref PubMed Scopus (4687) Google Scholar, 7Liang P. Averboukh L. Pardee A. Nucleic Acids Res. 1993; 21: 3269-3275Crossref PubMed Scopus (883) Google Scholar, 8Bauer D. Muller H. Reich J. Riedel H. Ahrenkiel V. Warthoe P. Strauss M. Nucleic Acids Res. 1993; 21: 4272-4276Crossref PubMed Scopus (501) Google Scholar). The primary difficulties with DD have been the large number of false positives generated and the requirement of closely matched cell types with which to compare. Mast cells arise from the multipotent bone marrow stem cells. There are two types of tissue mast cells in the mouse (mucosal mast cells and connective tissue mast cells) that are believed to be derived from the same precursor cells. A variety of researchers have demonstrated that immature mucosal-like and connective tissue-like mast cells can be derived in vitro by culturing mouse bone marrow with either IL-3 or SCF, respectively (9Mekori Y.A. Oh C.K. Metcalfe D.D. J. Immunol. 1993; 151: 3775-3783PubMed Google Scholar, 10Levi-Schaffer Austen K.F. Gravellese P.M. Stevens R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6485-6492Crossref PubMed Scopus (247) Google Scholar, 11Tsai M. Takeishi T. Thompson H. Langley K.E. Zsebo K.M. Metcalfe D.D. Geissler E.N. Galli S.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6382Crossref PubMed Scopus (375) Google Scholar, 12Gurish M.F. Ghildyal N. McNeil H.P. Austen K.F. Gillis S. Stevens R.L. J. Exp. Med. 1992; 175: 1003-1012Crossref PubMed Scopus (167) Google Scholar). The molecular events that drive the differentiation of these two cell types have remained an enigma despite the recent characterization of mast cell progenitor cells (13Rodewald H.R. Dessing M. Dvorak A.M. Galli S.J. Science. 1996; 271: 818-821Crossref PubMed Scopus (300) Google Scholar). Since these two cell types are very similar to one another and can, given the correct circumstances, shift their phenotype back and forth, they are ideal candidates for the utilization of DD to identify those gene products specific for each cell phenotype. In this work, we describe a modification of the original differential display protocol designed to identify transcripts implicated in bone marrow maturation. This protocol not only allows for a more rapid progression of the protocol but also appears to increase the specificity of the products such that the generation of false positives is greatly diminished. By using this protocol we identified a transcript preferentially expressed in SCF-derived mast cells but absent in those derived with IL-3. The gene fragment identified in this protocol was used to screen a mast cell cDNA library from which an apparent full-length transcript was obtained. This cDNA predicts a novel murine integrin β subunit-like molecule, Pactolus, that, via alternative splicing, would be expected to produce both a membrane-bound and truncated form. The expression of this gene is most pronounced in the murine bone marrow. The expression of Pactolus in cells derived in SCF but not the IL-3 cells suggests that the SCF-derived cells may represent a more immature mast cell type than those derived in IL-3 culture. DISCUSSIONIn this report, we describe the isolation of a novel murine gene (Pactolus) that shares a high degree of homology with the family of adhesion molecules known as the integrin β subunits. The Pactolus sequence was obtained from a differential display (DD) analysis of murine bone marrow-derived mast cells in which transcripts derived from cells cultured in IL-3 were compared with those derived in SCF. Based upon our data and data from others (11Tsai M. Takeishi T. Thompson H. Langley K.E. Zsebo K.M. Metcalfe D.D. Geissler E.N. Galli S.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6382Crossref PubMed Scopus (375) Google Scholar, 14Marietta E.V. Chen Y. Weis J.H. Eur. J. Immunol. 1996; 26: 49-56Crossref PubMed Scopus (40) Google Scholar, 32Ducharme L. Weis J.H. Eur. J. Immunol. 1992; 22: 2603-2608Crossref PubMed Scopus (37) Google Scholar), those cells derived solely in IL-3 possess a phenotype associated with mast cells found in the intestinal mucosa, whereas those derived in SCF possess characteristics of mast cells found in the skin and peritoneal cavity of the animal. Following this cell culture rationale and utilizing a modified DD protocol, we isolated a novel sequence from bone marrow cells cultured in SCF which was lacking, as an apparent transcript, from those cells cultured in IL-3. Subsequent cloning and sequence analysis indicated this novel gene transcript was very similar to the members of the integrin β subunit gene family.The protein predicted by the Pactolus sequence is related to the integrin β subunits but is clearly divergent in two important domains. The first of these is within the proposed I (inserted) domain-like structure (also termed A for activation domain) which is also present within the majority of the α integrin subunits (reviewed in Ref. 33Humpries M.J. Curr. Opin. Cell Biol. 1996; 8: 632-640Crossref PubMed Scopus (203) Google Scholar). This region resides at the amino terminus of the protein (thus extracellular) and is implicated in ligand binding, heteroduplex formation, and metal ion binding. Single amino acid mutations in this site abrogate stable expression of the integrin heterodimers and block ligand binding. Structural analysis of the I domain of the CD11b chain of the CR3 integrin complex suggested the presence of a MIDAS motif (metal ion-dependent adhesion site) that was formed in a three-dimensional fold utilizing a conserved DXSXS-(65 amino acids)-T-(25 amino acids)-D sequence, where the X residues and spacer amino acids are not conserved (26Lee J.-O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (798) Google Scholar). The analagous sequence present in Pactolus differs from that of the α and β integrin subunits. Not only does Pactolus lack the amino-terminal Asp residue, but the spacing between the Ser and Thr residues, which presumably is critical for the ternary folds of the protein, is 21 amino acids shorter than the consensus sequence. Of the more than 40 gene sequences that possess the proposed MIDAS motif, the shortest distance between the analogous Ser and Thr residues is 61 amino acids, compared with 44 for Pactolus. This deletion would, with respect to the proposed MIDAS motif structure, apparently delete the α2,α3 helices, thus placing the Thr residue in opposite orientation to the Mg2+ ion within the MIDAS structure. These two alterations in this region of Pactolus suggest that this site may not be functionally similar to those of the integrin β subunits.The other site of high homology within the integrin β subunit family is the cytoplasmic domain. Sequences have been defined in the cytoplasmic tail of these subunits that are implicated in the binding of the β chain to cytosolic proteins including α-actinin, talin, paxillin, and others (reviewed in Ref. 34Dedhar S. Hannigan G.E. Curr. Opin. Cell Biol. 1996; 8: 657-669Crossref PubMed Scopus (344) Google Scholar). Pactolus is lacking these conserved residues in its proposed cytoplasmic domain suggesting it may bind to a different set of cytoplasmic proteins than those described for the integrin β subunits.The Pactolus gene transcripts also differ from members of the integrin β subunit family in predicting two distinct forms of the protein, plus or minus the transmembrane and cytoplasmic domains. Attempts to generate an antisera specific for the truncated form of the Pactulus protein have so far been unsuccessful; thus we cannot conclude whether the protein produced by the truncated transcript is stably expressed in mammalian cells. Some integrin β subunits utilize alternative splicing to produce variant isoforms of the proteins. In particular, at least four distinct alternative cytoplasmic domains have been described for integrin subunit β1 which alter the functional characteristics of the protein (35Zhidkova N.I. Blkin A.M. Mayne R. Biochem. Biophys. Res. Commun. 1995; 214: 279-285Crossref PubMed Scopus (80) Google Scholar, 36Atruda F. Cervella P. Tarone G. Botta C. Balzac F. Stefunato G. Silengo L. Gene (Amst .). 1990; 95: 261-266Crossref PubMed Scopus (84) Google Scholar, 37Languino L.R. Ruoslahiti E. J. Biol. Chem. 1992; 267: 7116-7120Abstract Full Text PDF PubMed Google Scholar, 38Meridith Jr., J. Takada Y. Fornaro M. Languino L.R. Schwartz M.A. Science. 1995; 269: 1570-1572Crossref PubMed Scopus (117) Google Scholar). However, we do not know of any β integrin subunit that would, like Pactolus, predict a secreted form of the protein.The expression of the Pactolus gene appears to be limited to immature cells of bone marrow derivation. The murine tissue demonstrating the highest level of expression is the mouse marrow. Based upon β-actin transcript levels, the quantity of Pactolus transcripts in the splenic sample was 10% or less than that of the bone marrow. Since the spleen is primarily populated by mature cells of bone marrow origin (B cells, T cells, and macrophages), the absence of appreciable Pactolus transcripts in the spleen may suggest the down-regulation of this gene during cellular maturation.Two key findings were provided in the analysis of the Pactolus protein. First, the protein is expressed on the surface of the bone marrow cells with an apparent molecule mass of 95 kDa. Second, we cannot detect any significant association of Pactolus with any other cell-surface protein, as might be expected if Pactolus was to function as a β integrin subunit. Based upon the sequence of the Pactolus gene product and the lack of an apparent heterodimer complex, it is likely that Pactolus does not function as the typical β integrin subunit despite its sequence homology with β2. Therefore placing Pactolus within the integrin gene/protein family would imply a functionality of the protein that it may not possess.The major question left open by this study is the function of Pactolus. Its expression pattern suggests it is expressed by immature and maturing cells of bone marrow derivation. Its similarity in structure to the integrin β subunit gene family suggests it may be a receptor mediating adhesion of such cells within the marrow stroma. Previously the α4 integrin has been shown to be critical in marrow maintenance (39Arroyo A.G. Yang J.T. Rayburn H. Hynes R.O. Cell. 1996; 85: 997-1008Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 40Papayannopoulou T. Nakamoto B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9374-9378Crossref PubMed Scopus (415) Google Scholar). Alternatively, Pactolus may be playing a signaling role for cells within the marrow. For example, one of the alternative cytoplasmic domains for the integrin β1 subunit (β1c) acts to directly inhibit cell cycle progression (38Meridith Jr., J. Takada Y. Fornaro M. Languino L.R. Schwartz M.A. Science. 1995; 269: 1570-1572Crossref PubMed Scopus (117) Google Scholar). The marrow consists of many cell types, some of which are held in a state of low replicative activity. The two different forms of the Pactolus protein may, upon ligation with ligand, send two quite distinct signals into the cell. This model might be especially appropriate if a modulation of splicing between the two forms is evident during bone marrow cell maturation and if we can detect the stable expression of the truncated form of the Pactolus protein. In this report, we describe the isolation of a novel murine gene (Pactolus) that shares a high degree of homology with the family of adhesion molecules known as the integrin β subunits. The Pactolus sequence was obtained from a differential display (DD) analysis of murine bone marrow-derived mast cells in which transcripts derived from cells cultured in IL-3 were compared with those derived in SCF. Based upon our data and data from others (11Tsai M. Takeishi T. Thompson H. Langley K.E. Zsebo K.M. Metcalfe D.D. Geissler E.N. Galli S.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6382Crossref PubMed Scopus (375) Google Scholar, 14Marietta E.V. Chen Y. Weis J.H. Eur. J. Immunol. 1996; 26: 49-56Crossref PubMed Scopus (40) Google Scholar, 32Ducharme L. Weis J.H. Eur. J. Immunol. 1992; 22: 2603-2608Crossref PubMed Scopus (37) Google Scholar), those cells derived solely in IL-3 possess a phenotype associated with mast cells found in the intestinal mucosa, whereas those derived in SCF possess characteristics of mast cells found in the skin and peritoneal cavity of the animal. Following this cell culture rationale and utilizing a modified DD protocol, we isolated a novel sequence from bone marrow cells cultured in SCF which was lacking, as an apparent transcript, from those cells cultured in IL-3. Subsequent cloning and sequence analysis indicated this novel gene transcript was very similar to the members of the integrin β subunit gene family. The protein predicted by the Pactolus sequence is related to the integrin β subunits but is clearly divergent in two important domains. The first of these is within the proposed I (inserted) domain-like structure (also termed A for activation domain) which is also present within the majority of the α integrin subunits (reviewed in Ref. 33Humpries M.J. Curr. Opin. Cell Biol. 1996; 8: 632-640Crossref PubMed Scopus (203) Google Scholar). This region resides at the amino terminus of the protein (thus extracellular) and is implicated in ligand binding, heteroduplex formation, and metal ion binding. Single amino acid mutations in this site abrogate stable expression of the integrin heterodimers and block ligand binding. Structural analysis of the I domain of the CD11b chain of the CR3 integrin complex suggested the presence of a MIDAS motif (metal ion-dependent adhesion site) that was formed in a three-dimensional fold utilizing a conserved DXSXS-(65 amino acids)-T-(25 amino acids)-D sequence, where the X residues and spacer amino acids are not conserved (26Lee J.-O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (798) Google Scholar). The analagous sequence present in Pactolus differs from that of the α and β integrin subunits. Not only does Pactolus lack the amino-terminal Asp residue, but the spacing between the Ser and Thr residues, which presumably is critical for the ternary folds of the protein, is 21 amino acids shorter than the consensus sequence. Of the more than 40 gene sequences that possess the proposed MIDAS motif, the shortest distance between the analogous Ser and Thr residues is 61 amino acids, compared with 44 for Pactolus. This deletion would, with respect to the proposed MIDAS motif structure, apparently delete the α2,α3 helices, thus placing the Thr residue in opposite orientation to the Mg2+ ion within the MIDAS structure. These two alterations in this region of Pactolus suggest that this site may not be functionally similar to those of the integrin β subunits. The other site of high homology within the integrin β subunit family is the cytoplasmic domain. Sequences have been defined in the cytoplasmic tail of these subunits that are implicated in the binding of the β chain to cytosolic proteins including α-actinin, talin, paxillin, and others (reviewed in Ref. 34Dedhar S. Hannigan G.E. Curr. Opin. Cell Biol. 1996; 8: 657-669Crossref PubMed Scopus (344) Google Scholar). Pactolus is lacking these conserved residues in its proposed cytoplasmic domain suggesting it may bind to a different set of cytoplasmic proteins than those described for the integrin β subunits. The Pactolus gene transcripts also differ from members of the integrin β subunit family in predicting two distinct forms of the protein, plus or minus the transmembrane and cytoplasmic domains. Attempts to generate an antisera specific for the truncated form of the Pactulus protein have so far been unsuccessful; thus we cannot conclude whether the protein produced by the truncated transcript is stably expressed in mammalian cells. Some integrin β subunits utilize alternative splicing to produce variant isoforms of the proteins. In particular, at least four distinct alternative cytoplasmic domains have been described for integrin subunit β1 which alter the functional characteristics of the protein (35Zhidkova N.I. Blkin A.M. Mayne R. Biochem. Biophys. Res. Commun. 1995; 214: 279-285Crossref PubMed Scopus (80) Google Scholar, 36Atruda F. Cervella P. Tarone G. Botta C. Balzac F. Stefunato G. Silengo L. Gene (Amst .). 1990; 95: 261-266Crossref PubMed Scopus (84) Google Scholar, 37Languino L.R. Ruoslahiti E. J. Biol. Chem. 1992; 267: 7116-7120Abstract Full Text PDF PubMed Google Scholar, 38Meridith Jr., J. Takada Y. Fornaro M. Languino L.R. Schwartz M.A. Science. 1995; 269: 1570-1572Crossref PubMed Scopus (117) Google Scholar). However, we do not know of any β integrin subunit that would, like Pactolus, predict a secreted form of the protein. The expression of the Pactolus gene appears to be limited to immature cells of bone marrow derivation. The murine tissue demonstrating the highest level of expression is the mouse marrow. Based upon β-actin transcript levels, the quantity of Pactolus transcripts in the splenic sample was 10% or less than that of the bone marrow. Since the spleen is primarily populated by mature cells of bone marrow origin (B cells, T cells, and macrophages), the absence of appreciable Pactolus transcripts in the spleen may suggest the down-regulation of this gene during cellular maturation. Two key findings were provided in the analysis of the Pactolus protein. First, the protein is expressed on the surface of the bone marrow cells with an apparent molecule mass of 95 kDa. Second, we cannot detect any significant association of Pactolus with any other cell-surface protein, as might be expected if Pactolus was to function as a β integrin subunit. Based upon the sequence of the Pactolus gene product and the lack of an apparent heterodimer complex, it is likely that Pactolus does not function as the typical β integrin subunit despite its sequence homology with β2. Therefore placing Pactolus within the integrin gene/protein family would imply a functionality of the protein that it may not possess. The major question left open by this study is the function of Pactolus. Its expression pattern suggests it is expressed by immature and maturing cells of bone marrow derivation. Its similarity in structure to the integrin β subunit gene family suggests it may be a receptor mediating adhesion of such cells within the marrow stroma. Previously the α4 integrin has been shown to be critical in marrow maintenance (39Arroyo A.G. Yang J.T. Rayburn H. Hynes R.O. Cell. 1996; 85: 997-1008Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 40Papayannopoulou T. Nakamoto B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9374-9378Crossref PubMed Scopus (415) Google Scholar). Alternatively, Pactolus may be playing a signaling role for cells within the marrow. For example, one of the alternative cytoplasmic domains for the integrin β1 subunit (β1c) acts to directly inhibit cell cycle progression (38Meridith Jr., J. Takada Y. Fornaro M. Languino L.R. Schwartz M.A. Science. 1995; 269: 1570-1572Crossref PubMed Scopus (117) Google Scholar). The marrow consists of many cell types, some of which are held in a state of low replicative activity. The two different forms of the Pactolus protein may, upon ligation with ligand, send two quite distinct signals into the cell. This model might be especially appropriate if a modulation of splicing between the two forms is evident during bone marrow cell maturation and if we can detect the stable expression of the truncated form of the Pactolus protein. We thank Ina Karbegovic for excellent technical assistance and the preparation of the mast cell cultures. We also thank Dr. Eric Kofoid for the T overhang cloning vector and Dr. David Stillman for a clean bench in his lab. We would also like to thank all the members of the Weis laboratories for their valuable comments and discussions." @default.
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• W2034961094 title "Identification of Pactolus, an Integrin β Subunit-like Cell-surface Protein Preferentially Expressed by Cells of the Bone Marrow" @default.
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