G6b, a Novel Immunoglobulin Superfamily Member Encoded in the Human Major Histocompatibility Complex, Interacts with SHP-1 and SHP-2

The G6b gene, located in the class III region of the human major histocompatibility complex, has been suggested to encode a putative receptor of the immunoglobulin superfamily. Genomic sequence information was used as a starting point to clone the corresponding cDNA. Reverse transcriptase polymerase chain reaction showed that expression of the gene is restricted to certain hematopoietic cell lines including K562, Molt 4, and Jurkat. Several splice variants were detected, varying only in their C-terminal parts. One of the potential membrane-bound isoforms contained two immunoreceptor tyrosine-based inhibitory motifs in its cytoplasmic tail. Four of the isoforms were expressed as epitope-tagged proteins in the cell lines K562 and COS-7. The two splice isoforms lacking the hydrophobic transmembrane segment were secreted from the cell. Glycosidase treatment of the four recombinant proteins provided evidence for N- and O-glycosylation. Immunofluorescence studies indicated that the spliced isoforms having a transmembrane segment were directed to the cell membrane. The G6b isoform containing two immunoreceptor tyrosine-based inhibitory motifs in its cytoplasmic tail was found to be phosphorylated on tyrosine residues after pervanadate treatment of cells and, subsequently, interacts with the SH2-containing protein-tyrosine phosphatases SHP-1 and SHP-2. Mutagenesis studies showed that phosphorylation of tyrosine 211 is critical for the interaction of G6b with SHP-1 and SHP-2. The G6b gene, located in the class III region of the human major histocompatibility complex, has been suggested to encode a putative receptor of the immunoglobulin superfamily. Genomic sequence information was used as a starting point to clone the corresponding cDNA. Reverse transcriptase polymerase chain reaction showed that expression of the gene is restricted to certain hematopoietic cell lines including K562, Molt 4, and Jurkat. Several splice variants were detected, varying only in their C-terminal parts. One of the potential membrane-bound isoforms contained two immunoreceptor tyrosine-based inhibitory motifs in its cytoplasmic tail. Four of the isoforms were expressed as epitope-tagged proteins in the cell lines K562 and COS-7. The two splice isoforms lacking the hydrophobic transmembrane segment were secreted from the cell. Glycosidase treatment of the four recombinant proteins provided evidence for N- and O-glycosylation. Immunofluorescence studies indicated that the spliced isoforms having a transmembrane segment were directed to the cell membrane. The G6b isoform containing two immunoreceptor tyrosine-based inhibitory motifs in its cytoplasmic tail was found to be phosphorylated on tyrosine residues after pervanadate treatment of cells and, subsequently, interacts with the SH2-containing protein-tyrosine phosphatases SHP-1 and SHP-2. Mutagenesis studies showed that phosphorylation of tyrosine 211 is critical for the interaction of G6b with SHP-1 and SHP-2. immunoreceptor tyrosine-based inhibitory motif Src homology 2 domain containing protein-tyrosine phosphatase major histocompatibility complex reverse transcriptase polymerase chain reaction polyacrylamide gel electrophoresis nucleotide(s) monoclonal antibodies The Ig superfamily receptors constitute a large group of cell surface proteins involved in the immune system and cellular recognition (1Hunkapiller T. Hood L. Adv. Immunol. 1989; 44: 1-63Crossref PubMed Scopus (224) Google Scholar, 2Williams A.F. Barclay A.N. Annu. Rev. Immunol. 1988; 6: 381-405Crossref PubMed Scopus (1786) Google Scholar). Members of this family are characterized by an extracellular part containing at least one immunoglobulin domain, a transmembrane segment, and a cytoplasmic tail. A subset of the Ig superfamily is the inhibitory receptor characterized by the presence of one or more immunoreceptor tyrosine-based inhibitory motifs (ITIM)1 in their cytoplasmic tail. The consensus sequence of the ITIM is generally described as (L/V/I/S/T)XY XX(L/V) (3Vivier E. Daeron M. Immunol. Today. 1997; 18: 286-291Abstract Full Text PDF PubMed Scopus (328) Google Scholar). Following phosphorylation of the tyrosine residue within this motif, the SH2 domain containing protein-tyrosine phosphatases SHP-1 and/or SHP-2 can be recruited to the receptor, where they can dephosphorylate membrane-bound phosphoproteins, thus modulating the signaling cascade. SHP-1 is a non-transmembrane protein primarily expressed in hematopoietic cells and is considered to play a negative role in cell signaling (4Zhang J. Somani A.K. Siminovitch K.A. Semin. Immunol. 2000; 12: 361-378Crossref PubMed Scopus (290) Google Scholar). Identified substrates for SHP-1 are the linker of activated T-cells (5Valiante N.M. Phillips J.H. Lanier L.L. Parham P. J. Exp. Med. 1996; 184: 2243-2250Crossref PubMed Scopus (107) Google Scholar, 6Weber J.R. Orstavik S. Torgersen K.M. Danbolt N.C. Berg S.F. Ryan J.C. Tasken K. Imboden J.B. Vaage J.T. J. Exp. Med. 1998; 187: 1157-1161Crossref PubMed Scopus (128) Google Scholar) and the adapter protein Slp-76 (7Binstadt B.A. Billadeau D.D. Jevremovic D. Williams B.L. Fang N. Yi T. Koretzky G.A. Abraham R.T. Leibson P.J. J. Biol. Chem. 1998; 273: 27518-27523Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). In contrast, SHP-2 has been considered to act primarily as a positive signal transducer (8Feng G.S. Exp. Cell Res. 1999; 253: 47-54Crossref PubMed Scopus (252) Google Scholar). Possible substrates for this phosphatase are the platelet-derived growth factor beta receptor (9Klinghoffer R.A. Kazlauskas A. J. Biol. Chem. 1995; 270: 22208-22217Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) and PZR (10Zhao Z.J. Zhao R. J. Biol. Chem. 1998; 273: 29367-29372Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). SHP-2 modulates the signal strength of receptor-protein-tyrosine kinases and is also involved in cytokine and antigen signaling not involving receptors with intrinsic kinase activity (11Streuli M. Curr. Opin. Cell Biol. 1996; 8: 182-188Crossref PubMed Scopus (165) Google Scholar). Although the two phosphatases appear to have opposing roles, there are examples of ITIM-containing receptors that recruit both SHP-1 and SHP-2 (12Fournier N. Chalus L. Durand I. Garcia E. Pin J.J. Churakova T. Patel S. Zlot C. Gorman D. Zurawski S. Abrams J. Bates E.E. Garrone P. J. Immunol. 2000; 165: 1197-1209Crossref PubMed Scopus (86) Google Scholar, 13Cantoni C. Bottino C. Augugliaro R. Morelli L. Marcenaro E. Castriconi R. Vitale M. Pende D. Sivori S. Millo R. Biassoni R. Moretta L. Moretta A. Eur. J. Immunol. 1999; 29: 3148-3159Crossref PubMed Scopus (132) Google Scholar). Other receptors are reported to recruit primarily only one form (14Falco M. Biassoni R. Bottino C. Vitale M. Sivori S. Augugliaro R. Moretta L. Moretta A. J. Exp. Med. 1999; 190: 793-802Crossref PubMed Scopus (199) Google Scholar,15Xu M. Zhao R. Zhao Z.J. J. Biol. Chem. 2000; 275: 17440-17446Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Both phosphatases SHP-1 and SHP-2 contain two SH2 domains and a catalytic domain (16Ahmad S. Banville D. Zhao Z. Fischer E.H. Shen S.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2197-2201Crossref PubMed Scopus (196) Google Scholar, 17Shen S.H. Bastien L. Posner B.I. Chretien P. Nature. 1991; 352: 736-739Crossref PubMed Scopus (337) Google Scholar). The human major histocompatibility complex (MHC) is located on chromosome band 6p21.3 and spans ∼3.6 megabases of DNA. The complete sequence of this region has been determined (18The MHC Sequencing ConsortiumNature. 1999; 401: 921-923Crossref PubMed Scopus (926) Google Scholar). The central MHC region is termed the class III region and comprises 0.8 megabases. Of the 59 genes in this region, 40% are known or predicted to have a role in the immune system or inflammation, such as tumor necrosis factor, lymphotoxin-α, and lymphotoxin-β. Susceptibility to a wide range of diseases has been linked to the MHC, including insulin-dependent diabetes mellitus (19Todd J.A. Pathol. Biol. 1997; 45: 219-227PubMed Google Scholar), rheumatoid arthritis (20Svejgaard A. Platz P. Ryder L.P. Immunol. Rev. 1983; 70: 193-218Crossref PubMed Scopus (456) Google Scholar), and ankylosing spondylitis (21Brown M.A. Pile K.D. Kennedy L.G. Campbell D. Andrew L. March R. Shatford J.L. Weeks D.E. Calin A. Wordsworth B.P. Arthritis Rheum. 1998; 41: 588-595Crossref PubMed Google Scholar). Although disease susceptibility is often due to allelic differences in the class I and class II antigens, there is evidence that loci located in the class III region may also contribute (21Brown M.A. Pile K.D. Kennedy L.G. Campbell D. Andrew L. March R. Shatford J.L. Weeks D.E. Calin A. Wordsworth B.P. Arthritis Rheum. 1998; 41: 588-595Crossref PubMed Google Scholar, 22Schaffer F.M. Palermos J. Zhu Z.B. Barger B.O. Cooper M.D. Volanakis J.E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8015-8019Crossref PubMed Scopus (164) Google Scholar). For this reason the detailed characterization of genes located in the class III region with a potential role in the immune system is of great interest. G6b is an uncharacterized gene located in the class III region of the MHC that encodes a putative cell surface receptor of the Ig superfamily. Its predicted gene product was found to contain a potential signal peptide, a variable type Ig domain, and a transmembrane segment (23Ribas G. Neville M. Wixon J.L. Cheng J. Campbell R.D. J. Immunol. 1999; 163: 278-287PubMed Google Scholar). Interestingly, we have observed that the intracellular stretch also contains two tyrosine residues in ITIM consensus sequences. In this study, the available genomic sequence information was used as a starting point to obtain the cDNA encoding the G6b protein. By RT-PCR, several alternative splice variants of the G6b mRNA were identified in the bone marrow-derived cell lines K562, Molt4, and Jurkat but not in other hematopoietic and fibroblast cell lines studied. These mRNAs encode proteins with different C termini, some of which lacked a transmembrane segment. Expression of four of the proteins as epitope-tagged fusion proteins in mammalian cells allowed their further characterization. An association of the splice isoform containing the ITIM motifs with SHP-1 and SHP-2 was shown following pervanadate-induced tyrosine phosphorylation. RNA isolation from human cell lines was performed as described previously (24Aguado B. Campbell R.D. Biochem. J. 1999; 341: 679-689Crossref PubMed Google Scholar). cDNA synthesis was carried out using a Promega Reverse Transcription System and ∼1 μg of poly(A)+ RNA according to the manufacturer’s instructions. Control PCR reactions with β-actin primers were performed on each cDNA reaction (forward 5′-CTTCGCGGGCGACGATGC-3′ and reverse 5′-TGGTGGTGAAGCTGTAGCC-3′). PCR primers for G6b were designed based on the genomic sequence (GenBankTM accession number AF129756). To obtain the complete open reading frame, nested PCR was performed on the cDNA samples. In the first round, forward primer 5′-AGCTTCTCCTCACCACATCC-3′ (nt position 26694–26713) and reverse primer 5′-AAAGGGTCAGTCTCCTGACG-3′ (nt position 23402–23421) were used. In the second round, forward primer 5′-CCTAACCATGGCTGTGTTTC-3′ (nt position 26676–26695) was used in combination with reverse primer 5′-GGGAGGTTTGGAGTAAGGGC-3′ (nt position 24972–24991). In each round, 25 cycles were performed using an annealing temperature of 60 °C. PCR fragments were cloned into the pGEM-T vector (Promega). Clones were checked by sequencing on an Applied Biosystems (Applera) 377 automated DNA sequencer using Big Dye terminators. For expression of epitope-tagged proteins in mammalian cells, the open reading frames of the different splice isoforms were cloned into the pcDNA3 vector (Invitrogen) fused to a T7-epitope tag (MASMTGGQQMGRDP). To express the G6b isoforms fused at the C terminus to the T7-epitope tag, PCR copies were made of the open reading frames removing the stop codons. The forward primer 5′-TTATAAGCTTACCATGGCTGTGTTTCTGC-3′ was used, creating a HindIII The reverse primer G6b splice isoforms and or G6b splice isoforms A and was used. These two reverse primers the stop and fusion to the tag sequence in the vector M. Campbell R.D. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus (105) Google Scholar). PCR fragments were cloned into this vector using were checked by DNA sequencing as described The encoding the fusion proteins were cloned into the pcDNA3 vector using To express the G6b isoforms with the T7-epitope tag fused to the an expression vector was containing the human signal D. B. J. Immunol. 1988; Google Scholar) of the G6b signal by the T7-epitope tag in a fusion the signal and the T7-epitope tag was in The tag fusion was by PCR using primers a and containing a The was cloned into the pcDNA3 vector using and the The G6b splice isoforms were with primer a and removing the first of the potential signal and a and the stop The PCR fragments were with and and cloned into with and The were checked by DNA of tyrosine to in the cytoplasmic tail of the were made using the according to the manufacturer’s instructions. were expressed in cells using the as described M. Campbell R.D. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus (105) Google Scholar). after cells and were K562 cells were with according to the manufacturer’s instructions. after cells were in the presence of For on cells were with and in Nature. PubMed Scopus Google Scholar). were by was according to Nature. PubMed Scopus Google Scholar) on polyacrylamide was performed with the tag monoclonal proteins were with by with Immunofluorescence studies were performed as described B. Campbell R.D. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar), and was with a linked to a system G6b splice isoforms were expressed in cells as described and cell were in and For treatment of secreted G6b the was and and were To the both cell and were for 2 at °C. and were of were with or for at °C. were from were by the of and on polyacrylamide by as described was by and in to of and and the for at To was to a of were by a of pervanadate in for at °C. were in and with were by a at and were performed with the and protein at °C. were with at for 2 and by by The were with the tag to or In the of the and SHP-2 a to was used. were with as human expressed sequence tag were found that contained part of the region of was found to encode a putative there were a human that to the genomic sequence of the putative stop of some of indicated that they not contain the complete open reading out not to be nested was performed on cDNA from from human cell The from the bone marrow-derived cell lines K562 Molt4, and Jurkat cell a number of PCR fragments with the two A and of the PCR on the bone marrow-derived cell lines and the fibroblast cell lines and not of in a we were not to by PCR G6b mRNA from human cell lines and not of the showed that the band with the the form predicted by (23Ribas G. Neville M. Wixon J.L. Cheng J. Campbell R.D. J. Immunol. 1999; 163: 278-287PubMed Google Scholar) and also in AF129756). form contains two ITIM motifs at and in the 2 of the cDNA sequence of the band with the genomic sequence that this form an stretch of to due to different at the of stretch the reading of the C-terminal of with in the of the two ITIM motifs in The to variants or and the putative transmembrane segment is by variants encode proteins that are predicted to be secreted and not to be that showed the described and out to contain sequences. These encode proteins that contain the signal Ig domain, and transmembrane domain as the but with a different cytoplasmic tail. The only PCR band observed in cells contained the and which to a stop be from this cell Four splice isoforms were for further two transmembrane isoforms and and two isoforms and The C terminus of is to the C terminus of and the C terminus of to the C terminus of The four splice isoforms were cloned into a pcDNA3 expression vector as with a The fusion proteins were expressed in were observed on which be of each band the protein in a different of only the isoforms lacking transmembrane and were in the The for the G6b splice variants are and The observed of the proteins on gel are in in with the the band found in each An to this is the form found in the which at be due to the presence of large In this is that found within the cell has a to the proteins have not been the complete To the different G6b variants were cell and cell containing the of G6b were with of the and/or of in the of some of the that the at least in with both and in with different from the after only in the of the secreted form of that only this form contains a O-glycosylation. the of the proteins and with only of are with proteins with of both N- as as a in the is observed can be by the presence of the in the of secreted that the band observed the protein is with is not the protein is with that this band contains O-glycosylation. In the of the isoforms and the proteins secreted into the contain more is found in the corresponding cell The proteins found in the cell may also For that reason they may not be the and thus may not be the were performed on the were not studies with were performed using the with the C-terminal or T7-epitope tag the an of the cells was observed in the of the isoforms and which contain potential transmembrane is in with a at the membrane. In contrast, and showed a with and In the of the was performed both as as the observed were to the observed with the proteins indicated that the G6b containing a transmembrane segment and were at the with the T7-epitope tag the cell In contrast, and cells showed a different with cells showed the of the in to where this type of is be the protein to the cell after inhibitory of the Ig superfamily or contain at least two with a of in the sequence (3Vivier E. Daeron M. Immunol. Today. 1997; 18: 286-291Abstract Full Text PDF PubMed Scopus (328) Google Scholar) and are known to SHP-1 and/or SHP-2 after phosphorylation. contains two tyrosine residues in consensus ITIM in its cytoplasmic and the two is To this isoform was to SHP-1 and/or SHP-2 after cells the four G6b isoforms were with In the of the membrane-bound and the tag the is not to with which at the cytoplasmic tail. of by with antibodies showed that tyrosine phosphorylation in the cells was due to pervanadate treatment not The different G6b isoforms were with the from cells or with pervanadate of tyrosine phosphorylation on G6b was checked with a the isoform was found to be after pervanadate In the containing both SHP-1 and SHP-2 can be by The presence of SHP-1 and SHP-2 is on both the presence of the ITIM-containing isoform and on the of tyrosine phosphorylation by pervanadate on this To both cytoplasmic of or one of are were expressed in cells, and of to in a of tyrosine phosphorylation as as a of interaction of with SHP-1 and SHP-2 of to to a of tyrosine phosphorylation and SHP-1 and SHP-2 These that is the only tyrosine residue to be phosphorylated and to be involved in SHP-1 and SHP-2 is in an ITIM consensus To the interaction of with SHP-1 and SHP-2 in a human bone marrow-derived cell a was performed using the human cell K562, which G6b at the RNA The two membrane-bound of G6b and were expressed in this cell the isoform was phosphorylated pervanadate treatment Both SHP-1 and SHP-2 can be in of in K562 cells, the in have characterized the G6b gene located in the class III region of the human MHC, a region known to contain genes with in the immune on cDNA from human cell lines showed that the G6b gene is only expressed in a restricted of hematopoietic cell an was observed that the RNA from this gene is One spliced which encodes a protein containing two ITIM to be at the RNA the form lacking motifs other variants were encoding proteins lacking a transmembrane segment and expressed in cells, were that the hydrophobic segment as a signal Glycosidase treatment of the G6b isoforms provided evidence that the proteins are both N- as as The extracellular part of the membrane-bound of G6b contains only one consensus is located in the predicted of the Ig domain, and the chain of the residue to be is to be on the surface C. I. A. J. Biol. 1998; PubMed Scopus Google for a detailed of Ig a residue for contains a of with the other the and in the C-terminal is that there is at least one in this part of which the in two Although contains a C terminus to the C terminus of in the of this tail is cytoplasmic and, not available for Immunofluorescence with cells using the showed that the tag is the cells in the of and Although not their signal that the proteins are to the and not in the These that proteins are to in cells proteins that are not to are in the and Biochem. J. 2000; PubMed Scopus Google Scholar). For the can be that and are to in cells variants are secreted into the Of the G6b isoforms only to be phosphorylated and K562 cells this recombinant protein are with is in with as not contain cytoplasmic tyrosine residues and the isoforms not have a part to the cytoplasmic kinases contains ITIM to The interaction of with SHP-1 and SHP-2 be observed in two different cell one of G6b mRNA SHP-1 is known to be primarily expressed in hematopoietic cells (4Zhang J. Somani A.K. Siminovitch K.A. Semin. Immunol. 2000; 12: 361-378Crossref PubMed Scopus (290) Google Scholar), but we this protein in the fibroblast cell COS-7. Although contains two tyrosine residues in ITIM consensus in its cytoplasmic tail and suggested that only one of phosphorylated pervanadate of cells and that this tyrosine is for SHP-1 and SHP-2 was not studies with receptors containing two tyrosine residues in showed the of both residues in SHP-1 or SHP-2 M. Zhao R. Zhao Z.J. J. Biol. Chem. 2000; 275: 17440-17446Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, R. Zhao Z.J. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). is that in the SH2 domains of SHP-1 and SHP-2 the receptor with the of tyrosine phosphorylation of the not that not phosphorylated in the type be that the phosphorylation of tyrosine is on the phosphorylation of tyrosine has that inhibitory receptors often have that are expressed on the cell type and are to or extracellular Rev. 2000; Google Scholar). examples are the inhibitory receptor family as as the family Rev. 2000; Google Scholar). In inhibitory and receptors are by different In to the inhibitory the receptors generally have a cytoplasmic tail lacking ITIM sequences. they a residue in the transmembrane segment involved in to signaling such as and J. T. Phillips J.H. Lanier L.L. J. Exp. Med. 2000; PubMed Scopus Google Scholar, L.L. B. J. 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A. 1999; PubMed Scopus Google Scholar). to one can that the is the of the inhibitory receptor both the form of as as a residue in the transmembrane segment, which is in to the inhibitory receptor and family of In we have shown that the G6b gene located in the class III region of the human MHC is expressed and in cells at the RNA of the different G6b isoforms expressed in mammalian cells is in with G6b encoding a cell surface receptor, variants are found as The interaction of with SHP-1 and SHP-2 this at least as a of the family of inhibitory receptors of the Ig superfamily and the first one found in the the G6b Ig domain not contain homology other Ig proteins with known of the extracellular be in the role of the G6b gene product in cellular A. and C. for on the