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• W2018118610 abstract "SHP-1-mediated dephosphorylation of protein tyrosine residues is central to the regulation of several cell signaling pathways, the specificity of which is dictated by the intrinsic affinity of SH2 domains for the flanking sequences of phosphotyrosine residues. By using a modified yeast two-hybrid system and SHP-1 as bait, we have cloned a human cDNA, PILRα, encoding a 303-amino acid immunoglobulin-like transmembrane receptor bearing two cytoplasmic tyrosines positioned within an immunoreceptor tyrosine-based inhibitory motif. Substrate trapping in combination with pervanadate treatment of 293T cells confirms that PILRα associates with SHP-1 in vivo upon tyrosine phosphorylation. Mutation of the tyrosine residues in PILRα indicates the pivotal role of the Tyr-269 residue in recruiting SHP-1. Surface plasmon resonance analysis further suggests that the association between PILRα-Tyr-269 and SHP-1 is mediated primarily via the amino-terminal SH2 domain of the latter. Polymerase chain reaction amplification of cDNA in combination with genomic sequence analysis revealed a second gene, PILRβ, coding for a putative activating receptor as suggested by a truncated cytoplasmic tail and a charged lysine residue in its transmembrane region. The PILRα and PILRβ genes are localized to chromosome 7 which is in contrast with the mapping of known members of the inhibitory receptor superfamily. SHP-1-mediated dephosphorylation of protein tyrosine residues is central to the regulation of several cell signaling pathways, the specificity of which is dictated by the intrinsic affinity of SH2 domains for the flanking sequences of phosphotyrosine residues. By using a modified yeast two-hybrid system and SHP-1 as bait, we have cloned a human cDNA, PILRα, encoding a 303-amino acid immunoglobulin-like transmembrane receptor bearing two cytoplasmic tyrosines positioned within an immunoreceptor tyrosine-based inhibitory motif. Substrate trapping in combination with pervanadate treatment of 293T cells confirms that PILRα associates with SHP-1 in vivo upon tyrosine phosphorylation. Mutation of the tyrosine residues in PILRα indicates the pivotal role of the Tyr-269 residue in recruiting SHP-1. Surface plasmon resonance analysis further suggests that the association between PILRα-Tyr-269 and SHP-1 is mediated primarily via the amino-terminal SH2 domain of the latter. Polymerase chain reaction amplification of cDNA in combination with genomic sequence analysis revealed a second gene, PILRβ, coding for a putative activating receptor as suggested by a truncated cytoplasmic tail and a charged lysine residue in its transmembrane region. The PILRα and PILRβ genes are localized to chromosome 7 which is in contrast with the mapping of known members of the inhibitory receptor superfamily. Src homology 2 paired immunoglobulin-like receptor α paired immunoglobulin-like receptor β immunoreceptor tyrosine-based inhibitory motif polymerase chain reaction kilobase pair hemagglutinin polyacrylamide gel electrophoresis surface plasmon resonance fetal bovine serum monoclonal antibody polyclonal antibody glutathione S-transferase major histocompatibility complex peptide:N-glycosidase F The initiation of cell signaling pathways relies on a dynamic interaction between activating and inhibiting processes that can include, among other things, changes in the phosphorylation status of certain tyrosine residues within the target proteins. Dephosphorylation of these residues is mediated by such phosphatases as the cytosolic SHP-1 (also known as SHP, PTP1C, SH-PTP1, or PTPN6 (1.Adachi M. Fischer E.H. Ihle J. Imai K. Jirik F. Neel B. Pawson T. Shen S. Thomas M. Ullrich A. Zhao Z. Cell. 1996; 85: 15Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar)) which is expressed in hematopoietic cells, and to a lesser extent in non-hematopoietic cells, and which contains tandem amino-terminal Src homology 2 (SH2)1 domains. The importance of SHP-1 in cellular signal delivery is underscored by the motheaten mouse that carries a natural mutation in the SHP-1 locus and is characterized by widespread autoimmune phenomena resulting from an inability to modulate immune responses (2.Shultz L.D. Schweitzer P.A. Rajan T.V. Yi T. Ihle J.N. Matthews R.J. Thomas M.L. Beier D.R. Cell. 1993; 73: 1445-1454Abstract Full Text PDF PubMed Scopus (690) Google Scholar, 3.Tsui H.W. Siminovitch K.A. de Souza L. Tsui F.W. Nat. Genet. 1993; 4: 124-129Crossref PubMed Scopus (522) Google Scholar). Affinity for the SH2 domains is pivotal for interaction of substrates with SHP-1. The flanking sequence of the phosphotyrosine residue promotes the recruitment of specific SH2-containing phosphatases and, thus, determines the specificity of the signaling pathway. The consensus sequence (S/L/I/V)XYXX(L/V), based on sequences originally deduced from several receptors known to bind to the carboxyl-terminal SH2 domain of the protein tyrosine phosphatase SHP-1 (4.D'Ambrosio D. Hippen K.L. Minskoff S.A. Mellman I. Pani G. Siminovitch K.A. Cambier J.C. Science. 1995; 268: 293-297Crossref PubMed Scopus (507) Google Scholar, 5.Burshtyn D.N. Scharenberg A.M. Wagtmann N. Rajagopalan S. Berrada K. Yi T. Kinet J.P. Long E.O. Immunity. 1996; 4: 77-85Abstract Full Text Full Text PDF PubMed Scopus (559) Google Scholar, 6.Olcese L. Lang P. Vély F. Cambiaggi A. Marguet D. Blery M. Hippen K.L. Biassoni R. Moretta A. Moretta L. Cambier J.C. Vivier E. J. Immunol. 1996; 156: 4531-4534PubMed Google Scholar), defines all immunoreceptor tyrosine-based inhibitory motif (ITIM)-bearing receptors (7.Cambier J.C. Immunol. Today. 1995; 16: 110Abstract Full Text PDF PubMed Scopus (257) Google Scholar) including natural killer cell, B cell, and monocyte and dendritic cell inhibitory receptors (reviewed in Refs. 8.Burshtyn D.N. Long E.O. Trends Cell Biol. 1997; 7: 473-479Abstract Full Text PDF PubMed Scopus (62) Google Scholar and 9.Vély F. Vivier E. J. Immunol. 1997; 159: 2075-2077PubMed Google Scholar). Members of the inhibitory receptor superfamily (10.Lanier L.L. Annu. Rev. Immunol. 1998; 16: 359-393Crossref PubMed Scopus (1486) Google Scholar, 11.Long E.O. Annu. Rev. Immunol. 1999; 17: 875-904Crossref PubMed Scopus (847) Google Scholar) can be divided into two groups. The immunoglobulin (Ig) superfamily includes luminal amino-terminal (e.g. type I) transmembrane glycoproteins with two or more Ig-like domains such as p58/KIR2DL3, FcγRIIB, Ig-like transcripts as well as the mouse PIR-B. The other group is comprised of CD94/NKG-2, CD72, and the mouse Ly49 and NKR-P1 which are cytoplasmic amino-terminal (e.g. type II) transmembrane proteins expressing C-type lectin extracellular architecture. The existence of complementary proteins expressing similar extracellular domains as the inhibitory receptors, while having distinctive transmembrane and frequently truncated cytoplasmic tails, suggests similar ligand-binding specificities but contrasting signaling capabilities for each individual of the protein pair. The truncated protein would have cellular activating properties, in contrast with its ITIM-bearing counterpart which would inhibit cell signaling through recruitment of SHP-1, SHP-2, or SHIP phosphatases (9.Vély F. Vivier E. J. Immunol. 1997; 159: 2075-2077PubMed Google Scholar) seemingly via their respective carboxyl-terminal SH2 domains (12.Vély F. Nunes J.A. Malissen B. Hedgecock C.J. Eur. J. Immunol. 1997; 27: 3010-3014Crossref PubMed Scopus (19) Google Scholar, 13.Bruhns P. Marchetti P. Fridman W.H. Vivier E. Daeron M. J. Immunol. 1999; 162: 3168-3175PubMed Google Scholar). Human immunoreceptors map to the “complex” or “leukocyte receptor cluster” located on chromosome region 19q13.4 or to the natural killer complex on chromosome 12 (11.Long E.O. Annu. Rev. Immunol. 1999; 17: 875-904Crossref PubMed Scopus (847) Google Scholar). Mouse immunoreceptors are located on chromosomes 7 and 6 in regions syntenic to human chromosome 19 and 12, respectively (11.Long E.O. Annu. Rev. Immunol. 1999; 17: 875-904Crossref PubMed Scopus (847) Google Scholar), or to band B4 of mouse chromosome 10 (14.Kuroiwa A. Yamashita Y. Inui M. Yuasa T. Ono M. Nagabukuro A. Matsuda Y. Takai T. J. Biol. Chem. 1998; 273: 1070-1074Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) which does not appear to have any conserved linkage homology to either human chromosome 19 or 12. We now present evidence of a novel pair of receptors expressing a single extracellular Ig-like domain that we have designated as “paired immunoglobulin-likereceptor” (PILR)α and PILRβ to distinguish their putative inhibitory and activating gene products, respectively. The ITIM-bearing receptor PILRα recruits SHP-1 via its amino-terminal SH2 domain and is likely to have cellular inhibitory potential. The lack of a cytoplasmic tail and the presence of the transmembrane lysine residue in the second receptor PILRβ suggest its potential activating function. Both genes map cytogenetically to human chromosome 7. All cell lines were obtained from American Type Culture Collection except where indicated. The human embryonic kidney epithelial cell line 293T (Edge Biosystems) was maintained in Dulbecco's modified Eagle's medium, 10% fetal bovine serum (FBS). The T cell-derived KG-1, Jurkat and K562 cell lines were maintained in RPMI, 10% FBS. The B cell-derived cell lines WIL2-NS, Namalwa, Daudi, and Raji were maintained in RPMI, 10% inactivated FBS. The 6F11 B cell line was cultured in Iscove's modified Dulbecco's medium containing 2 mml-glutamine and 15% FBS. The macrophage 28SC cell line was maintained in Iscove's medium supplemented with 0.03 mm thymidine, 0.1 mmhypoxanthine, 0.05 mm β-mercaptoethanol, and 10% FBS. NK-92ci cells (ImmuneMedicine, Vancouver, British Columbia, Canada) transformed with a pCep-IL-2 construct were maintained by selection in interleukin-2-free Myelocult medium (Stem Cell Technologies, Vancouver, British Columbia, Canada). SHP-1 was precipitated and detected using a monoclonal antibody (mAb; P17320; Transduction Laboratories) or an in-house polyclonal antibody (pAb; 237, see Ref. 15.Bouchard P. Zhao Z. Banville D. Dumas F. Fischer E.H. Shen S.H. J. Biol. Chem. 1994; 269: 19585-19589Abstract Full Text PDF PubMed Google Scholar). PILRα cDNA was subcloned in-frame with a carboxyl-terminal triple HA tag. The HA epitope was precipitated with a high affinity rat anti-HA mAb (3F10; Roche Molecular Biochemicals) and detected with a mouse mAb (12CA5; Roche Molecular Biochemicals). Separate membranes were also probed with anti-phosphotyrosine 4G10 mAb (Upstate Biotechnology, Inc.) and anti-ubiquitin pAb (U-5379; Sigma). Secondary antibodies were horseradish peroxidase-conjugated to goat anti-mouse or goat anti-rabbit antibody (Bio-Rad) and detection relied on enhanced chemiluminescence (NEN Life Science Products). Screening of a human mammary gland cDNA library (CLONTECH) with the SHP-1-C455S catalytic mutant (16.Su L. Zhao Z. Bouchard P. Banville D. Fischer E.H. Krebs E.G. Shen S.H. J. Biol. Chem. 1996; 271: 10385-10390Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) was performed as follows. The full-length SHP-1-C455S was cloned into SalI-restricted pBTM116-Src by standard polymerase chain reaction (PCR) procedures and thus was fused in-frame with the carboxyl terminus of the DNA binding domain of the bacterial activator LexA. pBTM116-Src contains a mutant c-Src kinase expression cassette designed for the phosphorylation of protein tyrosine residues in yeast (17.Keegan K. Cooper J.A. Oncogene. 1996; 12: 1537-1544PubMed Google Scholar). The pBTM116-Src-SHP-1-C455S construct was then used to transform the yeast reporter strain, L40α (MATa trp1 leu2 his3 LYS2::lexA-HIS3 URA3::lexA-lacz). Following transformation using Li2+ acetate (18.Gietz D. St-Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425Crossref PubMed Scopus (2899) Google Scholar), L40α expressing pBTM116-Src-SHP-1-C455S was further transformed with the library. Positive interactors were isolated according to the manufacturer's specifications (CLONTECH Laboratories, Inc.). The positive control consisted of a pBTM-116-cnx1+ and pGADGH-hus5+ combination kindly provided by M. Pelletier and D.Y. Thomas 2M. Pelletier and D. Y. Thomas, unpublished data. (NRC Biotechnology Research Institute, Montreal, Quebec, Canada). cDNA sequences were determined and subjected to the BLAST search of the NCBI data bases. The target sequence of interest was used as a probe for screening of cDNA libraries. 32P-Labeled probes were prepared by random labeling of the PILRα PCR product using Ready-to-Go DNA Labeling Beads (Amersham Pharmacia Biotech) and [α-32P]dCTP. Placenta, acute myelocytoma leukemia (KG-1), bone marrow, and leukocyte cDNA libraries were screened using the labeled probes. Positive clones were subjected to PCR using λ GT11-specific primers. Single and double tyrosine to phenylalanine mutants of PILRα-HA, i.e. Y269F, Y298F, Y269F/Y298F, were generated by PCR-based site-directed mutagenesis. 293T cells were transfected either alone or in combination with SHP-1 by calcium phosphate precipitation. Forty-eight hours after transfection the cells were treated with pervanadate (100 μm, 30 min at 37 °C), a phosphatase inhibitor that induces maximal tyrosine phosphorylation (19.O'Shea J.J. McVicar D.W. Bailey T.L. Burns C. Smyth M.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10306-10310Crossref PubMed Scopus (144) Google Scholar), and then washed with phosphate-buffered saline and lysed on ice. Solubilized extracts were immunoprecipitated with the appropriate antibodies followed by protein A- or protein G-Sepharose. The immunoprecipitates were resolved by SDS-PAGE and revealed by standard immunoblotting techniques. 293T cells were transiently transfected with PILRα-HA. Following immunoprecipitation with an HA-directed antibody, immune complexes were washed and denatured, and N-glycosylation status was determined by overnight incubation at 37 °C with 5 milliunits ofN-glycosidase F (PNGase:F, Roche Molecular Biochemicals) while a parallel series of experiments determinedO-glycosylation status by incubation with 1 milliunit of neuraminidase (to remove sialic acid residues, Roche Molecular Biochemicals) and/or 1 milliunit of endo-α-N-acetylgalactosaminidase (O-glycosidase, Roche Molecular Biochemicals). The various reactions were resolved on 10% SDS-PAGE gels. To test for the presence of covalently bound ubiquitin in the 55-kDa expressed PILRα-HA species, transiently transfected 293T cells were treated with pervanadate, and HA-bound immunoprecipitates were collected, resolved by SDS-PAGE, and probed with an anti-ubiquitin pAb. A 300-base pair segment encoding either the amino-terminal SH2 domain (SH2(N)) or the carboxyl-terminal SH2 domain (SH2(C)) of SHP-1 were generated by PCR amplification of human SHP-1 cDNA (20.Yu Z. Hoglinger O. Jaramillo M.L. Banville D. Shen S.H. J. Biol. Chem. 1998; 273: 3687-3694Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) and subcloned into pGEX-2T (Amersham Pharmacia Biotech). The SHP-1.SH2(N) and SHP-1.SH2(C) GST fusion proteins were produced inEscherichia coli DH5α cultures transformed with the corresponding plasmid and induced with 25 μmisopropyl-β-d-thiogalactoside for 22 h at 28 °C. The GST fusion proteins were isolated using glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech). Protein expression and purity were determined by SDS-PAGE and Coomassie Blue staining. Surface plasmon resonance (SPR) was performed on a BIAcore apparatus using CM5-sensor chips (Biosensor AB, Uppsala, Sweden). The tyrosyl-phosphorylated (pY) synthetic peptides, KDDGIV(pY)ASLALSSSTS and PQNETL(pY)SVLKA, corresponding, respectively, to the Tyr-269- and Tyr-298-based motifs contained in PILRα were immobilized at 0.5 mg/ml on the Biosensor chip. Immobilization efficiency was verified using the anti-phosphotyrosine 4G10 mAb. GST-SH2 domain fusion proteins (see above) were initially dialyzed in 10 mm Hepes buffer (pH 7.4) containing 150 mm NaCl and 3.4 mm EDTA (HBS). The GST fusion proteins (0.5–2000 nm), diluted in HBS, 0.05% P20, were injected over the test surfaces at a flow rate of 5 μl/min. Regeneration of the Biosensor chip using 46 mmHCl, 1 m NaCl did not result in loss of subsequent signal. Dissociation constants (K D) were calculated by saturation (nonlinear regression) analysis as well as by kinetic analysis (e.g. the ratio of k off andk on determined using BIAevaluation software; Biosensor AB, Uppsala, Sweden). Human multiple tissue Northern blots (CLONTECH Laboratories, Inc.) containing 2 μg of poly(A)+ RNA per lane were hybridized consecutively with32P-labeled full-length PILRα cDNA and with32P-labeled β-actin cDNA probes. The level of expression in a series of human cells was also examined. Total RNAs from the individual cell lines were extracted by the TriZOL method (Life Technologies, Inc.) and enriched in poly(A)+ RNA by passage on oligo(dT) spun columns (Amersham Pharmacia Biotech). Ten μg of poly(A)+ RNA were loaded per lane, transferred to membrane, probed as above, and then washed (maximum stringency being 2 × 15 min, 0.1× SSC, 0.1% SDS, 55 °C). The sequence of the 5′-untranslated region was amplified by 5′-rapid amplification of cDNA ends from Marathon-Ready cDNA (CLONTECH), whereas the genomic organization of PILRα and PILRβ genes was partially determined using the proofreading Taqpolymerase GenomeWalkerTM kit (CLONTECH). A SHP-1-C455S catalytic mutant was used as bait in a yeast two-hybrid screening of a human mammary gland cDNA library. 4.5 × 106 clones were screened to reveal 24 interactors able to grow on Trp−/Leu−/His− medium and able to induce β-galactosidase activity (Fig.1). These positive clones were sequenced and identified as a novel ITIM-bearing protein, subsequently named PILRα, as well as known interactors of SHP-1 namely SHPS-1 (21.Veillette A. Thibaudeau E. Latour S. J. Biol. Chem. 1998; 273: 22719-22728Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), EGFRBP-Grb2 (22.Kon-Kozlowski M. Pani G. Pawson T. Siminovitch K.A. J. Biol. Chem. 1996; 271: 3856-3862Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), and the leukocyte-associated Ig-like receptor LAIR-1 (23.Meyaard L. Adema G.J. Chang C. Woollatt E. Sutherland G.R. Lanier L.L. Phillips J.H. Immunity. 1997; 7: 283-290Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). The sequence of the novel interactor was extended by PCR amplification of the target cDNA library and was ultimately confirmed following its use as a probe in the cDNA library screening which yielded the full-length sequence. The nucleotide sequence and deduced amino acid sequence, indicating a theoretical mass of 31.8 kDa, are depicted in Fig. 2. Structurally the gene product does not contain an IgG domain per se, although the single extracellular cysteine residue (Cys-125) is flanked by a motif, e.g.YXCXVXL, reminiscent of the carboxyl-terminal cysteine-based motif, e.g.(F/Y)XCX(V/A)XH, involved in the intradomain disulfide bond of immunoglobulin and major histocompatibility complex (MHC) proteins (PROSITE data base; reference number PS00290). This same segment bears a slight homology to extracellular regions of sialoadhesin (24.Crocker P.R. Mucklow S. Bouckson V. McWilliam A. Willis A.C. Gordon S. Milon G. Kelm S. Bradfield P. EMBO J. 1994; 13: 4490-4503Crossref PubMed Scopus (226) Google Scholar). In addition, there is a potential N-glycosylation site (NX(T/S)), numerous serine and threonine residues capable of beingO-glycosylated, a transmembrane domain, and three potential tyrosine phosphorylation sites, two of which, e.g. Tyr-269 and Tyr-298, reside within an immunoreceptor tyrosine-based inhibitory motif.Figure 2Nucleotide sequence and deduced amino acid sequence of human PILRα. The signal peptide (underlined), the transmembrane region (bold, underlined), a potential N-glycosylation site (boxed), and putative binding sites for SHP-1 SH2 domains (shaded) are indicated. The AATAAA polyadenylation signal is also shown (bold). The bold numbers to theright indicate the nucleotide position, and theitalicized numbers indicate the amino acid position.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To examine protein-protein interactions, 293T cells were co-transfected with PILRα-HA and SHP-1 and treated with pervanadate. Fig.3 demonstrates that PILRα-HA is phosphorylated in 293T cells (Fig. 3 A, top), that PILRα-HA and SHP-1 co-precipitate (Fig. 3 A, middle), and that tyrosine phosphorylation was essential for binding to SHP-1 (Fig.3 A, bottom). In addition, confirmation that the association occurred in vivo was accomplished by co-immunoprecipitation of PILRα-HA as part of an immune complex containing the SHP-1-D421A substrate-trapping mutant (25.Flint A.J. Tiganis T. Barford D. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1680-1685Crossref PubMed Scopus (686) Google Scholar) (Fig. 3 B). PILRα-HA migrated with an apparent molecular mass of 55 kDa rather than the expected theoretical molecular mass of 36 kDa. The 55-kDa molecular mass includes 4.6 kDa contributed by the triple HA tag. The significant difference between the deduced and the apparent molecular mass of PILRα-HA on Western blotting led to the examination of the glycosylation and/or ubiquitination status of this protein. Samples of PILRα-HA immunoblotted for ubiquitin did not reveal any signal (data not shown). Digestion of immunoprecipitates with either PNGase:F or with neuraminidase plus O-glycosidase revealed shifts in the migration of expressed PILRα-HA. The combination of PNGase:F and neuraminidase plus O-glycosidase reduced the molecular mass to approximately 42 kDa thus revealing that post-translation addition of N- and O-linked carbohydrate residues accounts for a substantial portion of expressed 55-kDa PILRα-HA (Fig. 4). The role of the ITIM-based tyrosine residues of PILRα-HA in recruiting SHP-1 was investigated by mutational analysis. Immunodetection of SHP-1 following co-immunoprecipitation with an HA-directed antibody indicated that the Tyr-269 motif, e.g.IVYASL, was the target for binding with SHP-1 (Fig.5 A). Indeed, whereas the Y298F substitution seems to have had only a slight effect on the association with SHP-1, the Y269F mutant and the Y269F/Y298F double mutant significantly decreased the SHP-1 signal. PILRα-HA (Fig.5 C) and SHP-1 (Fig. 5 B) were equally expressed in the corresponding lysates. The importance of the Tyr-269 motif in recruiting SHP-1 is supported using BIAcore detection. The immobilized phosphorylated peptide corresponding to this motif, e.g.KDDGI(pY)-ASLALSSSTS, demonstrated high affinity for the SHP-1-SH2(N) domain (Fig. 6, top) and intermediate affinity for the SHP-1-SH2(C) domain (Fig. 6,bottom). Interestingly, the SHP-1-SH2(C) domain selectively recognized the Tyr-298 phosphopeptide, e.g. PQNETL(pY)SVLKA, but with much lower affinity (Fig. 6, bottom). The dissociation constants (K D) for the various interactions were determined by saturation analysis (nonlinear regression analysis) and supported by those obtained by kinetic analysis (e.g. K D =k off/k on: TableI). Saturation analysis revealedK D values of 167 ± 4 and 1091 ± 196 nm following binding of the SHP-1-SH2(N) and -SH2(C) GST fusion proteins, respectively, to the Tyr-269 phosphopeptide. AK D value of 3777 ± 447 nm was obtained upon binding of the SHP-1-SH2(C) GST fusion protein to the Tyr-298 phosphopeptide. GST itself did not bind to any of the test surfaces (data not shown) and neither of the GST-SH2 domain fusion proteins bound significantly (e.g. less than 5 resonance units) to the control Biosensor chip surface.Table IBinding potential of the SHP-1 amino- and carboxyl-terminal SH2 domains with human PILRα tyrosyl-phosphorylated peptidesPhosphorylated peptidesKDDGIV(pY)ASLALSSSTSPQNETL(pY)SVLKASHP-1.SH2(N) k on (104m−1 s−1)5.10 ± 0.53ND k off(10−3 s−1)2.48 ± 0.65ND K D(nm)47.8 ± 10.5NDSHP-1.SH2(C) k on (104m−1 s−1)2.42 ± 0.990.46 ± 0.05 k off (10−3 s−1)3.78 ± 0.9612.9 ± 0.59 K D(nm)214.0 ± 77.73000 ± 431Values represent the mean (±S.E.) of five separate experiments. ND, no detectable binding. Open table in a new tab Values represent the mean (±S.E.) of five separate experiments. ND, no detectable binding. Rapid amplification of cDNA ends designed to clone the 5′ end of the PILRα cDNA revealed the presence of a second cDNA displaying long regions of near sequence identity to PILRα but differing in its 5′ non-coding sequence. The existence of a related gene was also suggested from long range PCR performed on human genomic DNA (results not shown) and was confirmed by the sequence of a 200-kilobase segment of human chromosome 7 deposited subsequently in GenBankTM under the accession number RG161A02. The PILRα gene consists of seven exons and six introns spanning approximately 26.7 kb. The second gene, designatedPILRβ, is located 5.6 kb upstream of thePILRα gene and consists of four exons and three introns spanning approximately 9.8 kb (Fig.7 B). The nucleotide sequence of the first three exons of the two genes is extremely similar, displaying more than 90% sequence identity (Fig. 7 A), and suggests that the two genes share a common origin. TablesII and IIIsummarize the nucleotide sequence of the splice sites of thePILRα and PILRβ genes, respectively, and indicate that in both cases the intron-exon boundaries conform to the GT-AG rule. PILRβ codes for a protein with similar extracellular features as those of PILRα but with a short cytoplasmic tail and a charged lysine residue within its transmembrane domain (Fig.7 A).Table IIIntron-exon splice junction sites of the human PILRα geneExoSizeSplice donorSplice acceptorIntron sizebpbp1276TTT CTG CAG CCT Agtgagtacccc......cctcctctagGT GGC TCC ACA GGA3232390TCC ATC ACC CAG Ggtgagtccagc......ctctccccagCT GTC ACG ACC ACC15,4553219AGG AGA AGG AAA Ggtaagtgccca......ccccctacagGT CAG CAG CGG ACT7772434ACA ACC CCA GCC AGgtgagtgctgg......tcccatacagG GAA CCC TTC CAA1378550ATC AGG AAT GAA Ggtgagtcctt......ttattcttagGA CAA AAT ACA GAT240632CTA AAT CCC AAGgtaagcaatca......tctcgcccagGAT GAC GGC ATC1727308The nucleotide positions within the RG161A02 human genome clone are as follows: exon 1, 170751–171026; exon 2, 171350–171739; exon 3, 187194–187412; exon 4, 195185–195218; exon 5, 196597–196646; exon 6, 196887–196918; exon 7, 197091–197398 (polyadenylation signal at 197381). The “gt-ag” delimiting each intron is underlined. Open table in a new tab Table IIIIntron-exon splice junction sites of the human PILRβ geneExoSizeSplice donorSplice acceptorIntron sizebpbp1≥352...CTG CAG CCT Ggtgagtaccca......cttcctctagGT GGC TCC AC...3232390...ATC ACC CAG Ggtgagtccagc......ggcccagcagCT GTC ACA AC...2573201...AGA AGG AAA Ggtaagtgccca......cctcctgcagGT AGC AGG GC...78114≥481The nucleotide positions within the RG161A02 human genome clone are as follows: exon 1, ∼155,321–155,672; exon 2, 155,996–156,385; exon 3, 156,643–156,843; exon 4, 164,655–165,135 (polyadenylation signal at 165,111). The “gt-ag” delimiting each intron is underlined. Open table in a new tab The nucleotide positions within the RG161A02 human genome clone are as follows: exon 1, 170751–171026; exon 2, 171350–171739; exon 3, 187194–187412; exon 4, 195185–195218; exon 5, 196597–196646; exon 6, 196887–196918; exon 7, 197091–197398 (polyadenylation signal at 197381). The “gt-ag” delimiting each intron is underlined. The nucleotide positions within the RG161A02 human genome clone are as follows: exon 1, ∼155,321–155,672; exon 2, 155,996–156,385; exon 3, 156,643–156,843; exon 4, 164,655–165,135 (polyadenylation signal at 165,111). The “gt-ag” delimiting each intron is underlined. Northern blot analysis of selected human tissues revealed a strong signal in peripheral blood leukocytes and lower intensity signals in lung, spleen, and placenta (Fig.8 A). Analysis of various human cell lines revealed a signal in a two B cell lines, e.g.WIL2-NS and 6F11 (Fig. 8 B), whereas all other cells tested were negative for PILR. Due to the high nucleotide sequence identity of the two genes and the similar length of the two transcripts, the Northern blots do not allow us to discriminate which of the two forms, e.g. PILRα orPILRβ, is expressed in these tissues and cell lines. To resolve this question, PCR with a forward oligonucleotide primer whose sequence was common to both PILRα and PILRβ(HUKF1, 5′-GGGAAGCTTATGGGTCGGCCCCTGCTGCTGCCC) and reverse primers specific for each form, HUKR3 for PILRα(5′-CCATCTCGAGTTTTGAGAGGGCTG) and HUKR5 for PILRβ(5′-TCCTCCGGGGCTAATACACATCC), were performed on reverse transcribed single-stranded cDNA from various tissues including colon, leukocyte, ovary, prostate, small intestine, spleen, testis, thymus, placenta, and mammary gland. The results indicate that bothPILRα and PILRβ were expressed,e.g. were paired, at the mRNA level in the individual samples (results not shown). The cytosolic phosphatase SHP-1 contains tandem SH2 domains that bind tyrosine-phosphorylated proteins and, thus, by virtue of its catalytic subunit, gets recruited as an effector enzyme in a signaling pathway initiated by activated tyrosine kinase receptors (26.Pawson T. Nature. 1995; 373: 477-478Crossref PubMed Scopus (41) Google Scholar). Specificity in tyrosine kinase signaling pathways is critical and is often dictated by the intrinsic affinity of SH2 domains for the flanking sequences of phosphotyrosine residues. The ITIM consensus sequence (S/L/I/V)- XYXX(L/V), via its recruitment" @default.
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