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• W2034125855 abstract "The present study was designed to identify novel membrane proteins that signal during platelet aggregation. Because one putative mechanism for signaling by a membrane protein involves phosphorylation, we used oligonucleotide-based microarray analyses and mass spectrometric proteomics techniques to specifically discover membrane proteins and also identify those proteins that become phosphorylated on tyrosine, threonine, or serine residues upon platelet aggregation. Surprisingly, both techniques converged to identify a novel membrane protein we have termed PEAR1 (platelet endothelial aggregation receptor 1). Sequence analysis of PEAR1 predicts a type-1 membrane protein, 15 extracellular epidermal growth factor-like repeats, and multiple cytoplasmic tyrosines. Analysis of the tissue distribution of PEAR1 showed that it was most highly expressed in platelets and endothelial cells. Upon platelet aggregation induced by physiological agonists, PEAR1 became phosphorylated on tyrosine (Tyr-925), and serine (Ser-953 and Ser-1029) residues. PEAR1 tyrosine phosphorylation was blocked by eptifibatide, an αIIbβ3 antagonist, which inhibits platelet aggregation. Immune clustering of PEAR1 resulted in PEAR1 phosphorylation. Aggregation-induced PEAR1 tyrosine phosphorylation lead to the subsequent association with the ShcB adaptor protein. Platelet proximity induced by centrifugation also induced PEAR1 tyrosine phosphorylation, a reaction not inhibited by eptifibatide. These data suggest that PEAR1 is a novel platelet receptor that signals secondary to αIIbβ3-mediated platelet-platelet contacts. The present study was designed to identify novel membrane proteins that signal during platelet aggregation. Because one putative mechanism for signaling by a membrane protein involves phosphorylation, we used oligonucleotide-based microarray analyses and mass spectrometric proteomics techniques to specifically discover membrane proteins and also identify those proteins that become phosphorylated on tyrosine, threonine, or serine residues upon platelet aggregation. Surprisingly, both techniques converged to identify a novel membrane protein we have termed PEAR1 (platelet endothelial aggregation receptor 1). Sequence analysis of PEAR1 predicts a type-1 membrane protein, 15 extracellular epidermal growth factor-like repeats, and multiple cytoplasmic tyrosines. Analysis of the tissue distribution of PEAR1 showed that it was most highly expressed in platelets and endothelial cells. Upon platelet aggregation induced by physiological agonists, PEAR1 became phosphorylated on tyrosine (Tyr-925), and serine (Ser-953 and Ser-1029) residues. PEAR1 tyrosine phosphorylation was blocked by eptifibatide, an αIIbβ3 antagonist, which inhibits platelet aggregation. Immune clustering of PEAR1 resulted in PEAR1 phosphorylation. Aggregation-induced PEAR1 tyrosine phosphorylation lead to the subsequent association with the ShcB adaptor protein. Platelet proximity induced by centrifugation also induced PEAR1 tyrosine phosphorylation, a reaction not inhibited by eptifibatide. These data suggest that PEAR1 is a novel platelet receptor that signals secondary to αIIbβ3-mediated platelet-platelet contacts. Platelet aggregation during arterial thrombosis results in ischemic complications precipitating in acute myocardial infarction and stroke. Platelet aggregation is known to be mediated by signaling events initiated by primary platelet agonists such as thrombin, ADP, and collagen, which induce a conformational change in the platelet integrin αIIbβ3, allowing it to bind soluble fibrinogen and von Willebrand factor, resulting in platelet cross-linking. Platelet-platelet contacts during aggregation subsequently initiate secondary signaling events. Aggregation-induced signaling can result in multiple platelet secondary signaling events such as calcium mobilization, protein tyrosine phosphorylations, cytoskeletal rearrangements, and the release of platelet-dense bodies and α-granules. Aggregation-induced signaling is key to the formation of stable aggregates, particularly when aggregation is induced by low concentrations of one or more primary agonists. Platelet activation also causes the release of ADP from dense bodies and the generation of thromboxane A2, both of which induce further platelet stimulation.Several mediators of aggregation-induced signals have been identified. One is αIIbβ3 itself, which becomes tyrosine-phosphorylated and also associates with numerous signaling and cytoskeletal proteins following platelet activation, allowing fibrinogen and/or von Willebrand factor binding and platelet aggregation. The importance of αIIbβ3 “outside-in” signaling in the enhancement of platelet aggregation was demonstrated by the generation of knock-in mice where tyrosine residues Tyr-747 and Tyr-759 were mutated to phenylalanine (1Law D.A. DeGuzman F.R. Heiser P. Ministri-Madrid K. Killeen N. Phillips D.R. Nature. 1999; 401: 808-811Crossref PubMed Scopus (269) Google Scholar). These so-called DiYF mice displayed selective impairment of outside-in signaling, resulting in the formation of unstable aggregates. Other mediators are released from the activated platelets. One is the soluble CD40 ligand, a hydrolytic product produced by metalloprotease cleavage of the CD40 ligand on activated platelets that subsequently binds to αIIbβ3. Another is GAS6, a protein released from α-granules that is involved in the stabilization of platelet-rich thrombi (2Andre P. Prasad K.S. Denis C.V. He M. Papalia J.M. Hynes R.O. Phillips D.R. Wagner D.D. Nat. Med. 2002; 8: 247-252Crossref PubMed Scopus (644) Google Scholar, 3Angelillo-Scherrer A. de Frutos P. Aparicio C. Melis E. Savi P. Lupu F. Arnout J. Dewerchin M. Hoylaerts M. Herbert J. Collen D. Dahlback B. Carmeliet P. Nat. Med. 2001; 7: 215-221Crossref PubMed Scopus (356) Google Scholar).Although the importance of each of the platelet secondary signaling reactions described above is well documented, these reactions are dependent upon platelet activation. It has also been well documented, however, that platelet stimulation can be induced by platelet-platelet contact. Signaling of platelet receptors induced by platelet-platelet proximity, independent of platelet aggregation, have not been described. One exception may be the Eph kinases and ephrins, specifically EphA4 and ephrinB1, which, through receptor-ligand interactions on the platelet surface, enhance the binding of αIIbβ3 to immobilized fibrinogen in the presence of physiological agonists (4Prevost N. Woulfe D. Tanaka T. Brass L.F. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9219-9224Crossref PubMed Scopus (110) Google Scholar, 5Prevost N. Woulfe D. Tognolini M. Brass L.F. J. Thromb. Haemost. 2003; 1: 1613-1627Crossref PubMed Scopus (42) Google Scholar); however, the importance of this mechanism in platelet-platelet signaling on unstimulated platelets is unknown.The present study was designed to identify novel platelet proteins involved in platelet proximity-induced activation. Reasoning that many signaling receptors become tyrosine-phosphorylated during signaling, we sought not only to identify novel receptors on platelets but to specifically identify those that become phosphorylated upon platelet-platelet interactions, independent of the activation state of the platelet. Initially, we used a bioinformatics approach and profiled platelet RNA on oligonucleotide-based microarrays to identify novel membrane proteins. We also used LC/MS/MS 1The abbreviations used are: LC/MS/MS, liquid chromatography tandem mass spectrometry; aa, amino acid(s); ECD, extracellular domain; EGF, epidermal growth factor; FITC, fluorescein isothiocyanate; HUVEC, human umbilical vein endothelial cell; HRP, horseradish peroxidase; ICD, intracellular domain; PEAR1, platelet endothelial aggregation receptor 1; hPEAR1, human PEAR1; mPEAR1, mouse PEAR1; PTB, phosphotyrosine binding; SREC, scavenger receptor expressed by endothelial cells; TRAP, thrombin receptor activating peptide. 1The abbreviations used are: LC/MS/MS, liquid chromatography tandem mass spectrometry; aa, amino acid(s); ECD, extracellular domain; EGF, epidermal growth factor; FITC, fluorescein isothiocyanate; HUVEC, human umbilical vein endothelial cell; HRP, horseradish peroxidase; ICD, intracellular domain; PEAR1, platelet endothelial aggregation receptor 1; hPEAR1, human PEAR1; mPEAR1, mouse PEAR1; PTB, phosphotyrosine binding; SREC, scavenger receptor expressed by endothelial cells; TRAP, thrombin receptor activating peptide. to identify platelet proteins that become phosphorylated upon platelet aggregation. Surprisingly, both techniques converged on a novel 1034-amino acid protein we termed PEAR1 (platelet-endothelial aggregation receptor 1). Bioinformatic analyses revealed that PEAR1 is a type 1 membrane protein containing fifteen EGF-like repeats in its extracellular domain. The intracellular domain contains five proline rich domains, which may interact with Src homology 3 domain-containing proteins, as well as four potential tyrosine phosphorylation sites (Tyr-804, Tyr-925, Tyr-943, Tyr-979). The LC/MS/MS data demonstrated that PEAR1 is, in fact, tyrosine-phosphorylated at Tyr-925. We further demonstrated that PEAR1 becomes tyrosine phosphorylated in response to receptor clustering and during platelet aggregation. Finally, we showed that the PEAR1 signaling is induced through platelet-platelet contacts independent of platelet activation. These data are the first demonstration of a platelet receptor to signal through platelet-platelet contacts independent of platelet activation.MATERIALS AND METHODSReagents—The following reagents were purchased from the suppliers named in parentheses: anti-Myc (9E10) monoclonal IgG (Covance; Princeton, NJ); goat anti-rabbit HRP and goat anti-mouse HRP (Bio-Rad); anti-PY99 monoclonal IgG (Santa Cruz Technologies, Santa Cruz, CA); anti-phospho-Src (Tyr-416) polyclonal IgG, anti-Shc polyclonal IgG, and anti-Shc monoclonal IgG (Cell Signaling Technology, Beverly, MA); 4G10 anti-PY monoclonal antibody (Upstate Cell Signaling Solutions); anti-CD62-P and anti-PY20 monoclonal antibodies and isotype-specific anti-mouse and anti-rabbit antibodies (BD Biosciences); IV.3 monoclonal antibody hybridoma (American Type Culture Collection, Manassas, VA); thrombin (Hematologic Technology, Essex Junction, VT); Collagen (Chronolog Corp., Havertown, PA); TRAP (SynPep Corp., Dublin, CA), eptifibatide, (Millennium Pharmaceuticals, Cambridge, MA); Easy-FITC kit (Pierce); Superscript II reverse transcriptase, platinum Pfx DNA polymerase, and pCR-BluntII-Topo vector (Invitrogen); goat anti-rabbit/mouse HRP (Bio-Rad); ECL chemiluminescent detection kit and protein G-Sepharose (Amersham Biosciences); Restore stripping buffer (Pierce); and Immobilon-P polyvinylidene difluoride membranes (Millipore).Purification of Platelet RNA—Platelets were collected from a healthy volunteer by apheresis, including a filtration step that removed lymphocyte contaminants following an Institutional Review Board-approved protocol. This procedure yielded ∼250 ml of platelets (∼1 × 109 platelets/ml). Platelets were diluted 1:4 with CGS (13 mm sodium citrate, 30 mm glucose, and 0.12 m sodium chloride) containing 0.1 μg/ml prostacyclin (PGI2). Platelets were pelleted by spinning for 20 min at 2200 rpm in a Beckman Coulter Allegra 6 centrifuge at room temperature. The supernatant was removed, and the platelet pellet was resuspended in RNA-STAT-60 (Tel-Test) using 1 ml of RNA-STAT-60 per 2.9 × 108 platelets and immediately vortexed to facilitate platelet lysis. The platelet lysate was further processed according to RNA-STAT-60 protocol specifications. Total RNA was then treated with RNA Easy (Qiagen) for further purification.Identification of PEAR1 cDNA and Full-length Cloning—5 μg of DNase-treated platelet RNA was converted to cRNA, fragmented, and hybridized to the Millennium Pharmaceuticals cardiovascular custom 60-mer oligonucleotide array per the manufacturer's suggested protocol (Affymetrix, Santa Clara, CA). The probe sets contained on the custom array represented 25,000 genes that were selected based on publicly available sequence information and in-house sequencing efforts of endothelial and platelet cDNA libraries. Data were analyzed with MASv5 (Affymetrix) and Rosetta Resolver (Rosetta Biosoftware). cRNA hybridized to probe sets with intensity p values of ≤0.01 were identified. Platelet cRNA hybridized to a probe set for FLJ00193, and through bioinformatic analyses it was determined that the FLJ00193 cDNA encoded an incomplete open reading frame that contained two EGF repeats and a potential transmembrane domain. A proprietary in-house DNA sequence and a publicly available DNA sequence were used to assemble a 3.6-kb predicted cDNA. Platelet cDNA was synthesized by using Superscript II reverse transcriptase. PEAR1 cDNA was amplified using Platinum Pfx DNA polymerase and the primers 5′-GCAGGCTTCATATCCTGAACGCTG-3′ (forward) and 5′-GCTCTAGATTAACGGTCCTGGCGTCGAAGTGGAGGTGATG-3′ (reverse). The resulting 3.2-kb cDNA was cloned into pCR-BluntII-Topo vector.Generation and Purification of Anti-peptide Antibodies—Anti-PEAR1 rabbit polyclonal antibodies were generated by BIOSOURCE using keyhole limpet hemocyanin-conjugated peptides derived from the N-terminal extracellular domain (aa 72–88; YRTVYRQVVKTDHRQRL) and the intracellular domain (aa 856–874, QGHDNHTTLPADWKHRREP). Serum was affinity-purified using the immunizing peptide coupled to a thiol reactive gel. The antibodies raised against peptide 71–85 are referred to as α-extracellular domain (α-ECD) PEAR1 IgGs. The antibodies raised against peptide 864–874 are referred to as α-intracellular domain (α-ICD) PEAR1 IgGs.In Situ Localization of PEAR1 mRNA Using Biotin-labeled Oligonucleotide Probes Detected by ABC Peroxidase—Formalin-fixed, paraffin-embedded human and rodent tissues were used in this study. Digoxigenin-labeled riboprobes were used to localize PEAR1 mRNA. The details of the non-isotopic in situ hybridization and the tyramide-mediated signal amplification, as well as the methods used for acquiring digital images, have been described earlier (6Komuves L.G. Feren A. Jones A.L. Fodor E. J. Histochem. Cytochem. 2000; 48: 821-830Crossref PubMed Scopus (66) Google Scholar, 7Wasserman S.M. Mehraban F. Komuves L.G. Yang R.B. Tomlinson J.E. Zhang Y. Spriggs F. Topper J.N. Physiol. Genomics. 2002; 12: 13-23Crossref PubMed Scopus (108) Google Scholar). Following the manufacturer's protocol (Roche Applied Science), digoxigenin-labeled antisense and sense riboprobes were generated from a DNA template containing nucleotides 2369–2896.TaqMan Analysis—TaqMan experiments were performed using an ABI PRISM 7700 system (Applied Biosystems). Primers were designed using Primer Express software. 50 ng of RNA from a variety of human tissues and primary cell lines were used for analysis. PEAR1 and β-macroglobulin probes were labeled with different fluorescent dyes as per the manufacturer's protocol. β-Macroglobulin was used as an endogenous control to allow for normalization of the amount of RNA added to each reaction. The differential labeling of the target gene (PEAR1) and the reference gene (β-macroglobulin) occurred in the same well.hPEAR1 Constructs, FLAG- and Myc-tagged—The primers 5′-CGGAATTCACCCAGTGATCCCAATACCTGC-3′ (forward) and 5′-GCTCTAGATTAACGGTCCTGGCGTCGAAGTGG-3′ (reverse) were used to PCR-amplify a PEAR1 fragment that coded for amino acids 23–1037. The resulting PCR fragment was digested with EcoR1 and XbaI and subcloned into pFLAG-CMV1 (Sigma) to express a membrane-localized, N-terminally FLAG-tagged PEAR1. A C-terminally Myc-tagged PEAR1 was constructed by PCR amplification using the primers 5′-CGGAATTCAATGTCACCGCCTCTGTGTCC-3′ and 5′-CCGCTCGAGACGGTCCTGGCGTCGAAGTGG-3′. The resulting PEAR1 PCR fragment, which coded for amino acids 1–1037, was digested with EcoR1 and XhoI and subcloned into pCMV-5A (Stratagene).Cell Culture and Transfection—COS-7 cells were maintained in Dulbecco's modified Eagle's media supplemented with 10% fetal bovine serum, 100 μg/ml penicillin, and 100 μg/ml streptomycin. Cells were plated in 6-well dishes (4 × 105 cells/well). After 24 h, transfections were performed using FuGENE 6 (2 μg of DNA per well) for 24 h. The vectors and constructs used in the transfection experiments were pCDNA3 (vector control), pFLAG-CMV-PEAR1 (N-terminal FLAG-tagged PEAR1), pCMV-5A-PEAR1 (C-terminal Myc-tagged PEAR1), and pcDNA3-v-Src.Immunoprecipitations and Western Blotting from COS-7 Cells— Transfected cells were lysed with 0.4 ml of lysis buffer (40 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EGTA, 5 mm MgCl2, 1% Triton X-100, 1 mm sodium orthovanadate, 10 mm NaF, 1 mm Pefabloc, 10 μg/ml leupeptin, and 10 μg/ml aprotinin). For immunoprecipitations, lysates were incubated with 2 μg of the indicated antibodies. For Western analysis, polyvinylidene difluoride membranes were incubated with the indicated antibodies (1 μg/ml) for 2 h at 4 °C. Membranes were then incubated with 1:10,000 dilution of horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG for 1 h at room temperature and developed with the ECL™ chemiluminescent detection kit. Methodologies used for SDS-PAGE, Western blotting, and immunoprecipitations have been described previously (8Cowan K.J. Law D.A. Phillips D.R. J. Biol. Chem. 2000; 275: 36423-36429Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar).Phosphopeptide Identification by In-gel Digestion and Mass Spectrometry—Washed human platelets were aggregated with 5 μm human TRAP as described below and solubilized in 2× lysis buffer (20 mm Tris-HCl, pH 8, 2% Triton X-100, 4 mm EDTA, 250 mm NaCl, 20% glycerol, 1 mm phenylmethylsulfonyl fluoride, 2 mm Na2VO4, 2 μg/ml leupeptin, and 2 μg/ml aprotinin). Solubilized platelet-aggregated proteins from 6 × 109 platelets were incubated with α-phosphotyrosine IgG beads (PY99; Santa Cruz Biotechnology) overnight at 4 °C. The phosphoproteins were eluted with 100 mm phenyl phosphate, resolved by SDS-PAGE, and visualized by Coomassie staining. All Coomassie-stained bands were excised and subjected to in-gel proteolytic digestion with trypsin and analyzed by LC/MS/MS (9Loyet K.M. Stults J.T. Arnott D. Mol. Cell. Proteomics. 2005; 4: 235-245Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Data-dependent LC/MS/MS was performed using electrospray ionization on an LCQ Deca XP ion trap mass spectrometer (Thermo Finnigan). An aliquot of each digest mixture was introduced to the mass spectrometer by reversed-phase chromatographic separation with a 75-μm inner diameter capillary column flowing at a rate of ∼350 nl/min and eluted using a 30-min acetonitrile, 0.1% formic acid gradient. Chromatographic separation yielded ∼30-s peak widths, and mass spectra were acquired in 9-s cycles. Each cycle was of the form consisting of one full mass spectrometry scan followed by four MS/MS scans on the most abundant precursor ions subjected to dynamic exclusion for a period of 1.5 min. The identity of each peptide sequenced was determined by interpreting the MS/MS spectra using the SpectrumMill software (Agilent Technologies).Platelet Preparation—Human venous blood from healthy drug-free donors was drawn into ACD (85 mm citrate, 111 mm glucose, 714 mm citric acid) supplemented with 50 ng/ml prostacyclin (Sigma) and washed as described previously (10Jantzen H.M. Gousset L. Bhaskar V. Vincent D. Tai A. Reynolds E.E. Conley P.B. Thromb. Haemost. 1999; 81: 111-117Crossref PubMed Scopus (124) Google Scholar). Washed platelets were resuspended in Tyrode's solution-Hepes buffer with 1 mm CaCl2 and MgCl2 at a cell density of 1–3 × 108 platelets/ml.Platelet Lysate Preparation—Aggregations were measured in a Chronolog lumiaggregometer. Human washed platelet aggregations (3 × 108 cells/ml) were initiated with 0.5 units/ml thrombin (Hematologic Technology), 10 μg/ml collagen (Chronolog Corp.), or 5 μm human TRAP (SynPep Corp.) and, in some cases, 2 μm eptifibatide (Integrelin®; Millennium Pharmaceuticals) was included. Spun platelet pellets were prepared by centrifugation of human washed platelets (3 × 108 cells/ml) at 14,000 × g for 5 min. Platelet pellets were lysed as described below after various time points. Platelets were lysed in an equal volume of 2× lysis buffer (20 mm Tris-HCl, pH 8, 2% Triton X-100, 4 mm EDTA, 250 mm NaCl, 20% glycerol, 1 mm phenylmethylsulfonyl fluoride, 2 mm Na2VO4, 2 μg/ml leupeptin, and 2 μg/ml aprotinin) and incubated on ice for 30 min. Samples were sonicated for 40 s, and the insoluble fraction was removed by centrifugation at 14,000 × g for 20 min at 4 °C.Immunoprecipitiation and Cross-linking of Surface PEAR-1—Immunoprecipitations were performed with Protein G beads and 2.5 μg/ml α-ICD PEAR1 IgG or isotype control overnight at 4 °C. Immunoblotting of the complexes were performed with α-pTyr-4G10 (Upstate Cell Signaling Solutions) and α-pTyr-PY20 (Santa Cruz Biotechnology) mouse IgGs or α-ECD PEAR1 IgG rabbit IgG. For cross-linking experiments, 3 × 108 platelets/ml were incubated with 5 μg/ml α-ECD PEAR1 IgG in the presence of 25 μg/ml IV.3 monoclonal antibody for 30 min at room temperature followed by cross-linking the bound IgG with α-rabbit secondary antibody for 10 min at 37 °C. Post cross-linking, the reactions were stopped by the addition of ice-cold 2× lysis buffer. Immunoprecipitation of PEAR1 was performed as described above.Flow Cytometry Analysis—α-ECD PEAR1 IgG was FITC-labeled using the EASY-FITC kit. Washed human platelets (1 × 106) were left resting or were activated with 5 μm TRAP for 5 min at 37 °C without stirring. Platelets were fixed with 2% (v/v) paraformaldehyde for 30 min at room temperature and stained with nonspecific rabbit-IgG-FITC, α-ECD PEAR1-FITC, or α-CD62-P (P-selectin)-phycoerythrin. Platelets were analyzed on a FACSort flow cytometer (BD Biosciences).RESULTSIdentification of PEAR1 through Gene Profiling—In an effort to identify novel receptors in human platelets, high throughput molecular profiling of RNA isolated from double-phoresed platelets was performed on a custom array that contained probe sets representing 25,000 genes. The sequences for the probe sets represented on the custom arrays were derived both from sequencing of platelet and HUVEC cDNA libraries as well as all the non-overlapping human genes present in the public gene data base. The protein coding sequences of the platelet cRNAs, which hybridized to the microarrays, were then classified based on PROSITE, Pfam, and SMART (11Apweiler R. Attwood T.K. Bairoch A. Bateman A. Birney E. Biswas M. Bucher P. Cerutti L. Corpet F. Croning M.D. Durbin R. Falquet L. Fleischmann W. Gouzy J. Hermjakob H. Hulo N. Jonassen I. Kahn D. Kanapin A. Karavidopoulou Y. Lopez R. Marx B. Mulder N.J. Oinn T.M. Pagni M. Servant F. Sigrist C.J. Zdobnov E.M. Nucleic Acids Res. 2001; 29: 37-40Crossref PubMed Scopus (804) Google Scholar) sequence-motif searches. The selected probe sets were assigned structural annotations and further subdivided into classes of interest. The following search terms were used to classify the protein sequences of the hybridized cRNAs: predicted signal sequences, potential transmembrane domains, GPCRs (G protein-coupled receptors, kinases, phosphatases, cadherins, EGF domains, fibronectins, Ig domains, SCR (short consensus repeat), leucine rich repeats, CUB (complement Clr/Cls, Uegf, and bone morphogenic protein-1), integrins, chemokines, thrombospondins, and proteases. A search for novel membrane proteins identified a 3282-bp cDNA that contained an open reading frame encoding a novel type 1 membrane protein of 1037 aa (Fig. 1). We termed the protein the platelet endothelial aggregation receptor 1 or PEAR1, based on its primary location on platelets and endothelial cells and its role in aggregation-induced signaling (see below). Homology searches revealed significant overall identity in the extracellular and intracellular domains of human PEAR1, mPEAR1, KIAA1780, and KIAA1781. PEAR1 also displays significant homology to the ECDs of CED-1 (12Zhou Z. Hartwieg E. Horvitz H.R. Cell. 2001; 104: 43-56Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar) and SREC (scavenger receptor expressed by endothelial cells) (13Adachi H. Tsujimoto M. Arai H. Inoue K. J. Biol. Chem. 1997; 272: 31217-31220Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). A bioinformatics analysis of the PEAR1 ECD revealed the presence of 15 EGF-like repeats that vary in length from 39 to 42 aa and contain a consensus sequence of CX1–2GX2GX2–4CX3CX1–3CX1–2GX1–2CX4GX1CX1CX2GX2GX2C. An alignment of the PEAR1 EGF-like repeats is presented in Fig. 2A. The chromosomal localizations and the degree of identities between hPEAR1 and mPEAR1, KIAA1780, KIAA1781, CED-1, and SREC-I are summarized in Table I. The domain structures present in PEAR1 and related proteins are shown in Fig. 2B. Putative signaling motifs present within the ICD of PEAR1 were identified by the Scansite program (14Obenauer J.C. Cantley L.C. Yaffe M.B. Nucleic Acids Res. 2003; 31: 3635-3641Crossref PubMed Scopus (1327) Google Scholar). Five potential Src homology 3-binding, proline-rich domains (Fig. 3) were identified in hPEAR1 and mPEAR1 but not in KIAA1780 or KIAA1781. Human PEAR1, mPEAR1, and KIAA1780 contain an NPXY motif (Fig. 3) that is known to act as interaction sites for PTB domain-containing proteins (15Barnes H. Larsen B. Tyers M. van Der Geer P. J. Biol. Chem. 2001; 276: 19119-19125Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The Scansite program also predicted that the tyrosine residues 804, 925, 943, and 979 present in hPEAR1 may be tyrosine-phosphorylated (Fig. 3). Similar tyrosine residues are also present in mPEAR1, KIAA1780, and KIAA1781. Based on overall homology, it is likely that PEAR1, KIAA1780, and KIAA1781 represent a novel family of EGF repeat-containing proteins. Because of the EGF repeats and their homology to SREC and CED-1, possible functions for this novel family of proteins include cell adhesion (16Bork P. Downing A.K. Kieffer B. Campbell I.D. Q. Rev. Biophys. 1996; 29: 119-167Crossref PubMed Google Scholar), the uptake of acetylated low density lipoprotein (13Adachi H. Tsujimoto M. Arai H. Inoue K. J. Biol. Chem. 1997; 272: 31217-31220Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar), and phagocytosis of apoptotic bodies (12Zhou Z. Hartwieg E. Horvitz H.R. Cell. 2001; 104: 43-56Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar).Fig. 2Alignment of the EGF repeats in hPEAR1 (A) and the domain structures of PEAR1 and related proteins (B). A, the predicted EGF repeats present in PEAR1 were aligned based on a ClustalW analysis. 15 EGF-like domains contain a consensus sequence of CX1–2GX2GX2–4CX3CX1–3CX1–2GX1–2CX4GX1CX1CX2GX2GX2C and vary in length from 39 to 42 aa. The consensus EGF repeat contains six conserved glycine residues and eight conserved cysteine residues, suggesting four disulfide-bonded cysteine pairs in each EGF repeat. The exception is EGF repeat number 4 (EGF#4), which contains six cysteines and, therefore, three potential pairs of disulfide-bonded cysteines. The conserved cysteines and glycine residues are listed underneath the aligned EGF repeats. B, hPEAR1, mPEAR1, KIAA1780, KIAA1781, CED-1, and SREC are predicted to be type 1 membrane proteins. The single predicted transmembrane domains are identified as downward slanting striped boxes. The EGF repeats, which were identified by the Pfam search program (11Apweiler R. Attwood T.K. Bairoch A. Bateman A. Birney E. Biswas M. Bucher P. Cerutti L. Corpet F. Croning M.D. Durbin R. Falquet L. Fleischmann W. Gouzy J. Hermjakob H. Hulo N. Jonassen I. Kahn D. Kanapin A. Karavidopoulou Y. Lopez R. Marx B. Mulder N.J. Oinn T.M. Pagni M. Servant F. Sigrist C.J. Zdobnov E.M. Nucleic Acids Res. 2001; 29: 37-40Crossref PubMed Scopus (804) Google Scholar), are presented as chains of solid black boxes. The intracellular domains of human and mPEAR1, KIAA1780, and KIAA1781 are shown as upward slanting striped boxes to indicate their sequence similarity. The intracellular domains of SREC and CED-1 do not share similarity with PEAR-1 and are represented as white (CED-1) and gray (SREC) boxes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IChromosomal localizations and degree of identities between hPEAR1 and mPEAR1, KIAA1780, KIAA1781, CED-1 and SREC-IChromosomeMessageAmino acidsECD aaICD aaEGF repeatsHomology of aa to PEAR1%hPEAR11q23.13282103775425915100mPEAR1 (AF444274)aGenBank™ accession number.3429010347532571576/77bFirst number is ECD percentage, second number is ICD percentage.hKIAA1780 (NM_032446)aGenBank™ accession number.5q33752211408852611748/34bFirst number is ECD percentage, second number is ICD percentage.hKIAA1781 (NM_032445)aGenBank™ accession number.15q22.257029697731721748/25bFirst number is ECD percentage, second number is ICD percentage.CED-1 (AF332568)aGenBank™ accession number.Unknown378411119102011638/0bFirst number is ECD percentage, second number is ICD percentage.SREC (NM_003693)aGenBank™ accession number.17P13.33457830420384738/0bFirst number is ECD percentage, second number is ICD percentage.a GenBank™ accession number.b First number is ECD percentage, second number is ICD percentage. Open table in a new tab Fig. 3Alignment of the intracellular domains of human PEAR1, mPEAR1, KIAA1780, and KIAA1781 and identification of potential tyrosine phosphorylation sites and interaction motifs. The intracellular domains were aligned based on a ClustalW analysis. Amino acid identi" @default.
• W2034125855 created "2016-06-24" @default.
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• W2034125855 date "2005-07-01" @default.
• W2034125855 modified "2023-10-14" @default.
• W2034125855 title "Platelet Endothelial Aggregation Receptor 1 (PEAR1), a Novel Epidermal Growth Factor Repeat-containing Transmembrane Receptor, Participates in Platelet Contact-induced Activation" @default.
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