Platelet Endothelial Aggregation Receptor 1 (PEAR1), a Novel Epidermal Growth Factor Repeat-containing Transmembrane Receptor, Participates in Platelet Contact-induced Activation

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 ␣ IIb ␤ 3 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 ␣ IIb ␤ 3 -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 ␣ IIb ␤ 3 , 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 ␣-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 ␣ IIb ␤ 3 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 o...

A functional genomics approach reveals novel quantitative trait loci associated with platelet signaling pathways

“…Interestingly however, it was postulated by Herrera-Galeano et al 44 that increased expression of PEAR1 might be an important cause of hyperactivity. Here, we have demonstrated that genetic variation within PEAR1, particularly rs41299597, which codes for the S802C amino acid substitution in a potential phosphorylation site in the cytoplasmic domain, 43 is associated with an increased expression of PEAR1 in activated platelets as well as increased responsiveness through the GPVI receptor. That 2 of the 4 SNPs in PEAR1 were identified as a result of exon sequencing highlights the importance of the identification of rare SNPs in genotype-phenotype studies.…”

“…The PEAR1 gene has only recently been described in platelets and endothelial cells. 43 Herrera-Galeano et al recently reported an association between rs2768759, a SNP 10 kb upstream of PEAR1 that maps to the NTRK1 gene, and platelet aggregation in response to collagen and epinephrine in a white American cohort and to ADP and epinephrine in a black American cohort. 44 Neither this SNP, nor SNPs that are in LD, were included in the genotyping.…”

PEAR1 attenuates megakaryopoiesis via control of the PI3K/PTEN pathway

“…Expression was quantified via the DDCt method 14 and expressed in arbitrary units (defined in figure legends) or in percentage compared with control. Expression of genes included in the PI3K/Akt (reference PAHS-058A) and Notch (reference PAHS-059A) RT 2 Profiler signaling was analyzed by qRT-PCR (SABioscience, Qiagen). Cell populations transduced by control lentivirus were used as a reference for LV_miR_PEAR1-transduced cells.…”

“…1,2 We recently reported that interactions between PEAR1, Src family kinases (C-src and Fyn), and the p85/phosphatidylinositol 3-kinase 3-kinase (PI3K) subunit constitute a signaling complex, causing sustained aIIbb3 activation via Akt phosphorylation. 1 In addition to its role in platelet function, a potential role for PEAR1 in hematopoietic cell regulation has been proposed.…”

Allele-specific DNA methylation reinforces PEAR1 enhancer activity

“…1 In platelets, PEAR1 is involved in cell activation through its engagement in sustaining activation of the platelet integrin, a IIb b 3 , 2 and platelet FceR1a has been identified as one of its activating ligands.…”