title: An investigation of hierachical protein recruitment to the inhibitory platelet receptor, G6B-b.
abstract: Platelet activation is regulated by both positive and negative signals. G6B-b is an inhibitory platelet receptor with an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). The molecular basis of inhibition by G6B-b is currently unknown but thought to involve the SH2 domain-containing tyrosine phosphatase SHP-1. Here we show that G6B-b also associates with SHP-2, as well as SHP-1, in human platelets. Using a number of biochemical approaches, we found these interactions to be direct and that the tandem SH2 domains of SHP-2 demonstrated a binding affinity for G6B-b 100-fold higher than that of SHP-1. It was also observed that while SHP-1 has an absolute requirement for phosphorylation at both motifs to bind, SHP-2 can associate with G6B-b when only one motif is phosphorylated, with the N-terminal SH2 domain and the ITIM being most important for the interaction. A number of other previously unreported SH2 domain-containing proteins, including Syk and PLCγ2, also demonstrated specificity for G6B-b phosphomotifs and may serve to explain the observation that G6B-b remains inhibitory in the absence of both SHP-1 and SHP-2. In addition, the presence of dual phosphorylated G6B-b in washed human platelets can reduce the EC(50) for both CRP and collagen.
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom. carmen.coxon@dpag.ox.ac.uk
authors.forename: Carmen H
authors.initials: CH
authors.lastname: Coxon
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: Sadler
authors.forename: Jiandong
authors.initials: J
authors.lastname: Huo
authors.forename: R Duncan
authors.initials: RD
authors.lastname: Campbell
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D019921:Biotinylation; D001792:Blood Platelets / Q000378:metabolism; D019556:COS Cells; D006224:Cricetinae; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010455:Peptides / Q000737:chemistry; D051966:Phospholipase C gamma / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11 / Q000737:chemistry; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6 / Q000737:chemistry*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011971:Receptors, Immunologic / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D000072377:Syk Kinase; D014443:Tyrosine / Q000737:chemistry
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; C119883:MPIG6B protein, human; D010455:Peptides; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D051966:Phospholipase C gamma