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.