In recent years it has become increasingly clear that adapter proteins play a critical role in regulating and integrating signal transduction cascades. Our laboratory has been investigating the biochemistry and molecular biology of SH2 domain containing leukocyte phosphoprotein of 76 kD (SLP-76). This molecule, a substrate of syk family protein tyrosine kinases, has been shown to play a critical role in integrating signals elicited by engagement of the T cell antigen receptor. Recently, we have found that in addition to a T cell defect, mice made deficient in SLP-76 expression by homologous recombination exhibit fetal hemorrage and perinatal mortality. Followup studies indicate that although the mice are only moderately thrombocytopenic, stimulation of platelets from SLP-76-deficient mice fails to result in phosphorylation of PLCgamma2, platelet aggregation, or granule release following stimulation with collagen. The focus of the current proposal is on the role of SLP-76 in platelet function. Experiments are proposed which make use of cell lines, normal human and murine platelets, and the SLP-76-deficient mice to define the structure-function relationships of SLP-76 both in vitro and in vivo. To achieve this overall goal, three specific aims are presented. The first is to determine what ligands induce phosphorylation of SLP-76 following their incubation with platelets. Followup studies are presented to ascertain the sites of phosphorylation of SLP-76 in platelets and to analyze what other proteins associate with this adapter protein following various stimuli. The second specific aim describes experiments to address the consequences of overexpression of wild-type or mutant SLP-76 in megakaryocyte cell lines. Studies are then described to evaluate more fully platelet function in SLP-76 deficient mice and to compare and contrast signaling defects with those found in platelets from mice deficient in the kinase which phosphorylates SLP-76. The final specific aim describes an in vivo approach to define structure-function relationships of SLP-76 by selective reconstitution of wild-type or mutant SLP-76 in the platelet compartment using a megakaryocyte-specific transgenic promoter construct. It is anticipated that these studies collectively will provide new insights into the biology of SLP-76 as a regulator of signaling events in platelets.
