Integrins are a large family of alpha-beta heterodimeric cell surface receptors that mediate cell adhesion to extracellular matrix proteins and to other cells. In the immune system, integrins play a critical role in lymphocyte recirculation, leukocyte influx into inflammatory sites, lymphocyte migration through lymph nodes and other extravascular tissues, and in cell-cell interactions critical for the development of cellular and humoral immunity. The long-term objective of this application is to understand the molecular basis by which the beta1 integrin subfamily of receptors contributes to lymphocyte function. Specifically, this request for continuing support will address the interplay between beta1 integrins and chemokines in regulating beta1 integrin-dependent T cell migration. The specific hypothesis to be tested is that T cell migration in response to a chemotactic gradient is regulated by beta1 integrin subunit-dependent and chemokine-dependent tyrosine phosphorylation of the hematopoietic cell- specific adapter proteins SLAP-130 and SLP-76 respectively. The resulting SLAP-130/SLP-76 phosphoprotein complex subsequently serves to regulate the actin cytoskeleton.
In Aim 1, a novel beta1 integrin- deficient T cell line will be used in laminar shear flow assays and in in vitro migration assays to define the motifs in the beta1 integrin cytoplasmic domain that are critical for T cell migration, as well as T cell accumulation, shear resistance and rolling/arrest under shear flow.
In Aim 2, the functional significance of beta1 integrin-mediated tyrosine phosphorylation of SLAP-130 will be addressed by determining the role of the fyn tyrosine kinase in phosphorylating SLAP-130 in response to beta1 integrin stimulation, and the domains of SLAP-130 that are critical for SLAP-130-mediated regulation of T cell migration and adhesion under shear flow.
In Aim 3, the role of chemokine signaling to the tyrosine kinase ZAP-70 and SLP-76 in regulating of leukocyte migration and adhesion under shear flow will be addressed using over-expression strategies and SLP-76-deficient macrophages. The role of chemokines and beta1 integrins in regulating the actin cytoskeleton via SLAP-130 and SLP-76 will also be addressed. These studies will identify novel pathways of beta1 integrin and chemokine signaling in lymphocytes, as well as novel functions for SLAP-130 and SLP-76. Identification of signaling pathways that regulate cell migration is critical to the development of therapeutic strategies that can modulate lymphocyte recirculation and leukocyte influx into inflammatory sites.
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