In order to mediate an efficient response to a foreign pathogen, T lymphocytes utilize adhesion receptors to transiently interact with and respond to other cells and proteins found in the external environment. Beta 1 or VLA integrins mediate the adhesion of cells to other cells and to extracellular matrix components. Beta 1-mediated interactions play an important role in embryogenesis and tumor growth and metastasis as well as T cell activation and localization of specific anatomic sites. Since T cell movement dictates that T cells be able to alternate between adhesive and nonadhesive states, beta 1 integrin functional activity on T cells is tightly regulated. Of particular importance to the understanding of the role of integrins in T cell function is the finding that stimulation of several different T cell antigens (integrin regulators) upregulates integrin functional activity within minutes without corresponding increases in integrin cell surface expression. The broad objective of this competing renewal application remains the elucidation of intracellular responses triggered by T cell integrin regulators that mediate rapid upregulation of beta 1 integrin functional activity.
Aims 1 and 2 of this proposal address the hypothesis that the intracellular lipid kinase phosphatidylinositol 3-kinase (PI 3-K) is a common downstream mediator of integrin upregulation mediated by three different integrin regulators, the CD2, CD7 and CD28 antigens. The experimental approach will be to use gene transfer to assess the effect of cytoplasmic domain mutations on: 1) integrin upregulation mediated by antibody stimulation of these molecules, 2) association with PI 3-K, 3) PI 3-K enzymatic activity, and 4) protein kinase C activity.
Aim 1 will specifically test the hypothesis that CD2 association with PI 3-K occurs via a novel association between a currently undefined proline-rich sequence in the CD2 cytoplasmic domain and the SH3 domain on the p85 subunit of PI 3-K.
Aim 2 will use this approach to test the hypothesis that the PI 3-K binding motifs found in the CD7 and CD28 cytoplasmic domains are required for these molecules to regulate beta 1 integrin function. In both aims, the emphasis will be on investigation of the functional significance of PI 3-K on the ability of these molecules to regulate integrin activity.
Aim 3 exploits a panel of T cell mutants that have been isolated that express normal levels of beta 1 integrins but fail to show increased beta 1 integrin function upon stimulation. Expression cloning and panning will be used to identify and subsequently characterize genes that can restore activation-dependent upregulation of integrin function in these mutants. These studies will provide new insights into the biochemical mechanism mediating activation-dependent integrin upregulation in T cells. In addition to demonstrating a role of PI 3-K in integrin regulation, these studies will also idenify novel molecules involved in this signaling event. These studies also provide important model systems capable of further elucidation of the precise sequence of intracellular events that ultimatlely culminate in increased integrin function. Finally, these studies identify intracellular targets that might serve for the development of therapeutics capable of specific modulation of integrin functions.
Showing the most recent 10 out of 12 publications
