Integrin adhesion receptors play a vital role in cell function and development by mediating the adhesion of cells to other cells and to extracellular matrix proteins. In the immune system, the functional activity of integrins expressed on T lymphocytes is dynamically regulated by the activation state of the T cell in order to promote transient periods of adhesion that facilitate T cell trafficking and antigen recognition in tissue. Stimulation of the antigen-specific CD3/T cell receptor (TCP) complex initiates a signaling cascade that results in a rapid increase in the functional activity of beta l and beta 2 integrins without increases in integrin levels on the cell surface. TCR-mediated integrin activation can be mimicked by phorbol esters such as PMA, implicating effectors downstream of protein kinase C (PKC) in this process. During the prior funding period, we identified a novel adapter protein function for the PKC effector protein kinase D1 (PKD1) in PMA- and TCR-mediated enhancement of beta l integrin function that is associated with increased beta l integrin clustering and activation of the small GTPase Rap1. We propose that the carboxy-terminal end of the beta l integrin cytoplasmic domain regulates integrin function in response to T cell activation by nucleating the formation of a membrane-localized complex consisting of PKD1, Rap1 and the Rap1 guanine nucleotide exchange factor C3G that controls Rap1 activation and subsequent integrin clustering. We propose the use of novel in vitro cell systems and genetically modified mice in order to test this hypothesis and its relevance to T cell function during immune responses in intact animals.
In Aim 1, we will define the structural basis for interactions between the beta l integrin cytoplasmic domain and the PKD1/Rap1/C3G complex and determine if the beta l integrin tail is sufficient to control Rap1 activity.
In Aim 2, we will use conditional gene targeting and adoptive transfer approaches to define the function of beta l integrin expression in controlling integrin- dependent responses of T cells to antigen challenge in mice. We will specifically determine if beta2 integrin function is dependent on expression of a beta l integrin subunit that couples effectively to PKD1 and Rap1.
In Aim 3, we will use RNA interference and adenoviral-mediated gene transfer techniques to elucidate the function of PKD1 in regulating integrin function in vitro and T cell activation responses to antigen in vivo. Together, these studies will define a novel function for both PKD1 and the beta l integrin cytoplasmic domain in regulating integrin function in T cells and will enhance our ability to develop novel therapies that can specifically modulate T cell immune responses.
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