Integrins mediate cell adhesion and signaling during physiological responses to vascular injury, as exemplified by the requirement for platelet ?IIb?3 in hemostasis. Furthermore, evidence indicates that integrins may function abnormally in pathological circumstances, such as arterial thrombosis and neoplasia. A key regulatory step in integrin function is receptor activation, manifest by conversion from a low- to a high-affinity conformational state, and from an unclustered to a clustered, high-avidity state. Integrin activation is regulated by "inside-out" signals that are triggered by cellular agonists, although te precise nature of these signals remains incompletely understood. The long-term objectives of this proposal are to 1) fully understand the molecular basis of inside-out ?IIb?3 signaling in platelets;and 2) extend these fundamental concepts to integrins in human cancer cells, since these integrins have been implicated in tumor progression, including the metastatic cascade.
Two specific aims will address major unresolved questions pertaining to these objectives.
Aim 1 will test the hypothesis that a specific molecular adapter protein, ADAP, interacts functionally if not physically with one or more recently identified integrin-proximal regulatory proteins (e.g., talin, kindlin), to activate ?IIb?3 in response to platelet agonists. Accordingly, biochemical and advanced imaging techniques will be used to determine whether and how ADAP interacts with such integrin-proximal regulators in platelets, in gene-targeted primary murine megakaryocytes, and in model cells engineered to recapitulate ?IIb?3 activation. These experimental systems will also be used to assess the role of ADAP in promoting changes in ?IIb?3 clustering as a mechanism of ?IIb?3 activation complementary to affinity modulation.
Aim 2 will test the hypothesis that activation of ?1 integrins is required for tumor cell extravasation from blood vessels and for the development of metastatic foci. Preliminary data indicate the presence of activated ?1 integrins in human cancers. Therefore, human melanoma and breast cancer cell lines and a well-characterized vertebrate experimental metastasis model system will be used to address which ?1 integrins must become activated to promote metastasis, whether specific integrin-interacting proteins such as talin, kindlin and Src family kinases are involved, and whether deletion of a specific tumor suppressor gene, DLC-1, promotes metastasis by activating ?1 integrins. The proposed studies will establish common and unique mechanisms of integrin activation in physiological and pathological circumstances, with diagnostic and therapeutic implications for vascular biology and beyond.
Integrin adhesion receptors are regulated by inside-out signals, a form of regulation exemplified by activation of platelet ?IIb?3, an integrin that promotes physiological hemostasis and pathological thrombosis following blood vessel injury. Abnormal inside-out signaling may also lead to ?1 integrin activation in tumor cells and promote the metastatic spread of these cells to distant organs. Thus, by determining molecular mechanisms of inside-out integrin signaling, these studies have potential diagnostic and therapeutic implications for vascular biology and neoplasia.
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