We have previously shown that malignant astrocytoma cells synthesize vitronectin (VN) and express two VN binding integrins alpha(v)beta3 and alpha(v)beta5, in vivo and in vitro, which promote VN-dependent adhesion and migration, and mark the malignant astrocyte phenotype. We now have data demonstrating that these integrins internalize VN, and that platelet derived growth factor (PDGF) rapidly and specifically stimulates both 125I VN degradation and VN-directed migration of malignant astrocytoma cells. In addition, PDGF induces localization of integrin alpha(v)beta5 focal contacts to the cell cortex without changing integrin density or stimulating mitosis. Therefore, we hypothesize that astrocytoma integrin alpha(v)beta3 and alpha(v)beta5 functions are PDGF-driven and promote astrocytoma cell sampling of and movement through the extracellular matrix. In this proposal, we will examine 1) the role of autocrine and exogenous PDGF in regulating 125I-VN turnover and internalization by astrocytoma cell monolayers, utilizing 125I-VN-binding and internalization assays, 2) the role of PDGF in regulating integrin alpha(v)beta3 and alpha(v)beta5-dependent VN-directed motility and focal contact localization, utilizing migration and scrape motility assays, and immunofluorescent analysis, and 3) the role of the beta3 subunit NPLY and NITY cytoplasmic tail sequences, and the alpha(v) cytoplasmic tail, in internalization of ligated alpha(v)beta3 by creating beta3 cDNA constructs containing trunk and point mutations in the NPLY and NITY beta3 sequences, as well as trunk mutations of the alpha(v) cytoplasmic tail, transfecting the beta3 constructs into an astrocytoma cell line lacking beta3 and the alpha(v) constructs into a melanoma cell line lacking alpha(v), followed by internalization assays. As the incidence of malignant astrocytoma is increasing and the prognosis is very poor, understanding how these tumors invade is crucial. Completion of these studies will delineate the role of PDGF in regulating malignant astrocytoma cell VN-binding integrin internalization and motility, and should be relevant to understanding VN-binding integrin function in other malignancies as well as potentially the developing brain.
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