Cancer cells in vivo differ from normal cells in that they have both altered growth which leads to tumor formation and altered interactions with extracellular matrix (ECM) which leads to invasion and metastasis. In vitro, tumor cells which differ in that their growth is independent of both growth factors and adhesion to ECM proteins such as fibronectin (FN). Our previous work, aimed at understanding link between growth and ECM, demonstrated that local clustering of the FN receptor (integrin alpha5 beta1), activates the plasma membrane Na+/H+ exchanger and elevates intracellular PH (pHi). This effect has been implicated in control of growth by growth factors, oncogenes and several second messengers, and strongly suggests that ECM receptors control cell growth in part via chemical signaling pathways. This proposal aims to use pHi as a marker to understand signaling by the alpha5 beta1 integrin, focusing on the functions of their short cytoplasmic domains. First, foreign alpha5 and beta1 integrins will be expressed in mouse 3TIO 1/2 cells. Sequences necessary for their function will be identified by assaying the ability of integrins with mutations in their cytoplasmic domains to elevate pHi, and promote DNA synthesis, cell spreading and organization of cytoskeletal proteins. Second, sequences sufficient for interactions with intracellular proteins will be identified by microinjecting cytoplasmic domain peptides into cells, then assaying effects on pHi, DNA synthesis, spreading and cytoskeletal proteins. Third, intracellular proteins that interact with these sequences will be identified by using immobilized peptides for affinity chromatography of cell extracts. Understanding the sequences and intracellular proteins involved in signaling by integrins is important for understanding both growth in normal cells, and growth, invasion and metastasis by cancer cells.
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