Malignant melanoma cells must penetrate the basement membrane barrier both when they escape from the primary tumor and when they arrest in the microvasculature at distant sites. As tumor cells invade basement membrane, they interact with laminin, a major basement-membrane-specific glycoprotein that promotes cell attachment and migration. This interaction is mediated by multiple types of adhesion receptors. A novel integrin receptor complex that specifically binds laminin has recently been identified on human and mouse melanoma cells. This heterodimer complex has been tentatively designated alpha7beta1. The overall objectives of this proposal are to isolate and further characterize this unique melanoma- associated integrin, and to determine how this receptor modulates cell behavior on laminin and basement membrane substrates. The alpha7beta1 receptor complex will be purified from human melanoma cells by preparative ligand affinity chromatography for detailed immunochemical and biochemical analysis. The expression and distribution of the receptor in various tissues will be determined. Levels of the alpha7 receptor in normal and dysplastic nevi, and in primary and metastatic melanoma will be compared in order to establish a possible link between receptor expression and tumor progression. The distinctive alpha subunit will be compared with the other known integrin beta1-associated alpha subunits by peptide mapping and N- terminal amino acid sequence analysis. How the alpha7 and beta1 subunits are processed by post-translational modifications and when ligand binding activity is acquired will be followed. Studies with isolated subdomains of laminin will identify the specific sites that bind the alpha7 receptor. Antibodies that block receptor function will be developed and tested for their effects on adhesion, migration, and invasion. Integrin receptor profiles will be compared in sets of established human and mouse melanoma cell lines with high and low metastatic potential. Panels of variant tumor cell lines with altered receptor profiles will be selected from parental cell populations by flow cytometry and tested for their invasive behavior. These studies should increase our understanding of how laminin-binding receptors, such as the novel alpha7 complex, influence the metastatic behavior of malignant melanoma cells.
Showing the most recent 10 out of 14 publications
