Journal Article

Molecular proximity of seprase and the urokinase-type plasminogen activator receptor on malignant melanoma cell membranes: dependence on β<sub>1</sub> integrins and the cytoskeleton

Vira V. Artym, Andrei L. Kindzelskii, Wen-Tien Chen and Howard R. Petty

in Carcinogenesis

Volume 23, issue 10, pages 1593-1602
Published in print October 2002 | ISSN: 0143-3334
Published online October 2002 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/23.10.1593
Molecular proximity of seprase and the urokinase-type plasminogen activator receptor on malignant melanoma cell membranes: dependence on β1 integrins and the cytoskeleton

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Previous studies have shown that several proteolytic enzymes are associated with membrane protrusions at the leading edge of migrating tumor cells. In this study we demonstrate that seprase and the urokinase plasminogen activator receptor (uPAR), co-localize in the plasma membrane of LOX malignant melanoma cells. Cells were labeled with fluorochrome-conjugated monoclonal antibodies (mAb) directed against seprase and uPAR. Proximity between these two molecules was detected with resonance energy transfer (RET) imaging, single-cell emission spectrophotometry, and single-cell excitation spectrophotometry. Significant RET signals were detected on LOX cells when adherent to uncoated and extracellular matrix (ECM)-coated surfaces. This indicates that seprase and uPAR are within ∼7 nm in the plasma membrane of LOX cells. When LOX cells adhered to a 3D extracellular-like matrix, seprase–uPAR complexes were found to be associated with invadopodia. Further microscopy experiments demonstrated gelatinolytic activity, a functional attribute of seprase, in association with seprase–uPAR membrane domains. Formation of seprase–uPAR membrane complexes is dependent upon both the cytoskeleton and integrins. Specifically, the involvement of β1-integrins was demonstrated by the inhibition of RET by an inhibitory anti-β1-integrin mAb. Based on these findings, we speculate that formation of heterogeneous lytic domains in the invading membranes of LOX cells increases the efficiency of directed pericellular proteolysis.

Keywords: AMCA, 7-amino-4-methylcoumarin-3-acetic acid; ECM, extracellular matrix; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; RET, resonance energy transfer; TRITC, tetramethylrhodamine isothiocyanate; uPAR, urokinase plasminogen activator receptor

Journal Article.  6041 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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