Regulation of α5β1 integrin conformation and function by urokinase receptor binding

Department of Medicine and Pulmonary and Critical Care Division, University of California, San Francisco, San Francisco, CA 94143, USA.
The Journal of Cell Biology (Impact Factor: 9.69). 02/2005; 168(3):501-11. DOI: 10.1083/jcb.200404112
Source: PubMed

ABSTRACT Urokinase-type plasminogen activator receptors (uPARs), up-regulated during tumor progression, associate with beta1 integrins, localizing urokinase to sites of cell attachment. Binding of uPAR to the beta-propeller of alpha3beta1 empowers vitronectin adhesion by this integrin. How uPAR modifies other beta1 integrins remains unknown. Using recombinant proteins, we found uPAR directly binds alpha5beta1 and rather than blocking, renders fibronectin (Fn) binding by alpha5beta1 Arg-Gly-Asp (RGD) resistant. This resulted from RGD-independent binding of alpha5beta1-uPAR to Fn type III repeats 12-15 in addition to type III repeats 9-11 bound by alpha5beta1. Suppression of endogenous uPAR by small interfering RNA in tumor cells promoted weaker, RGD-sensitive Fn adhesion and altered overall alpha5beta1 conformation. A beta1 peptide (res 224NLDSPEGGF232) that models near the known alpha-chain uPAR-binding region, or a beta1-chain Ser227Ala point mutation, abrogated effects of uPAR on alpha5beta1. Direct binding and regulation of alpha5beta1 by uPAR implies a modified "bent" integrin conformation can function in an alternative activation state with this and possibly other cis-acting membrane ligands.

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    ABSTRACT: Binding of type-1 plasminogen activator inhibitor (PAI-1) to cell surface urokinase (uPA) promotes inactivation and internalization of adhesion receptors (e.g., urokinase receptor (uPAR), integrins) and leads to cell detachment from a variety of extracellular matrices. In this report, we begin to examine the mechanism of this process. We show that neither specific antibodies to uPA, nor active site inhibitors of uPA, can detach the cells. Thus, cell detachment is not simply the result of the binding of macromolecules to uPA and/or of the inactivation of uPA. We further demonstrate that another uPA inhibitor, protease nexin-1 (PN-1), also stimulates cell detachment in a uPA/uPAR-dependent manner. The binding of both inhibitors to uPA leads to the specific inactivation of the matrix-engaged integrins and the subsequent detachment of these integrins from the underlying extracellular matrix (ECM). This inhibitor-mediated inactivation of integrins requires direct interaction between uPAR and those integrins since cells attached to the ECM through integrins incapable of binding uPAR do not respond to the presence of either PAI-1 of PN-1. Although both inhibitors initiate the clearance of uPAR, only PAI-1 triggers the internalization of integrins. However, cell detachment by PAI-1 or PN-1 does not depend on the endocytosis of these integrins since cell detachment was also observed when clearance of these integrins was blocked. Thus, PAI-1 and PN-1 induce cell detachment through two slightly different mechanisms that affect integrin metabolism. These differences may be important for distinct cellular processes that require controlled changes in the subcellular localization of these receptors.
    Journal of Cellular Physiology 09/2009; 220(3):655-63. DOI:10.1002/jcp.21806 · 3.87 Impact Factor
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    ABSTRACT: Integrins are cell adhesion receptors that have many important roles in organ development and tissue integrity, functioning to mediate interactions between cells and the ECM. The entire repertoire of integrins is vast, and the specific roles of each are determined by unique integrin-ligand interactions. These interactions allow for dynamic regulation of multiple processes. Despite intense efforts to elucidate individual integrin ligands, existing methods have been limiting. In this chapter, we describe methods developed in our laboratory to identify new integrin ligands that should be useful for characterizing novel integrin functions. These methods are applicable for studies on a variety of integrins, and may be extended to other cell surface receptors as well.
    Methods in Enzymology 02/2007; 426:223-37. DOI:10.1016/S0076-6879(07)26011-2 · 2.19 Impact Factor
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    ABSTRACT: Urokinase-type plasminogen activator (uPA) binding to uPAR induces migration, adhesion, and proliferation through multiple interactions with G proteins-coupled receptor FPRL1, integrins, or the epidermal growth factor (EGF) receptor (EGFR). At least two forms of uPAR are present on the cell surface: full-length and cleaved uPAR, each specifically interacting with one or more transmembrane proteins. The connection between these interactions and the effects on the signaling pathways activation is not clear. We have exploited an uPAR mutant (hcr, human cleavage resistant) to dissect the pathways involved in uPA-induced cell migration. This mutant is not cleaved by proteases, is glycosylphosphatidylinositol anchored, and binds uPA with a normal K(d). Both wild-type (wt) and hcr-uPAR are able to mediate uPA-induced migration, are constitutively associated with the EGFR, and associate with alpha3beta1 integrin upon uPA binding. However, they engage different pathways in response to uPA. wt-uPAR requires both integrins and FPRL1 to mediate uPA-induced migration, and association of wt-uPAR to alpha3beta1 results in uPAR cleavage and extracellular signal-regulated kinase (ERK) activation. On the contrary, hcr-uPAR does not activate ERK and does not engage FPRL1 or any other G protein-coupled receptor, but it activates an alternative pathway initiated by the formation of a triple complex (uPAR-alpha3beta1-EGFR) and resulting in the autotyrosine phosphorylation of EGFR.
    Molecular Biology of the Cell 02/2006; 17(1):367-78. DOI:10.1091/mbc.E05-07-0635 · 4.55 Impact Factor

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