Mapping of the vitronectin-binding site on the urokinase receptor: involvement of a coherent receptor interface consisting of residues from both domain I and the flanking interdomain linker region

TY - JOUR

T1 - Mapping of the vitronectin-binding site on the urokinase receptor

T2 - involvement of a coherent receptor interface consisting of residues from both domain I and the flanking interdomain linker region

AU - Gårdsvoll, Henrik

AU - Ploug, Michael

PY - 2007/5/4

Y1 - 2007/5/4

N2 - The urokinase-type plasminogen activator receptor (uPAR) has been implicated as a modulator of several biochemical processes that are active during tumor invasion and metastasis, e.g. extracellular proteolysis, cell adhesion, and cell motility. The structural basis for the high affinity interaction between the urokinase-type plasminogen activator (uPA) and uPAR, which focuses cell surface-associated plasminogen activation in vivo, is now thoroughly characterized by site-directed mutagenesis studies and x-ray crystallography. In contrast, the structural basis for the interaction between uPAR and the extracellular matrix protein vitronectin, which is involved in the regulation of cell adhesion and motility, remains to be clarified. In this study, we have identified the functional epitope on uPAR that is responsible for its interaction with the full-length, extended form of vitronectin by using a comprehensive alanine-scanning library of purified single-site uPAR mutants (244 positions tested). Interestingly, the five residues identified as "hot spots" for vitronectin binding form a contiguous epitope consisting of two exposed loops connecting the central fourstranded beta-sheet in uPAR domain I (Trp(32), Arg(58), and Ile(63)) as well as a proximal region of the flexible linker peptide connecting uPAR domains I and II (Arg(91) and Tyr(92)). This binding topology provides the molecular basis for the observation that uPAR can form a ternary complex with uPA and vitronectin. Furthermore, it raises the intriguing possibility that the canonical receptor and inhibitor for uPA (uPAR and PAI-1) may have reached a convergent solution for binding to the somatomedin B domain of vitronectin.

AB - The urokinase-type plasminogen activator receptor (uPAR) has been implicated as a modulator of several biochemical processes that are active during tumor invasion and metastasis, e.g. extracellular proteolysis, cell adhesion, and cell motility. The structural basis for the high affinity interaction between the urokinase-type plasminogen activator (uPA) and uPAR, which focuses cell surface-associated plasminogen activation in vivo, is now thoroughly characterized by site-directed mutagenesis studies and x-ray crystallography. In contrast, the structural basis for the interaction between uPAR and the extracellular matrix protein vitronectin, which is involved in the regulation of cell adhesion and motility, remains to be clarified. In this study, we have identified the functional epitope on uPAR that is responsible for its interaction with the full-length, extended form of vitronectin by using a comprehensive alanine-scanning library of purified single-site uPAR mutants (244 positions tested). Interestingly, the five residues identified as "hot spots" for vitronectin binding form a contiguous epitope consisting of two exposed loops connecting the central fourstranded beta-sheet in uPAR domain I (Trp(32), Arg(58), and Ile(63)) as well as a proximal region of the flexible linker peptide connecting uPAR domains I and II (Arg(91) and Tyr(92)). This binding topology provides the molecular basis for the observation that uPAR can form a ternary complex with uPA and vitronectin. Furthermore, it raises the intriguing possibility that the canonical receptor and inhibitor for uPA (uPAR and PAI-1) may have reached a convergent solution for binding to the somatomedin B domain of vitronectin.

KW - Amino Acid Substitution

KW - Animals

KW - CHO Cells

KW - Cell Adhesion

KW - Cell Movement

KW - Cricetinae

KW - Cricetulus

KW - Epitope Mapping

KW - Epitopes

KW - Humans

KW - Mutagenesis, Site-Directed

KW - Mutation, Missense

KW - Neoplasm Metastasis

KW - Neoplasms

KW - Protein Binding

KW - Protein Structure, Quaternary

KW - Protein Structure, Secondary

KW - Protein Structure, Tertiary

KW - Receptors, Cell Surface

KW - Receptors, Urokinase Plasminogen Activator

KW - Somatomedins

KW - Vitronectin

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1074/jbc.M610184200

DO - 10.1074/jbc.M610184200

M3 - Journal article

C2 - 17355965

SN - 0021-9258

VL - 282

SP - 13561

EP - 13572

JO - The Journal of Biological Chemistry

JF - The Journal of Biological Chemistry

IS - 18

ER -