Mapping part of the functional epitope for ligand binding on the receptor for urokinase-type plasminogen activator by site-directed mutagenesis

TY - JOUR

T1 - Mapping part of the functional epitope for ligand binding on the receptor for urokinase-type plasminogen activator by site-directed mutagenesis

AU - Gårdsvoll, H

AU - Danø, K

AU - Ploug, M

PY - 1999/12/31

Y1 - 1999/12/31

N2 - The urokinase-type plasminogen activator receptor (uPAR) is a glycolipid anchored multidomain member of the Ly-6/uPAR protein domain superfamily. Studies by site-directed photoaffinity labeling, chemical cross-linking, and ligand-induced protection against chemical modification have highlighted the possible involvement of uPAR domain I and particularly loop 3 thereof in ligand binding (Ploug, M. (1998) Biochemistry 37, 16494-16505). Guided by these results we have now performed an alanine scanning analysis of this region in uPAR by site-directed mutagenesis and subsequently measured the effects thereof on the kinetics of uPA binding in real-time by surface plasmon resonance. Only four positions in loop 3 of uPAR domain I exhibited significant changes in the contribution to the free energy of uPA binding (DeltaDeltaG >/= 1.3 kcal mol(-1)) upon single-site substitutions to alanine (i.e. Arg(53), Leu(55), Tyr(57), and Leu(66)). The energetic impact of these four alanine substitutions was not caused by gross structural perturbations, since all monoclonal antibodies tested having conformation-dependent epitopes on this domain exhibited unaltered binding kinetics. These sites together with a three-dimensional structure for uPAR may provide an appropriate target for rational drug design aimed at developing new receptor binding antagonists with potential application in cancer therapy.

AB - The urokinase-type plasminogen activator receptor (uPAR) is a glycolipid anchored multidomain member of the Ly-6/uPAR protein domain superfamily. Studies by site-directed photoaffinity labeling, chemical cross-linking, and ligand-induced protection against chemical modification have highlighted the possible involvement of uPAR domain I and particularly loop 3 thereof in ligand binding (Ploug, M. (1998) Biochemistry 37, 16494-16505). Guided by these results we have now performed an alanine scanning analysis of this region in uPAR by site-directed mutagenesis and subsequently measured the effects thereof on the kinetics of uPA binding in real-time by surface plasmon resonance. Only four positions in loop 3 of uPAR domain I exhibited significant changes in the contribution to the free energy of uPA binding (DeltaDeltaG >/= 1.3 kcal mol(-1)) upon single-site substitutions to alanine (i.e. Arg(53), Leu(55), Tyr(57), and Leu(66)). The energetic impact of these four alanine substitutions was not caused by gross structural perturbations, since all monoclonal antibodies tested having conformation-dependent epitopes on this domain exhibited unaltered binding kinetics. These sites together with a three-dimensional structure for uPAR may provide an appropriate target for rational drug design aimed at developing new receptor binding antagonists with potential application in cancer therapy.

KW - Amino Acid Sequence

KW - Base Sequence

KW - DNA Primers

KW - Epitope Mapping

KW - Humans

KW - Kinetics

KW - Ligands

KW - Models, Molecular

KW - Molecular Sequence Data

KW - Mutagenesis, Site-Directed

KW - Receptors, Cell Surface

KW - Receptors, Urokinase Plasminogen Activator

KW - Thermodynamics

KW - Urokinase-Type Plasminogen Activator

KW - Journal Article

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

M3 - Journal article

C2 - 10608868

SN - 0021-9258

VL - 274

SP - 37995

EP - 38003

JO - The Journal of Biological Chemistry

JF - The Journal of Biological Chemistry

IS - 53

ER -