Binding of discoidin domain receptor 2 to collagen I: an atomic force microscopy investigation.
Collagens have recently been identified as ligands for discoidin domain receptors (DDR1 and DDR2), generating an interest in studying the properties of binding of DDR to its ligand. We are interested in the interaction of DDR2 with collagen I because of its potential role in liver fibrosis. Our in vitro binding assay utilizes DDR2-Fc fusion proteins, which can be clustered (multimerized) by use of antibodies to form DDR2 complexes. Binding of DDR2 complexes to collagen I coated on plastic plates was established by a microplate-based assay using Eu(3+)-labeled proteins and time-resolved fluorometry. Clustering of the DDR2-Fc with antibody was found to be requisite for binding to collagen in vitro. Using atomic force microscopy (AFM) in an aqueous environment, we characterized the surface topographies of DDR2 complexes and collagen I, and investigated binding of this receptor-ligand pair. We were able to image and identify binding of DDR2 complexes onto individual molecules of triple-helical collagen and provide insight into the number and locations of binding sites on collagen I. In most cases, a single receptor complex bound to a single collagen molecule and there were preferred DDR2 binding sites on the collagen I triple helix. These data were validated by rotary-replication transmission electron microscopy (TEM) of glycerol-sprayed samples.
Agarwal
Gunjan
G
Procter & Gamble Pharmaceuticals, 8700 Mason Montgomery Road, Mason, Ohio 45040, USA. Gunjan.Agarwal@wpafb.af.mil
Kovac
Lubomir
L
Radziejewski
Czeslaw
C
Samuelsson
Steven J
SJ
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D024042:Collagen Type I / Q000737:chemistry* / Q000648:ultrastructure*; D000070818:Discoidin Domain Receptors; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics; D007074:Immunoglobulin G / Q000235:genetics; D008024:Ligands; D051379:Mice; D018625:Microscopy, Atomic Force / Q000379:methods*; D011485:Protein Binding / Q000235:genetics; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure*; D011975:Receptors, Mitogen / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000648:ultrastructure
D024042:Collagen Type I; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D008024:Ligands; D011975:Receptors, Mitogen; D011993:Recombinant Fusion Proteins; D000070818:Discoidin Domain Receptors; D020794:Receptor Protein-Tyrosine Kinases