Mechanism of platelet-derived growth factor (PDGF) AA, AB, and BB binding to alpha and beta PDGF receptor.
The biological effects of platelet-derived growth factor (PDGF) are mediated by cell surface alpha and beta PDGF receptors, which, as a result of ligand binding, undergo dimerization in a manner consistent with PDGF being bivalent. In order to directly demonstrate PDGF bivalency and to define the binding of PDGF AB to isolated beta receptor, we developed solid-phase binding assays using purified recombinant extracellular domain of human PDGF receptors. PDGF AA, AB, and BB were prepared from the monomeric chains expressed in Escherichia coli, and each was purified to homogeneity; PDGF AB contained < 0.5% of either homodimer. The interactions of these isoforms with immobilized PDGF receptors were examined by several approaches. Scatchard analysis revealed high affinity binding (Kd = 0.5-1.0 nM) of radiolabeled PDGF AA and AB to alpha receptor and of PDGF BB to both receptor subtypes. Contrary to previous reports, PDGF AB also bound beta receptor with high affinity (Kd = 0.9 nM). When a B-chain-specific monoclonal antibody that recognizes the putative binding domain of PDGF BB was used for ligand detection, we found that PDGF AB binding to beta receptor occurred exclusively through the B-chain subunit, whereas binding to alpha receptor occurred through either subunit. In addition, site-directed mutagenesis was used to specifically inactivate the B chain of PDGF AB, which eliminated binding to the beta receptor without affecting alpha receptor binding. These results establish that PDGF is bivalent and that monovalent ligand retains high affinity receptor binding.
Fretto
L J
LJ
COR Therapeutics Inc., South San Francisco, California 94080.
Snape
A J
AJ
Tomlinson
J E
JE
Seroogy
J J
JJ
Wolf
D L
DL
LaRochelle
W J
WJ
Giese
N A
NA
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004247:DNA / Q000235:genetics; D000939:Epitopes / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010982:Platelet-Derived Growth Factor / Q000235:genetics / Q000378:metabolism*; D017479:Receptors, Platelet-Derived Growth Factor / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D003062:Codon; D000939:Epitopes; D046911:Macromolecular Substances; D010982:Platelet-Derived Growth Factor; D011994:Recombinant Proteins; D004247:DNA; D017479:Receptors, Platelet-Derived Growth Factor