Binding of biotinylated thrombin receptor peptide to cloned human thrombin receptor overexpressed in baby hamster kidney cells.
Baby hamster kidney (BHK) cells transfected with an expression vector for the human thrombin receptor, and then treated with basic fibroblast growth factor, were found to express specific and saturable binding sites for biotinylated thrombin receptor peptide (SFLLRNPNDKYEPF). Analysis of the binding to live BHK cells yielded an equilibrium dissociation constant (Kd) of 3.0 +/- 0.3 mu mol/l and a maximal binding capacity (Bmax) of 31.0 +/- 0.5 nmol/mg of protein. In competitive binding experiments, the thrombin receptor agonist peptide (SFLLRN), which is a strong inducer of human platelet aggregation, was the most potent competitor. In contrast, position 1 to 2 inverted peptides such as FSLLRNPNDKYEPF and FSLLRNP, which fail to induce for the platelet aggregation, were less potent. This simple and convenient binding assay system using the biotinylated thrombin receptor peptide as a labeled ligand and the cloned thrombin receptor overexpressed in BHK cells may be useful for exploring specific antagonists of the receptor.
Tsukuba Research Laboratories, Ibaraki, Japan.
M
M
Takada
O
O
Ito
M
M
Kogushi
H
H
Kobayashi
T
T
Yamada
H
H
Tanaka
S
S
Yoshitake
I
I
Saito
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D001710:Biotin; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects; D011485:Protein Binding; D018179:Receptors, Thrombin / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D010446:Peptide Fragments; D018179:Receptors, Thrombin; D011994:Recombinant Proteins; C082835:thrombin receptor peptide (42-47); C072032:thrombin receptor peptide (42-55); D001710:Biotin