Heidaran M A, Mahadevan D, Larochelle W J
National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
FASEB J. 1995 Jan;9(1):140-5. doi: 10.1096/fasebj.9.1.7821753.
To localize human beta PDGFR binding determinants, we constructed a fusion protein comprising beta PDGFR Ig-like domains 1 to 3 and an IgG1 Fc domain (beta PDGFR-HFc). beta PDGFR-HFc was expressed as a 200 kDa dimeric molecule and contained Fc epitopes as demonstrated by anti-mouse Fc antibody recognition. Scatchard analysis revealed that PDGF BB possessed a dissociation constant of 1.5 nM for beta PDGFR-HFc. Thus, beta PDGFR Ig-like domains 1 to 3 are sufficient for high affinity PDGF BB binding. We exploited this fusion protein technology to identify and characterize beta PDGFR antagonists using a sensitive beta PDGFR immunosorbent assay. In this assay, beta PDGFR-HFc half-maximally bound to PDGF BB with an affinity of around 150 pM. Suramin, as well as bacterially expressed and refolded human alpha PDGFR domains 1-3, inhibited beta PDGFR-HFc binding to PDGF BB half-maximally at 25 microM and 10 nM respectively. Therefore, alpha PDGFR D1-3, like beta PDGFR D1-3, are sufficient for high affinity PDGF BB binding. Furthermore, the beta PDGFR-HFc immunosorbent assay will be useful to identify beta PDGFR antagonists as well as to study alpha and beta PDGFR substitution mutants which further map receptor binding determinants.
为了定位人β血小板衍生生长因子受体（β PDGFR）的结合决定簇，我们构建了一种融合蛋白，其包含β PDGFR的免疫球蛋白样结构域1至3和一个IgG1 Fc结构域（β PDGFR-HFc）。β PDGFR-HFc表达为一个200 kDa的二聚体分子，并含有Fc表位，这可通过抗小鼠Fc抗体识别得以证明。Scatchard分析表明，血小板衍生生长因子BB（PDGF BB）与β PDGFR-HFc的解离常数为1.5 nM。因此，β PDGFR的免疫球蛋白样结构域1至3足以实现与PDGF BB的高亲和力结合。我们利用这种融合蛋白技术，通过一种灵敏的β PDGFR免疫吸附测定法来鉴定和表征β PDGFR拮抗剂。在该测定法中，β PDGFR-HFc与PDGF BB的半数最大结合亲和力约为150 pM。苏拉明以及细菌表达并复性的人α血小板衍生生长因子受体结构域1-3分别在25 μM和10 nM时对β PDGFR-HFc与PDGF BB的结合产生半数最大抑制作用。因此，α PDGFR D1-3与β PDGFR D1-3一样，足以实现与PDGF BB的高亲和力结合。此外，β PDGFR-HFc免疫吸附测定法将有助于鉴定β PDGFR拮抗剂，以及研究α和β PDGFR替代突变体，从而进一步定位受体结合决定簇。
