CHARACTERIZATION OF PDGF RECEPTORS IN A BACULOVIRUS EXPRESSION SYSTEM

杆状病毒表达系统中 PDGF 受体的表征

• 批准号: 3874776
• 负责人: S A AARONSON
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3874776
• 关键词: affinity chromatography; antireceptor antibody; genetic manipulation; growth factor receptors; human tissue; insect virus; membrane proteins; monoclonal antibody; mutant; platelet derived growth factor; protein structure function; protein tyrosine kinase; receptor binding; transposon /insertion element; western blottings

Platelet-derived growth factor (PDGF) exerts its effects by interaction with two specific receptor molecules, alphaPDGFR and betaPDGFR. In an effort to better characterize this interaction and produce sufficient quantities of native and mutant receptors for structural, functional and biochemical studies, I have utilized the baculovirus expression system to produce recombinant alphaPDGFR and betaPDGFR at levels 50- to 100-fold greater than found in NIH/3T3 cells. The expression of recombinant receptor proteins was characterized by western blot analysis and ligand binding studies, and revealed that both receptors were produced as transmembrane proteins with molecular weights of approximately 160 kilodaltons. Ligand-binding analysis with radiolabelled PDGF-AA or PDGF-BB revealed saturable, high affinity binding to alphaPDGF in intact. virally infected Sf9 cells. In contrast, betaPDGFR expressed at similar levels in intact Sf9 cells showed high affinity binding only for PDGF-BB. Time course experiments demonstrated that early in the course of infection, both receptors were present in low numbers, in a nonphosphorylated state, and exhibited ligand-stimulated tyrosine protein kinase activity. However, with increasing time postinfection (and increasing receptor number). both receptors became constitutively tyrosine phosphorylated as demonstrated by antiphosphotyrosine signal on western blotting. This property has been utilized to purify each receptor to near homogeneity by antiphosphotyrosine immunoaffinity chromatography. Future studies will concentrate on the production of monoclonal antibodies to receptor and on localization of the ligand binding domain(s) for each receptor.

血小板衍生生长因子（PDGF）通过相互作用发挥作用