CHARACTERIZATION OF PDGF RECEPTORS IN A BACULOVIRUS EXPRESSION SYSTEM

血小板衍生生长因子(PDGF)通过相互作用发挥作用 结合两个特定的受体分子，α-PDGFR和β-PDGFR。在一个 努力更好地描述这种相互作用并产生足够的 结构的、功能的和突变的受体的数量 生化研究，我已经利用杆状病毒表达系统 生产水平为50-100倍的重组α-PDGFR和β-PDGFR 比在NIH/3T3细胞中发现的更大。 重组受体蛋白的表达通过 Western印迹分析和配基结合研究表明，两者 受体是以跨膜蛋白的形式产生的，其分子量为 大约160千道尔顿。放射性标记配基结合分析 PDGF-AA或PDGF-BB显示与αPDGF可饱和、高亲和力结合 完好无损。病毒感染的Sf9细胞。相比之下，BetaPDGFR在 在完整的Sf9细胞中，类似的水平显示出高亲和力结合 PDGF-BB。 时间进程实验表明，在感染过程的早期， 这两种受体都以低数量存在，处于非磷酸化状态， 并表现出配体刺激的酪氨酸蛋白激酶活性。然而， 随着感染后时间的延长(以及受体数量的增加)。两者都有 受体被结构性酪氨酸磷酸化，如以下所示 蛋白质印迹显示抗磷酸酪氨酸信号。这处房产一直是 用反磷酸酪氨酸纯化各受体，使其接近均一 免疫亲和层析。未来的研究将集中在 受体单抗的制备及受体定位的研究 每个受体的配体结合域(S)。

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.