Fenstermaker R A, Poptic E, Bonfield T L, Knauss T C, Corsillo L, Piskurich J F, Kaetzel C S, Jentoft J E, Gelfand C, DiCorleto P E
Department of Veterans Affairs Medical Center, Cleveland, Ohio 44106.
J Biol Chem. 1993 May 15;268(14):10482-9.
Platelet-derived growth factor-AA and -BB homodimers and -AB heterodimers bind with high affinity to the platelet-derived growth factor (PDGF) alpha-receptor. Basic polypeptides such as polylysine and protamine sulfate compete with PDGF for receptor binding, suggesting a role for ligand positive charge in the binding interaction. A pentapeptide amino acid sequence with a cationic tripeptide core is perfectly conserved between the A- and B-chains (Val158-Arg159-Lys160-Lys161-Pro162) and was therefore considered as a possible alpha-receptor-binding domain. We have investigated the functional importance of positive charge within this region of the PDGF A-chain by using site-directed mutagenesis to convert the cationic core amino acids to the acidic sequence triglutamic acid. cDNAs encoding wild-type (PDGF-AAwt) and charge mutant (PDGF-AAcm) proteins were expressed following stable transfection of Chinese hamster ovary cells. Proper assembly and secretion of PDGF-AAcm was verified by metabolic labeling with [35S]cysteine, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis analysis under nonreducing and reducing conditions. PDGF-AAcm was secreted as two major species of disulfide-linked A-chain homodimers identical in molecular mass to those observed for PDGF-AAwt (32 and 35 kDa). Secreted PDGF-AAwt and PDGF-AAcm proteins were purified to homogeneity and subjected to structural and functional analyses. Compared to purified PDGF-AAwt, PDGF-AAcm displayed a marked reduction in both binding affinity for PDGF alpha-receptors and mitogenic activity in Swiss 3T3 cells. Large reductions were also observed in the ability of semipurified PDGF-AAcm to stimulate calcium influx and the production of inositol phosphates. Measurement of circular dichroism spectra of highly purified PDGF-AAcm and PDGF-AAwt revealed no significant difference in secondary structure. Collectively, these results indicate that the cationic Arg159-Lys160-Lys161 region plays a critical role in the biological activity of PDGF-AA by direct participation in ligand binding to the PDGF alpha-receptor.
血小板衍生生长因子 - AA和 - BB同二聚体以及 - AB异二聚体以高亲和力与血小板衍生生长因子（PDGF）α受体结合。诸如聚赖氨酸和硫酸鱼精蛋白等碱性多肽可与PDGF竞争受体结合，这表明配体正电荷在结合相互作用中起作用。在A链和B链之间，具有阳离子三肽核心的五肽氨基酸序列（Val158 - Arg159 - Lys160 - Lys161 - Pro162）完全保守，因此被认为是一个可能的α受体结合域。我们通过定点诱变将阳离子核心氨基酸转化为酸性序列三谷氨酸，研究了PDGF A链该区域内正电荷的功能重要性。在中国仓鼠卵巢细胞稳定转染后，表达了编码野生型（PDGF - AAw t）和电荷突变型（PDGF - AAcm）蛋白的cDNA。通过用[35S]半胱氨酸进行代谢标记、免疫沉淀以及在非还原和还原条件下的SDS - 聚丙烯酰胺凝胶电泳分析，验证了PDGF - AAcm的正确组装和分泌。PDGF - AAcm以两种主要的二硫键连接的A链同二聚体形式分泌，其分子量与PDGF - AAw t观察到的相同（32和35 kDa）。将分泌的PDGF - AAw t和PDGF - AAcm蛋白纯化至同质，并进行结构和功能分析。与纯化的PDGF - AAw t相比，PDGF - AAcm对PDGFα受体的结合亲和力和在瑞士3T3细胞中的促有丝分裂活性均显著降低。在半纯化的PDGF - AAcm刺激钙内流和肌醇磷酸产生的能力方面也观察到大幅降低。对高度纯化的PDGF - AAcm和PDGF - AAw t的圆二色光谱测量显示二级结构无显著差异。总体而言，这些结果表明阳离子的Arg159 - Lys160 - Lys161区域通过直接参与配体与PDGFα受体的结合，在PDGF - AA的生物活性中起关键作用。
