Ostman A, Thyberg J, Westermark B, Heldin C H
Ludwig Institute for Cancer Research, Biomedical Center, Uppsala, Sweden.
J Cell Biol. 1992 Aug;118(3):509-19. doi: 10.1083/jcb.118.3.509.
Platelet-derived growth factor is a potent mitogen for cells of mesenchymal origin. It is made up of two polypeptide chains (A and B) combined in three disulfide-linked dimeric forms (AA, AB, and BB). Here, the biosynthesis and proteolytic processing of the two homodimeric forms of PDGF (AA and BB) were studied in CHO cells stably transfected with A-chain (short splice version) or B-chain cDNA. PDGF-AA was processed to a 30-kD molecule which was secreted from the cells. In contrast, PDGF-BB formed two structurally distinct end products; a minor secreted 30-kD form and a major cell-associated 24-kD form. Immunocytochemical studies at light- and electron-microscopical levels revealed presence of PDGF in the Golgi complex, in lysosomes, and to a smaller extent in the ER. From analysis of cells treated with brefeldin A, an inhibitor of ER to Golgi transport, it was concluded that dimerization occurs in the ER, whereas the proteolytic processing of PDGF-AA and PDGF-BB precursors normally occurs in a compartment distal to the ER. Exposure of the cultures to the lysosomal inhibitor chloroquine led to an increased cellular accumulation of PDGF-BB, as determined both by metabolic labeling experiments and immunocytochemical methods, indicating that the retained form of PDGF-BB is normally degraded in lysosomes. Structural analysis of the two end products of PDGF-BB revealed that the secreted 30-kD form is a dimer of peptides processed as the B-chain of PDGF purified from human platelets, and that the retained 24-kD form is made up of subunits additionally processed in the NH2-terminus. Also, the 24-kD form was shown to be composed of proteolytic fragments held together by disulfide bridges. Taken together these findings suggest that the newly synthesized PDGF A- and B-chains are dimerized in the ER and thereafter transferred to the Golgi complex for proteolytic processing. From there, PDGF-AA is carried in vesicles to the cell surface for release extracellularly by exocytosis. A smaller part of PDGF-BB (the 30-kD form) is handled in a similar way, whereas the major part (the 24-kD form) is generated by additional proteolysis in the Golgi complex, from which it is slowly carried over to lysosomes for degradation.
血小板衍生生长因子是一种对间充质来源细胞有强大作用的促分裂原。它由两条多肽链（A链和B链）组成，以三种二硫键连接的二聚体形式（AA、AB和BB）结合。在此，研究了在稳定转染A链（短剪接变体）或B链cDNA的CHO细胞中血小板衍生生长因子（PDGF）两种同型二聚体形式（AA和BB）的生物合成和蛋白水解加工过程。PDGF-AA被加工成一种30-kD的分子并从细胞中分泌出来。相比之下，PDGF-BB形成了两种结构不同的终产物；一种少量分泌的30-kD形式和一种主要与细胞相关的24-kD形式。光镜和电镜水平的免疫细胞化学研究显示，PDGF存在于高尔基体、溶酶体中，在内质网中的含量较少。通过对用布雷菲德菌素A（一种内质网到高尔基体转运的抑制剂）处理的细胞进行分析得出，二聚化发生在内质网中，而PDGF-AA和PDGF-BB前体的蛋白水解加工通常发生在内质网远端的一个区室中。用溶酶体抑制剂氯喹处理培养物后，通过代谢标记实验和免疫细胞化学方法测定发现，PDGF-BB在细胞内的积累增加，这表明保留形式的PDGF-BB通常在溶酶体中被降解。对PDGF-BB的两种终产物进行结构分析发现，分泌的30-kD形式是从人血小板中纯化的PDGF B链加工而成的肽二聚体，而保留的24-kD形式由在NH2末端额外加工的亚基组成。此外，24-kD形式显示为由通过二硫键连接在一起的蛋白水解片段组成。综上所述，这些发现表明新合成的PDGF A链和B链在内质网中发生二聚化，然后转移到高尔基体进行蛋白水解加工。从那里，PDGF-AA通过囊泡运输到细胞表面，通过胞吐作用释放到细胞外。PDGF-BB的一小部分（30-kD形式）以类似方式处理，而大部分（24-kD形式）是在高尔基体中通过额外的蛋白水解产生的，然后从高尔基体缓慢转运到溶酶体进行降解。
