Pietzko D, Zohlnhöfer D, Graeve L, Fleischer D, Stoyan T, Schooltink H, Rose-John S, Heinrich P C
Institut für Biochemie, Rheinisch-Westfälische Technische Hochschule Aachen, Federal Republic of Germany.
J Biol Chem. 1993 Feb 25;268(6):4250-8.
Recombinant human 125I-interleukin-6 (IL-6) was cross-linked with the homobifunctional reagent disuccinimidyl suberate to human hepatoma cells (HepG2). Three recombinant human 125I-IL-6-containing complexes of apparent molecular masses of 100, 120, and 200 kDa were immunoprecipitated with specific antibodies to human IL-6 or to the 80-kDa IL-6 receptor subunit. We show by immunoprecipitation, peptide mapping, and by the use of a cleavable heterobifunctional cross-linker (Denny-Jaffe reagent) that different polypeptides are involved in the formation of the 100- and 120-kDa IL-6-containing complexes. The molecular compositions of the 100- and 120-kDa cross-linked complexes were identified. The 100-kDa complex consisted of one ligand and one IL-6 receptor subunit, glycoprotein 80 (gp80), whereas the 120-kDa complex was found to be composed of one ligand and a polypeptide which was immunoprecipitable with the monoclonal antibody AM64 directed against gp130. Exposure of HepG2 cells to phorbol 12-myristate 13-acetate (PMA) or PMA-dexamethasone led to an increase in the 80-kDa IL-6 receptor mRNA and functional receptor protein. Whereas treatment of HepG2 cells with PMA led to an increase in the formation of gp80.gp130.IL-6 complexes determined by cross-linking, no corresponding increase in high affinity binding sites was found. The existence of a third IL-6 receptor subunit present in limiting amounts on HepG2 cells is proposed to explain this discrepancy. Evidence is presented that the 80-kDa IL-6 receptor up-regulation by PMA-dexamethasone is caused by the depletion of protein kinase C since the protein kinase C inhibitor staurosporine mimics the effect of PMA-dexamethasone.
重组人125I-白细胞介素-6（IL-6）与同型双功能试剂辛二酸二琥珀酰亚胺酯交联于人肝癌细胞（HepG2）。用抗人IL-6或80 kDa IL-6受体亚基的特异性抗体免疫沉淀出三种表观分子量分别为100、120和200 kDa的含重组人125I-IL-6的复合物。我们通过免疫沉淀、肽图谱分析以及使用可裂解的异双功能交联剂（丹尼-贾菲试剂）表明，不同的多肽参与了100 kDa和120 kDa含IL-6复合物的形成。确定了100 kDa和120 kDa交联复合物的分子组成。100 kDa复合物由一个配体和一个IL-6受体亚基糖蛋白80（gp80）组成，而120 kDa复合物被发现由一个配体和一种可被针对gp130的单克隆抗体AM64免疫沉淀的多肽组成。将HepG2细胞暴露于佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）或PMA-地塞米松会导致80 kDa IL-6受体mRNA和功能性受体蛋白增加。虽然用PMA处理HepG2细胞会导致通过交联测定的gp80.gp130.IL-6复合物形成增加，但未发现高亲和力结合位点有相应增加。有人提出HepG2细胞上存在第三种含量有限的IL-6受体亚基来解释这种差异。有证据表明，PMA-地塞米松对80 kDa IL-6受体的上调是由于蛋白激酶C的消耗，因为蛋白激酶C抑制剂星形孢菌素模拟了PMA-地塞米松的作用。
