Lehmann M, André F, Bellan C, Remacle-Bonnet M, Garrouste F, Parat F, Lissitsky J C, Marvaldi J, Pommier G
Unité Interactions entre Systèmes Protéiques et Différenciation dans la Cellule Tumorale, UPRES-A CNRS 6032, Université d'Aix-Marseille I, Faculté de Pharmacie, France.
Endocrinology. 1998 Sep;139(9):3763-71. doi: 10.1210/endo.139.9.6184.
To investigate endoproteolytic processing of the type I insulin-like growth factor receptor (IGF-IR), we have examined its structure and activity in the furin-deficient LoVo-C5 cell line. Immunoprecipitation experiments using the monoclonal anti-IGF-IR antibody (alpha-IR3) showed that LoVo-C5 cells expressed a major high molecular mass receptor (200 kDa) corresponding to the unprocessed alpha/beta pro-receptor. A small amount of successfully cleaved alpha/beta heterodimers was also produced, indicating a residual endoproteolytic cleavage activity in these cells. In vitro, a soluble form of recombinant furin was able to cleave the pro-IGF-IR (200 kDa) into alpha-subunit (130 kDa) and beta-subunit (97 kDa). Measurement of IGF binding parameters in LoVo-C5 cells indicated a low number of typical type I IGF-binding sites (binding capacity, 5 x 10(3) sites/cell; Kd, 1.9 nM for IGF-I and 7.0 nM for IGF-II). These findings in LoVo-C5 contrast with those in HT29-D4 cells, which have active furin, and where IGF-IR (2.8 x 10(4) sites/cell) was fully processed. Moreover, the 200-kDa pro-IGF-IR of LoVo-C5 was unable to induce intracellular signaling, such as beta-subunit tyrosine autophosphorylation and insulin-related substrate-1 tyrosine phosphorylation. Flow immunocytometry analysis using alpha-IR3 antibody indicated that LoVo-C5 cells expressed 40% more receptors than HT29-D4 cells, suggesting that in LoVo-C5 cells only the small amount of mature type I IGF-IR binds IGFs with high affinity. To provide evidence for this idea, we showed that mild trypsin treatment of living LoVo-C5 cells partially restored alpha/beta cleavage of IGF-IR, and greatly enhanced (6-fold) the IGF-I binding capacity of LoVo-C5 cells, but did not restore IGF-IR signaling activity. Moreover, LoVo-C5 cells were totally unresponsive to IGF-I in terms of cell migration, in contrast to fully processed IGF-IR-HT29-D4 cells. Our data indicate that furin is involved in the endoproteolytic processing of the IGF-IR and suggest that this posttranslational event might be crucial for its ligand binding and signaling activities. However, our data do not exclude that other proprotein convertases could participate to IGF-IR maturation.
为了研究I型胰岛素样生长因子受体（IGF-IR）的内切蛋白水解加工过程，我们检测了其在弗林蛋白酶缺陷型LoVo-C5细胞系中的结构和活性。使用抗IGF-IR单克隆抗体（α-IR3）进行的免疫沉淀实验表明，LoVo-C5细胞表达一种主要的高分子量受体（200 kDa），对应未加工的α/β前体受体。同时也产生了少量成功切割的α/β异二聚体，表明这些细胞中存在残余的内切蛋白水解切割活性。在体外，可溶性重组弗林蛋白酶能够将前体IGF-IR（200 kDa）切割成α亚基（130 kDa）和β亚基（97 kDa）。对LoVo-C5细胞中IGF结合参数的测量表明，典型的I型IGF结合位点数量较少（结合能力为5×10³个位点/细胞；IGF-I的解离常数为1.9 nM，IGF-II的解离常数为7.0 nM）。LoVo-C5细胞的这些发现与具有活性弗林蛋白酶的HT29-D4细胞不同，在HT29-D4细胞中IGF-IR（2.8×10⁴个位点/细胞）已被完全加工。此外，LoVo-C5细胞的200 kDa前体IGF-IR无法诱导细胞内信号传导，如β亚基酪氨酸自磷酸化和胰岛素相关底物-1酪氨酸磷酸化。使用α-IR3抗体进行的流式免疫细胞术分析表明，LoVo-C5细胞表达的受体比HT29-D4细胞多40%，这表明在LoVo-C5细胞中，只有少量成熟的I型IGF-IR能以高亲和力结合IGF。为了证明这一观点，我们发现对活的LoVo-C5细胞进行温和的胰蛋白酶处理可部分恢复IGF-IR的α/β切割，并显著提高（6倍）LoVo-C5细胞的IGF-I结合能力，但不能恢复IGF-IR信号传导活性。此外，与完全加工的IGF-IR-HT29-D4细胞相比，LoVo-C5细胞在细胞迁移方面对IGF-I完全无反应。我们的数据表明弗林蛋白酶参与了IGF-IR的内切蛋白水解加工过程，并表明这一翻译后事件可能对其配体结合和信号传导活性至关重要。然而，我们的数据并不排除其他前体蛋白转化酶可能参与IGF-IR成熟的可能性。
