DEFICIENT PROCESSING AND ACTIVITY OF TYPE-I INSULIN-LIKE-GROWTH-FACTOR RECEPTOR IN THE FURIN-DEFICIENT LOVO-C5 CELLS

To investigate endoproteolytic processing of the type I insulin-like g rowth factor receptor (IGF-IR), we have examined its structure and act ivity in the furin-deficient LoVo-C5 cell line. Immunoprecipitation ex periments using the monoclonal anti-IGF-IR antibody (alpha-IR3) showed that LoVo-C5 cells expressed a major high molecular mass receptor (20 0 kDa) corresponding to the unprocessed alpha/beta pro-receptor. A sma ll amount of successfully cleaved alpha/beta heterodimers was also pro duced, indicating a residual endoproteolytic cleavage activity in thes e cells. In vitro, a soluble form of recombinant furin was able to cle ave the pro-IGF-IR (200 kDa) into alpha-subunit (130 kDa) and beta-sub unit (97 kDa). Measurement of IGF binding parameters in LoVo-C5 cells indicated a low number of typical type I IGF-binding sites (binding ca pacity, 5 x 10(3) sites/cell; K-d, 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 unab le to induce intracellular signaling, such as beta-subunit tyrosine au tophosphorylation and insulin-related substrate-1 tyrosine phosphoryla tion. Flow immunocytometry analysis using alpha-IR3 antibody indicated that LoVo-C5 cells expressed 40% more receptors than HT29-D4 cells, s uggesting 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 id ea, we showed that mild trypsin treatment of living LoVo-C5 cells part ially 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 unres ponsive to IGF-I in terms of cell migration, in contrast to fully proc essed IGF-IR-HT29-D4 cells. Our data indicate that furin is involved i n the endoproteolytic processing of the IGF-IR and suggest that this p osttranslational event might be crucial for its ligand binding and sig naling activities. However, our data do not exclude that other proprot ein convertases could participate to IGF-IR maturation.