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Deficient Processing and Activity of Type I Insulin-Like Growth Factor Receptor in the Furin-Deficient LoVo-C5 Cells¹

Unité Interactions entre Systèmes Protéiques et Différenciation dans la Cellule Tumorale, UPRES-A CNRS 6032, Université d’Aix-Marseille I et II, Faculté de Pharmacie, 13385 Marseille; and INSERM U-387 (J.-C.L.), 13009 Marseille, France

Address all correspondence and requests for reprints to: Dr. Jacques Marvaldi, Laboratoire de Biochimie Cellulaire, UPRES-A CNRS 6032, Faculté de Pharmacie, 27 boulevard Jean Moulin, 13385 Marseille Cedex 05, France. E-mail: marvaldi{at}newsup.univ-mrs.fr

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 (-IR3) showed that LoVo-C5 cells expressed a major high molecular mass receptor (200 kDa) corresponding to the unprocessed /ß pro-receptor. A small amount of successfully cleaved /ß 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 -subunit (130 kDa) and ß-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 10³ 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 10⁴ sites/cell) was fully processed. Moreover, the 200-kDa pro-IGF-IR of LoVo-C5 was unable to induce intracellular signaling, such as ß-subunit tyrosine autophosphorylation and insulin-related substrate-1 tyrosine phosphorylation. Flow immunocytometry analysis using -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 /ß 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.

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