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 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.
Unité Interactions entre Systèmes Protéiques et Différenciation dans la Cellule Tumorale, UPRES-A CNRS 6032, Université d'Aix-Marseille I, Faculté de Pharmacie, France.
M
M
Lehmann
F
F
André
C
C
Bellan
M
M
Remacle-Bonnet
F
F
Garrouste
F
F
Parat
J C
JC
Lissitsky
J
J
Marvaldi
G
G
Pommier
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D002465:Cell Movement / Q000187:drug effects; D004351:Drug Resistance; D005098:Exotoxins / Q000494:pharmacology; D005434:Flow Cytometry; D045683:Furin; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000378:metabolism* / Q000494:pharmacology; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017451:Receptors, Somatomedin / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D013381:Subtilisins / Q000172:deficiency*; D014357:Trypsin / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D045683:Furin; D007334:Insulin-Like Growth Factor I; D017451:Receptors, Somatomedin; D013381:Subtilisins; D014357:Trypsin; D014443:Tyrosine; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A