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

Abstract

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.

Mutation at Cleavage Site of Insulin-Like Growth Factor Receptor in a Short-Stature Child Born with Intrauterine Growth Retardation

“…[ 125 I]IGF-I binding study using these LoVo-C5 cells showed that the affinity for IGF-I of each IGF-IR on these cells was not decreased, but the number of IGF-I binding sites on the cells was significantly lower. Therefore, these LoVo-C5 cells expressing proIGF-IR could not induce intracellular signaling such as ␤-subunit tyrosine autophosphorylation (33). These reports strongly suggest that mutation at the cleavage site of proIGF-IR, as observed in our patients, may cause failure of intracellular signaling transduction and abolish IGF-IR functions such as cell growth, transformation, and development.…”

“…Lehmann et al (33) reported that LoVo-C5 cells lacking furin, which cleaves proIGF-IR into ␣-and ␤-subunits, expressed a major high-molecular-mass proIGF-IR (200 kDa) instead of mature IGF-IR on the cell surface. [ 125 I]IGF-I binding study using these LoVo-C5 cells showed that the affinity for IGF-I of each IGF-IR on these cells was not decreased, but the number of IGF-I binding sites on the cells was significantly lower.…”

Propeptide cleavage conditions sortilin/neurotensin receptor-3 for ligand binding

“…In these receptors, cleavage of the precursor proteins serves to generate the subunits that constitute the mature two-chain receptors and does not directly involve exposure of sites for ligand binding. Thus, the proforms of the insulin receptor, the hepatocyte growth factor receptor and the LRP are all capable of ligand binding, and major dysfunction of uncleaved receptors, as seen with the insulin-like growth factor-1 receptor, most likely results from a general malconformation (Mark et al, 1992;Sugibayashi et al, 1992;Komada et al, 1993;Willnow et al, 1996a;Lehmann et al, 1998). Thus, sortilin is the first example of furin-mediated receptor activation.…”

The cAMP-responsive element binding protein (CREB) transcription factor regulates furin expression during human trophoblast syncytialization

“…However, the expression levels of furin vary among different tissue and cell types [13,19,23] and different stages of cell differentiation [24], indicating that precise regulation of spatial and temporal expression of furin is important for development. As detailed previously, furin plays an important role in processing important factors involved in trophoblast syncytialization, including IGF-I, IGF1R [25,26] VEGF [27] and syncytins [17,18]. More importantly, deletion of the FUR gene in mice leads to early embryonic death at E10.5 due to developmental defects and failure of allantois-chorion fusion [28], a key step and a pre-requisite for trophoblast syncytialization.…”