Up-regulation of insulin/insulin-like growth factor-I hybrid receptors during differentiation of HT29-D4 human colonic carcinoma cells.
To assess the autocrine function of insulin-like growth factor II (IGF-II) in the balance of proliferation and differentiation in HT29-D4 human colonic cancer cells, we studied the expression of IGF-I receptors (IGF-IR) and insulin receptors (IR) in relation to the state of cell differentiation. IGF-IR and IR were expressed in both undifferentiated and enterocyte-like differentiated HT29-D4 cells. IGF-IR had two isoforms with a 97-kDa and a 102-kDa beta-subunit. In addition, HT29-D4 cells expressed hybrid receptors (HR) formed by the association of two alphabeta heterodimers from both IR and IGF-IR. HR were evidenced through 1) inhibition of IGF-I binding by the B6 anti-IR antibody and 2) immunoprecipitation with the alpha-IR3 anti-IGF-IR antibody, which revealed an additional 95-kDa IR beta-subunit that disappeared when the heterotetrameric receptor was dissociated by disulfide reduction into alphabeta heterodimers before immunoprecipitation. Like IGF-IR, HR had a high affinity for IGF-I (Kd, approximately 1.5 nM), but did not bind insulin significantly; the latter interacted with the native IR only (Kd, approximately 4 nM). In the differentiated HT29-D4 cell monolayer, all receptor species were strongly polarized (>97%) toward the basolateral membrane. Moreover, HT29-D4 cell differentiation was accompanied by an approximately 2-fold increase in the number of IR, whereas the number of IGF-I-binding sites was unaltered. However, in differentiated HT29-D4 cells, approximately 55% of the latter were involved in HR vs. approximately 20% in undifferentiated HT29-D4 cells. Thus, HT29-D4 cell differentiation is characterized by an up-regulation (approximately 3-fold) of the level of HR coupled to a down-regulation (approximately 40%) of the level of native tetrameric IGF-IR. Alterations were induced early during the cell differentiation process, i.e. 5 days postconfluence, and remained unchanged for at least 21 days. Taken together, these results suggest that the IGF-II autocrine loop in HT29-D4 cells may trigger distinct signaling pathways if it activates native IGF-IR, which predominate in undifferentiated cells, or if it activates HR, which are up-regulated in differentiated cells.
Garrouste
F L
FL
Unité Interactions entre Systèmes Protéiques et Différenciation dans la Cellule Tumorale, CNRS URA 1924, Faculté de Médecine, Marseille, France.
Remacle-Bonnet
M M
MM
Lehmann
M M
MM
Marvaldi
J L
JL
Pommier
G J
GJ
D001667:Binding, Competitive; D002454:Cell Differentiation; D003432:Cross-Linking Reagents; D005434:Flow Cytometry; D019073:HT29 Cells; D006801:Humans; D007163:Immunosorbent Techniques; D007328:Insulin / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D007457:Iodine Radioisotopes; D017526:Receptor, IGF Type 1 / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism*
D003432:Cross-Linking Reagents; D007328:Insulin; D007457:Iodine Radioisotopes; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1; D011972:Receptor, Insulin