title: Receptor-mediated activation of a proinsulin-transferrin fusion protein in hepatoma cells.
abstract: A proinsulin-transferrin (ProINS-Tf) recombinant fusion protein was designed and characterized for the sustained release of an active form of insulin (INS) by hepatoma cells. During incubation with H4IIE hepatoma cells, a gradual decline of ProINS-Tf concentration, with a concomitant generation of the immuno-reactive insulin-transferrin (irINS-Tf), was detected in the culture medium by using INS- or proinsulin (ProINS)-specific radioimmunoassay (RIA) system. Further studies indicated that the conversion of ProINS-Tf to irINS-Tf was a transferrin receptor (TfR) mediated process that was pH-sensitive, and temperature- and microtubule-dependent. These results suggest that the conversion occurred during the slow recycling route of transferrin (Tf)-TfR pathway, possibly processed by proteases in the slow recycling compartments juxtaposed to the trans-Golgi network (TGN). ProINS-Tf exhibited little activity in the short-term promotion of glucose uptake in adipocytes, indicating that it was in an inactive form similar to ProINS. Stimulation of Akt phosphorylation by ProINS-Tf was detected only after prolonged incubation with H4IIE cells. On the other hand, ProINS-Tf pre-incubated with H4IIE cells for 24h acquired an immediate activity of stimulating Akt phosphorylation. Furthermore, ProINS-Tf elicited a strong activity in the inhibition of glucose production following 24h incubation with H4IIE cells. Based on these findings, we conclude that the Tf-TfR endocytosis and recycling pathway enables the conversion and release of ProINS-Tf in an active form of irINS-Tf. Results from this study suggest that the Tf-TfR pathway can be exploited for the design of prohormone-Tf fusion proteins as protein prodrugs for their sustained and targeted activation.
authors.affiliation: Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, United States.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.forename: Yu-Sheng
authors.initials: YS
authors.lastname: Chen
authors.forename: Jennica L
authors.initials: JL
authors.lastname: Zaro
authors.forename: Wei-Chiang
authors.initials: WC
authors.lastname: Shen
mesh_terms: D041721:3T3-L1 Cells; D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D016895:Culture Media, Serum-Free; D004705:Endocytosis; D005947:Glucose / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D057809:HEK293 Cells; D006801:Humans; D007004:Hypoglycemic Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D007328:Insulin / Q000378:metabolism; D051379:Mice; D011355:Prodrugs / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D011384:Proinsulin / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology*; D011863:Radioimmunoassay; D051381:Rats; D011990:Receptors, Transferrin / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology*; D014162:Transfection; D014168:Transferrin / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D016895:Culture Media, Serum-Free; D007004:Hypoglycemic Agents; D007328:Insulin; D011355:Prodrugs; D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; D014168:Transferrin; D011384:Proinsulin; D005947:Glucose