Transfer of functional insulin receptors to receptor-deficient target cells.
Purified human insulin receptors incorporated into phospholipid vesicles have previously been shown to retain insulin binding as well as insulin-stimulated beta-subunit autophosphorylating activity. These vesicles were used as a vehicle to transfer receptors to the insulin receptor-deficient Madin-Darby canine kidney (MDCK) cell line. Fusion of control [14C] dioleoylphosphatidylcholine-labeled phospholipid vesicles with MDCK cells was found to be dependent on both the amount of time and the concentration of polyethylene glycol used for fusion. Optimal insulin receptor transfer, as determined by recovery of [125I]insulin binding, occurred when MDCK cells were incubated for 45 min at 37 C in the presence of 15% polyethylene glycol plus receptor-containing vesicles. Scatchard analyses for insulin receptor binding before and after vesicle fusion demonstrated no postfusion alteration in insulin receptor affinity and a 10-fold increase in the number of insulin receptors present in the MDCK cells. Fusion transfer of insulin receptors to MDCK cells rendered the cells sensitive to insulin (10-100 nM) for stimulation of glycogen synthesis. Chloroquine (0.1 mM) was found to block endosomal processing of receptor-bound [125I] insulin within 1 h. These findings indicate that insulin receptors function as dissociable units which can be inserted into target plasma membranes with resultant recoupling to cellular systems.
Hofmann
C
C
Department of Biochemistry, Loyola University Stritch School of Medicine, Maywood, Illinois 60153.
Thys
R
R
Sweet
L J
LJ
Spector
A A
AA
Pessin
J E
JE
D000818:Animals; D002460:Cell Line; D002738:Chloroquine / Q000494:pharmacology; D004285:Dogs; D004705:Endocytosis; D006003:Glycogen / Q000096:biosynthesis; D006801:Humans; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology; D007668:Kidney; D008081:Liposomes / Q000378:metabolism; D008561:Membrane Fusion; D010713:Phosphatidylcholines; D011092:Polyethylene Glycols; D011972:Receptor, Insulin / Q000378:metabolism*
D007328:Insulin; D008081:Liposomes; D010713:Phosphatidylcholines; D011092:Polyethylene Glycols; D002738:Chloroquine; D006003:Glycogen; D011972:Receptor, Insulin; C017251:1,2-oleoylphosphatidylcholine