Abnormal insulin binding and membrane physical properties of a Friend erythroleukemia clone resistant to dimethylsulfoxide-induced differentiation.
We have compared insulin binding, plasma membrane fluidity, and phospholipid composition of three different Friend erythroleukemia clones, a wild type (FLC) a mutant (R3) and the revertant to wild type F+. The R3 clone is a non-differentiating DMSO-resistant clone (R3) and has altered membrane fluidity and dramatically altered insulin-binding properties. The receptor of R3 bound insulin as if it possessed a single class of low affinity receptors that lacks the property of negative cooperativity. The Scatchard plot is linear and there is no ligand-induced acceleration of dissociation. The Hill coefficient for R3 is 1, implying 'no cooperativity', whereas the Hill coefficient for the two DMSO-inducible clones, (FLC and F+) is 0.3, implying 'negative cooperativity'. In addition, the insulin receptor of R3 has a decreased affinity for insulin, manifested as a 40-fold increase in the amount of insulin required to compete for half of the tracer binding (41 nM for R3 vs. 1 nM for FLC and F+). Computer-fitted Scatchard plots analyzed by the negative cooperativity model reveal that R3 has 95 000 receptor sites/cell, with a high affinity constant Ke of 0.016 nM-1, and a low affinity constant, Kf of 0.012 nM-1. Both DMSO-inducible clones have about 40 000 receptor sites/cell with Ke of 0.11 nM-1 and Kf of 0.02 nM-1. Electron spin resonance measurements with the 5-nitroxy stearate spin probe demonstrate that R3 had a more fluid plasma membrane than the FLC and F+ clones. The lipid composition of R3 is different from that of the DMSO-inducible clones. The weight ratio for unsaturated fatty acids to saturated fatty acids for R3 is 2.5, and the FLC clone has a lower ratio of 1.9. These results are consistent with our earlier findings in FLC that very high membrane fluidity is associated with alterations in the binding properties of the insulin receptor.
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology*; D004351:Drug Resistance; D005227:Fatty Acids / Q000032:analysis; D005622:Friend murine leukemia virus; D007328:Insulin / Q000378:metabolism*; D004915:Leukemia, Erythroblastic, Acute / Q000473:pathology*; D008560:Membrane Fluidity; D008563:Membrane Lipids / Q000032:analysis; D051379:Mice; D011972:Receptor, Insulin / Q000378:metabolism
D005227:Fatty Acids; D007328:Insulin; D008563:Membrane Lipids; D011972:Receptor, Insulin; D004121:Dimethyl Sulfoxide