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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.

Turkey erythrocytes possess insulin receptors with binding properties very similar to those of mammalian insulin receptors. In the present study, the insulin receptor of the avian erythrocyte has been solubilized in Triton X-100, extensively characterized and partially purified, and its properties compared to those of the membrane-bound receptor. The solubilized insulin receptor has a Stokes radius of 70 A and an apparent molecular weight of 300 000 in 0.05% Triton. The binding of insulin to the soluble receptor was very similar to the binding observed with the membrane-bound receptor. Thus, binding was markedly temperature dependent for both the soluble and membrane-bound forms, although the kinetics of binding were slower with the soluble receptor. Both forms of the receptor also showed a sharp pH optimum; however, solubilization produced a shift from maximal binding at pH 7.8 to pH 7.3. The soluble receptor also retained insulin analog specificity, ion sensitivity and negative cooperativity. The soluble receptor did not appear to degrade either bound or free insulin. On DEAE-cellulose chromatography the receptor eluted as a single peak. The specific activity of this partially purified preparation was 25--30 pmol/mg protein (about 500-fold enrichment over crude extract and 5-fold over highly purified membranes). Extensive attempts to purify further the receptor by gel filtration, carboxymethyl-cellulose chromatography and affinity chromatography resulted in either a very low yield or only modest enrichment. Purification was also complicated because the receptor was easily denatured; about 40% of the activity was lost after a 90-min exposure to 3 M urea or pH 4.5. These data suggest that the insulin receptor retains its properties in the absence of the lipid bilayer of the membrane. Complete purification will be difficult due to a lack of stability under a number of conditions.

We have altered the phospholipid composition of the plasma membranes of Ehrlich ascites cells, grown in mice and studied the effects on the properties of the insulin receptor of this cell. The insulin receptor of the Ehrlich cell demonstrated all of the binding characteristics of mammalian insulin receptors: specificity for insulin and insulin analogs, saturability, inverse relationship of steady-state binding levels to temperature, and negative cooperativity. Cellular phospholipids enriched in monounsaturated fatty acyl groups were produced by growth in animals that were maintained on a diet rich in coconut oil; cellular phospholipids enriched in polyunsaturated fatty acyl groups were produced in animals fed sunflower oil. Insulin receptors were present in the normal cells at 180,000 sites/cell but this fell to 125000 (P less than 0.001) in cells enriched in monounsaturated fatty acids and rose to 386,000 (P less than 0.001) in cells enriched in polyunsaturated fatty acids. The normal cells had affinity constants (Ke and Kf) of 0.03 and 0.01 nM-1. The cells enriched in monounsaturated fatty acids had an increase in these affinity constants to 0.06 and 0.03 nM-1 whereas values of 0.01 and 0.005 mM-1 were obtained in the cells enriched in polyunsaturated fatty acids (all comparison P less than 0.001). Thus, increased unsaturation of plasma membrane phospholipids, produced by dietary manipulations, was associated with an increase in insulin receptor number but a decrease in binding affinity. In contrast, increased saturation of the phospholipids of the plasma membrane was associated with a decrease in receptor number and an increase in affinity. The results can be explained by a model in which the insulin receptor is assumed to be multimeric.

The effect of low serum concentration on plasma membrane fluidity and lipid composition, differentiation and insulin binding was investigated in three Friend erythroleukemia clones. Both FLC (clones No. 745) and F(+) (Ostertag F4N) Friend erythroleukemia cells can be induced to differentiate and to produce hemoglobin when exposed to DMSO. Clone R(3) (Ostertag F4-D5-1) is a DMSO-resistant clone when grown under normal conditions (15% serum) but could undergo differentiation with accumulation of protoporphyrin IX upon induction with DMSO when grown in low serum concentration (2.5% serum). Electron spin resonance measurements of the order parameters (S) and S(T parallel) demonstrate that R(3) has a more fluid plasma membrane than the FLC and F(+). The order parameters of the outer hyperfine splittings S(T parallel) at 37 degrees C are 0.60 +/- 0.009, 0.62 +/- 0.008 and 0.64 +/- 0.009 for R(3), F(+) and FLC, respectively. We have used the insulin receptors as a model for a polypeptide hormone receptor associated with the plasma membrane of the Friend clones. Insulin binding studies demonstrated that the receptor of R(3) had a decreased affinity for insulin manifest as a 10-fold increase in the amount of insulin required to compete for half of the tracer binding (18 nM for R(3) vs. 2 nM for FLC and F(+)). Computer-fit Scatchard plot analysis by the negative cooperativity model reveal that R(3) possessed a similar number of sites/cell (about 70,000) as the FLC or F(+) cells, with similar high and low affinities. Growing the DMSO-resistant clone R(3) in low serum concentration caused a decrease in receptor number by 35%, and an increase in receptor affinity to that seen with the differentiable clones. Thus, the abnormal properties of the plasma membrane and insulin receptor of the DMSO-resistant clone in our earlier report (Simon et al. (1984) Biochim. Biophys. Acta 803, 39-47) were partially reversed by growing the cells in a low serum concentration, restoring the cellular response to the differentiation agent.

The insulin receptor of the turkey erythrocyte has previously been shown to be very similar to that of the mammalian insulin receptors. As a first step in the isolation of this receptor a highly purified plasma membrane fraction has been prepared. The binding characteristics of the purified membrane-bound receptor were identical to those found with intact erythrocytes, but the membrane preparation had very little insulin-degrading activity. Isolation of the membrane by the methods described gave a 100-fold purification of the insulin receptor with 67% yield.