Insulin receptors of chicken liver and brain

Receptors on membranes of chicken liver and brain bound porcine 125 I‐insulin in a specific and temperature‐dependent manner. Competition with unlabeled insulin derivatives exhibited typical insulin potency ratios, i.e. chicken > porcine insulin > human proinsulin (2.1/1/0.02). Apparent binding affinity was higher in brain with a 50% inhibition of tracer binding of 1.3 ± 0.2 nM porcine insulin as compared to 2.8 ± 0.3 nM in liver. The apparent molecular mass of the 125 I‐insulin cross‐linked α subunit of the insulin receptor was 139 ± 2 kDa for chicken liver and 127 ± 2kDa for chicken brain. These molecular masses were similar to those of rat liver and brain insulin receptors. Neuraminidase treatment of the cross‐linked insulin receptor increased the mobility of the α subunit from liver but did not affect that from brain, suggesting a difference in the glycosylation of the chicken brain α subunit as previously described in the rat. Despite this change, both receptors could be purified on wheat germ agglutinin (WGA) chromatography after Triton solubilization. In the presence of CTP and vanadate (phosphatase inhibitors) insulin‐stimulatable tyrosine‐specific phosphorylation of exogenous substrates was demonstrated with chicken liver and brain receptors. The reaction was dependent on Mg 2+ and Mn 2+ . As noted with other insulin receptors, the best artificial substrate for phosphorylation was poly(Glu, Tyr) 4: 1 . In both chicken liver and brain the smallest effective insulin dose as well as maximal stimulation of phosphorylation of the substrate was similar to that seen with rat liver, and in all three tissues chicken insulin was more potent than porcine insulin. In chicken liver an active ATP hydrolytic activity copurified with the insulin receptors during WGA chromatography. Further purification using S‐300 Sephacryl filtration or affinity (insulin‐biotin‐avidin) chromatography could dissociate the phosphorylation and the hydrolytic activities. Gel electrophoresis, under reducing conditions revealed β subunits with apparent M r of 97–99 kDa in chicken liver and brain, which were phosphorylated in the presence of insulin. Similar apparent molecular masses have been described for the β subunit of rat liver receptors. These studies suggest that both chicken brain and liver insulin receptors exhibit coupling of α and β subunits with fully active tyrosine kinase and that the structural difference of the brain insulin receptor is widespread and phylogenetically old.