DIFFERENTIAL INSERTION OF INSULIN-RECEPTOR COMPLEXES INTO TRITON-X-114 BILAYER-MEMBRANES - EVIDENCE FOR A DIFFERENTIAL ACCESSIBILITY OF THE MEMBRANE-EXPOSED RECEPTOR DOMAIN

In the present study, the Triton X-114 phase-separation system has been used to characterize molecular properties of the membrane-exposed domain of an integral-membrane hormone receptor. This approach provides novel details of the structure/function relationship of insulin receptors. Upon raising the temperature of a micellar Triton X-1 14 solution above the cloud-point, a detergent enriched phase pellets and coprecipitates 95% of the purified insulin-free (alphabeta)2 receptors. In contrast, 83% of the hormone bound (alphabeta)2 receptor complexes prefer the detergent-depleted phase, exhibiting prominent properties of non-membraneous proteins. Kinetic studies show that, following insulin binding, the amphiphilicity of the receptor complexes is immediately altered. Only monodisperse (alphabeta)2 complexes were detected when receptor/insulin complexes of the detergent-depleted phase were analyzed by detergent-free sucrose density centrifugation in the presence of 10 nM insulin. These results can be explained in the light of the lipid-bilayer-like organization of the precipitating Triton X-114; hormone-induced intramolecular alterations of (alphabeta)2 receptors appear to fundamentally restrict access to the membrane-exposed receptor domain. Basically, different molecular properties are found for alphabeta receptors. Only 67% of the insulin-free alphabeta receptors coprecipitate with the Triton-X-114-enriched phase; following insulin binding the coprecipitation is only decreased to 42%. In contrast to (alphabeta)2 receptors, formation of noncovalently aggregated receptor complexes, which are detected by sucrose density centrifugation, could account for the exclusion of alphabeta receptor species from Triton X-114 membranes.