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-114 solution above the cloud-point, a detergent enriched phase pellets and coprecipitates 95% of the purified insulin-free (alpha beta)2 receptors. In contrast, 83% of the hormone bound (alpha beta)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 (alpha beta)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 (alpha beta)2 receptors appear to fundamentally restrict access to the membrane-exposed receptor domain. Basically, different molecular properties are found for alpha beta receptors. Only 67% of the insulin-free receptors coprecipitate with the Triton-X-114-enriched phase; following insulin binding the coprecipitation is only decreased to 42%. In contrast to (alpha beta)2 receptors, formation of noncovalently aggregated receptor complexes, which are detected by sucrose density centrifugation, could account for the exclusion of alpha beta receptor species from Triton X-114 membranes.
Flörke
R R
RR
Diabetes-Forschungsinstitut an der Heinrich Heine-Universität, Düsseldorf, Federal Republic of Germany.
Klein
H W
HW
Reinauer
H
H
D011232:Chemical Precipitation; D006801:Humans; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism; D008051:Lipid Bilayers; D008562:Membrane Glycoproteins / Q000737:chemistry; D008567:Membranes, Artificial; D011092:Polyethylene Glycols / Q000737:chemistry; D011972:Receptor, Insulin / Q000737:chemistry* / Q000378:metabolism; D012995:Solubility
D007328:Insulin; D008051:Lipid Bilayers; D008562:Membrane Glycoproteins; D008567:Membranes, Artificial; D011092:Polyethylene Glycols; D011972:Receptor, Insulin