Reconstitution studies of lipid effects on insulin-receptor kinase activation.
Insulin receptors extracted from human placenta were reconstituted by dialysis into well-characterized lipid vesicles. For all types of lipids studied, vesicles were shown to be unilamellar, about 120 nm in diameter. The incorporation of lectin-purified insulin receptors was assessed by cosedimentation of 125I-insulin binding and [32P]phospholipids in a sucrose gradient. The insulin-binding activity was not modified by the composition of the lipid vesicles. However, tyrosine kinase activation appeared to be more sensitive to its lipid environment. Mixtures of phosphatidylcholine/phosphatidylserine or phospholipids/phosphatidylserine, in ratios of 1-4, increased the insulin-induced tyrosine kinase activation in a dose-dependent manner. In contrast, experiments performed in the presence of phosphatidylinositol showed a decrease in the enzyme stimulation. These results indicate an opposing involvement of these two anionic phospholipids in the kinase activation. Inclusion of cholesterol (10-30%) into phosphatidylcholine vesicles reduced kinase activation, which was drastically inhibited by 30% cholesterol. The effect of a total extract of brain gangliosides was biphasic, stimulatory at low concentration (5-10%), but with a reverse effect at higher concentrations. These results stress the importance of the lipid environment for insulin-receptor signaling, particularly for the insulin-induced activation of its beta-subunit kinase.
Leray
V
V
Institut National de la Santé et de la Recherche Médicale, Unité 338, Centre de Neurochimie, Strasbourg, France.
Hubert
P
P
Burgun
C
C
Staedel
C
C
Crémel
G
G
D000818:Animals; D002417:Cattle; D002784:Cholesterol / Q000494:pharmacology; D005732:Gangliosides / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000378:metabolism; D008055:Lipids / Q000494:pharmacology*; D010743:Phospholipids / Q000494:pharmacology; D010920:Placenta / Q000201:enzymology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011972:Receptor, Insulin
D005732:Gangliosides; D007328:Insulin; D008055:Lipids; D010743:Phospholipids; D002784:Cholesterol; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin