Internalization of insulin receptors in the isolated rat adipose cell. Demonstration of the vectorial disposition of receptor subunits

The internalization of the insulin receptor in the isolated rat adipose cell and the spatial orientation of the α (M(r) = 135,000) and β (M(r) = 95,000) subunits of the receptor in the plasma membrane have been examined. The receptor subunits were labeled by lactoperoxidase/Na ¹²⁵I iodination, a technique which side-specifically labels membrane proteins in intact cells and impermeable membrane vesicles. Internalization was induced by incubating cells for 30 min at 37°C in the presence of saturating insulin. Plasma, high density microsomal (endoplasmic reticulum-enriched), and low density microsomal (Golgi-enriched) membrane fractions were prepared by differential ultracentrifugation. Receptor subunit iodination was analyzed by immunoprecipitation with anti-receptor antibodies, sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and autoradiography. When intact cells were surface-labeled and incubated in the absence of insulin, the α and β receptor subunits were clearly observed in the plasma membrane fraction and their quantities in the microsomal membrane fractions paralleled plasma membrane contamination. Following receptor internalization, however, both subunits were decreased in the plasma membrane fraction by 20-30% and concomitantly and stoichiometrically increased in the high and low density microsomal membrane fractions, without alterations in either their apparent molecular size or proportion. In contrast, when the isolated particulate membrane fractions were directly iodinated, both subunits were labeled in the plasma membrane fraction whereas only the β subunit was prominently labeled in the two microsomal membrane fractions. Iodination of the subcellular fractions following their solubilization in Triton X-100 again clearly labeled both subunits in all three membrane fractions in identical proportions. These results suggest that 1) insulin receptor internalization comprises the translocation of both major receptor subunits from the plasma membrane into at least two different intracellular membrane compartments associated, respectively, with the endoplasmic reticulum and Golgi-enriched membrane fractions, 2) this translocation occurs without receptor loss or alterations in receptor subunit structure, and 3) the α receptor subunit is primarily, if not exclusively, exposed on the extracellular surface of the plasma membrane while the β receptor subunit traverses the membrane, and this vectorial disposition is inverted during internalization.