Biogenesis, transit, and functional properties of the insulin proreceptor and modified insulin receptors in 3T3-L1 adipocytes. Use of monensin to probe proreceptor cleavage and generate altered receptor subunits.

The biogenesis, intracellular transport, and functional properties of the insulin proreceptor and modified insulin receptors were studied in hormone-responsive 3T3-L1 adipocytes. After control cells were labeled with [35S]Met for 7 min, the principal polypeptide that was precipitated by anti-insulin receptor antibodies had a molecular weight (Mr) of 180 000. This initial precursor was rapidly converted (t1/2 = 35 min) to a 200-kilodalton (kDa) polypeptide, designated the insulin proreceptor, by the apparent posttranslational addition of N-linked, high mannose core oligosaccharide units. Mature alpha (Mr 130 000) and beta (Mr 90 000) subunits were derived from sequences within the proreceptor by proteolytic cleavage and late processing steps, and these subunits appeared on the cell surface 2-3 h after synthesis of the 180-kDa precursor. The cation ionophore monensin was used in combination with metabolic labeling, affinity cross-linking, and external proteolysis to probe aspects of proreceptor function, transit, and the development of insulin sensitivity at the target cell surface. At 5 micrograms/mL, monensin potently inhibited the proteolytic cleavage step, and the 200-kDa polypeptide accumulated. Lower concentrations of the ionophore selectively blocked late processing steps in 3T3-L1 adipocytes so that apparently smaller alpha' (Mr 120 000) and beta' (Mr 85 000) subunits were produced. Proreceptor and alpha' and beta' subunits were translocated to the cell surface, indicating that the signal for intracellular transit occurs in the 200-kDa polypeptide and is independent of the posttranslational proteolysis and late processing steps. The alpha' subunit bound insulin both at the surface of intact cells and after solubilization with Triton X-100; the beta' subunit was phosphorylated in an insulin-stimulated manner. The detergent-solubilized 200-kDa proreceptor also exhibited both functional properties. However, the proreceptor that was transported to and exposed on the cell surface was incapable of binding insulin in intact adipocytes. Thus, late processing is not essential for the expression of functions associated with mature alpha and beta subunits. In contrast, it appears that the proteolytic generation of subunits is required for the correct orientation of the hormone binding site in the plasma membrane bilayer and the development of insulin responsiveness in 3T3-L1 adipocytes.