Posttranslational processing of the insulin proreceptor.

Following translation of the insulin proreceptor by 3T3-L1 adipocytes, about 1.5 hours is required for its conversion into active receptor; an additional 1.5 hours is needed for the active receptor to reach the plasma membrane. During this 3-hour period the proreceptor undergoes a complex series of processing events, glycosylation being an essential processing step. Thus, treatment of 3T3-L1 adipocytes with tunicamycin causes the depletion of cellular insulin binding activity and the accumulation of an inactive aglyco proreceptor. To investigate posttranslational processing of normal proreceptor and the role of glycosylation in active receptor formation, metabolic labeling experiments were conducted. The first 35S-labeled intermediate detected is a 190-kDa polypeptide (proreceptor) which is rapidly (t1/2 = 15 minutes) processed into a 210-kDa species. Both polypeptides contain N-linked core oligosaccharide chains, but in the latter case these chains appear to contain terminal N-acetylglucosamine. The 210-kDa precursor is converted slowly (t1/2 = 2 hours) by proteolytic processing into a 125-kDa (alpha') and 83-kDa (beta') species. Immediately prior to insertion into the plasma membrane, 3 hours after its synthesis, the alpha' and beta' precursors are converted to mature receptor composed of alpha (135 kDa) and beta (95 kDa) subunits. The 125-kDa alpha' and 83-kDa beta' precursors are endoglycosidase H-sensitive and their oligosaccharide chains do not contain terminal sialic acid. Just prior to insertion into the plasma membrane the alpha' and beta' precursors are sialylated, giving rise to the 135-kDa alpha and 95-kDa beta receptor subunits and becoming Endo H resistant and neuraminidase sensitive. In the presence of tunicamycin, a 180-kDa aglyco receptor polypeptide accumulates which is not further processed and does not reach the cell surface. It is concluded that N-linked oligosaccharide chains on the proreceptor are required either for its intracellular translocation to the proteolytic cleavage site or for its identification as a target of the cleavage enzyme. Thus, glycosylation of the insulin proreceptor is crucial for proper processing and formation of functional receptor.