Post-translational processing and activation of insulin and EGF proreceptors

We have investigated the role of glycosylation on the post-translational processing of the insulin, and EGF proreceptor polypeptides. Following translation of the insulin proreceptor, by 3T3-L1 adipocytes, about 1.5 h are required for its conversion into active receptor; an additional 1.5 h are 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 caused the loss of cellular insulin binding activity and the accumulation of an inactive aglyco-proreceptor. Similarly, it was demonstrated in human A431 epidermoid carcinoma cells that the initial EGF-proreceptor (160 kDa) translation product undergoes a slow (t_1/2 = 30 min) processing step by which ligand (EGF) binding activity was acquired. It was shown that N-linked core oligosaccharide addition is essential for this critical processing step and the acquisition of EGF binding activity. This was found not to require the conversion of high mannose chains to complex chains which have been capped with fucose and sialic acid. Possible explanations for this activation in terms of translocation of intermediates and/or formation of disulfide bonds are discussed. To investigate post-translational processing of normal insulin proreceptor and the role of glycosylation in active receptor formation, metabolic labeling experiments were conducted. The first ³⁵S-methionine-labeled intermediate detected is a 190 kDa polypeptide (proreceptor) which is rapidly (t_1/2 = 15 min) 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 (t_1/2 = 2 h) by proteolytic processing into a 125 kDa (α′) and 83 kDa (β′) species. Immediately prior to insertion into the plasma membrane, 3 h after its synthesis, the α′ and β′ precursors are converted to mature receptor comprised of α-(135 kDa) and β-(95 kDa) subunits. The 125 kDa α′- and 83 kDa β′-subunit precursors are endoglycosidase H-sensitive and their oligosaccharide chains do not contain terminal sialic acid. Just prior to insertion into the plasma membrane the α′ and β′ precursors are sialylated, apparently in the Golgi apparatus, giving rise to the 135 kDa α and 95 kDa β receptor subunits and become Endo H-resistant and neuraminidase-sensitive. A proposed sequence of post-translational processing events for the insulin proreceptor is shown in Figure 10. 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 proposed that N-linked oligosaccharide chains on the proreceptor are required either for this 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.