Post-translational Acquisition of Insulin Binding Activity

The post-translational acquisition of ligand binding activity by the insulin receptor was examined in 3T3L1 adipocytes. In pulse-chase experiments with [35S] methionine, labeled receptor species were separated into “active” and “inactive” forms by affinity chromatography on insulin-agarose. and then were characterized and quantitated. It was found that the newly translated high molecular weight proreceptor lacks the capacity to bind insulin. The acquisition of binding activity is relatively slow (tllz = 45 min) and occurs prior to conversion of the proreceptor to the mature aand @-subunits by proteolytic cleavage and maturation of its N-linked oligosaccharide chains (tl, = 3 h). Glycosylation appears to be required for thls activation since the aglycoproreceptor, synthesized in the presence of tunicamycin, does not acquire insulin binding activity. However, once the proreceptor has acquired ligand binding activity, removal of its N-linked oligosaccharide chains with endoglycosidase H has no effect on the ability of the proreceptor to bind insulin. The modification of the proreceptor that gives rise to insulin binding activity most likely involves a conformational change in the binding domain. A human autoimmune antibody that recognizes only the active insulin binding site does not interact with the inactive proreceptor, whereas a rabbit polyclonal antireceptor antibody recognizes all forms. Thus, the autoimmune antibody must recognize a new epitope created during conversion of the inactive proreceptor to the active form.