Post-translational changes in tertiary and quaternary structure of the insulin proreceptor. Correlation with acquisition of function.

摘要: Abstract Tertiary and quaternary structural changes that occur during post-translational processing of the insulin proreceptor were examined in 3T3-L1 adipocytes. In pulse-chase experiments with [35S]methionine, labeled receptor species, isolated by immuno- insulin-affinity adsorption, analyzed sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis under conditions where intra- intermolecular disulfide bonds remained intact or cleaved reduction. Reducing SDS-polyacrylamide confirmed is synthesized as a long-lived (t1/2 = 3 h) precursor 210 kDa which undergoes proteolytic cleavage carbohydrate maturation to form alpha- beta-subunits mature receptor. The acquires binding activity through subtle change 45 min) detected only an autoimmune antibody specific for epitope active site. Analysis species nonreducing revealed two additional not reducing electrophoresis. monomer (M1) apparent molecular mass 170 converted rearrangement another monomeric 190-kDa (M2). N-Linked glycosylation required this transition, since aglycoproreceptor, presence tunicamycin, does undergo any detectable tertiary changes. M2 self-associates disulfide-linked dimer (D) subsequently proteolytically processed, forming mature, alpha 2 beta tetramer. was distinguished from three (M1, M2, D) its cell surface location ability bind tightly wheat germ agglutinin-agarose, indicating complex oligosaccharide chains. Subcellular fractionation indicated both M1 D conversions endoplasmic reticulum. Separation nonreduced into "active" "inactive" forms affinity chromatography on insulin-agarose neither transition nor D, correlated acquisition function. Rather, life-time, recognized site