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J. Biol. Chem., Vol. 263, Issue 15, 7342-7351, May, 1988
TS Olson, MJ Bamberger and MD Lane
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
insulin receptor species, isolated by immuno- and insulin- affinity
adsorption, were analyzed by sodium dodecyl sulfate (SDS)- polyacrylamide
gel electrophoresis under conditions where intra- and intermolecular
disulfide bonds remained intact or were cleaved by reduction. Reducing
SDS-polyacrylamide gel electrophoresis confirmed that the insulin receptor
is synthesized as a long-lived (t1/2 = 3 h) proreceptor precursor of 210
kDa which undergoes proteolytic cleavage and carbohydrate maturation to
form the alpha- and beta-subunits of the mature receptor. The proreceptor
acquires insulin binding activity through a subtle structural change (t1/2
= 45 min) detected only by an autoimmune antibody specific for an epitope
of the active insulin binding site. Analysis of insulin receptor species by
nonreducing SDS- polyacrylamide gel electrophoresis revealed that the
proreceptor undergoes two additional structural changes not detected by
reducing SDS-polyacrylamide gel electrophoresis. The proreceptor is
synthesized as a monomer (M1) with an apparent molecular mass of 170 kDa
that is converted by disulfide rearrangement to another monomeric form of
190- kDa apparent molecular mass (M2). N-Linked glycosylation is required
for this transition, since aglycoproreceptor, synthesized in the presence
of tunicamycin, does not undergo any detectable tertiary or quaternary
structural changes. M2 self-associates to form a disulfide- linked
proreceptor dimer (D) which is subsequently proteolytically processed,
forming the mature, disulfide-linked alpha 2 beta 2 receptor tetramer. The
mature receptor was distinguished from the three proreceptor species (M1,
M2, and D) by its cell surface location and its ability to bind tightly to
wheat germ agglutinin-agarose, indicating the presence of complex
oligosaccharide chains. Subcellular fractionation indicated that both the
M1 to M2 and M2 to D conversions occur in the endoplasmic reticulum.
Separation of the nonreduced proreceptor species into "active" and
"inactive" forms by affinity chromatography on insulin-agarose revealed
that neither the transition of M1 to M2, nor of M2 to D, is correlated with
the acquisition of insulin binding function. Rather, during its life-time,
the M2 species acquires insulin binding activity and an epitope recognized
by a binding site specific autoimmune antibody through a subtle structural
change not detected by reducing or nonreducing SDS-polyacrylamide gel
electrophoresis.
Post-translational changes in tertiary and quaternary structure of the insulin proreceptor. Correlation with acquisition of function
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
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