J. Biol. Chem., Vol. 263, Issue 10, 4789-4794, Apr, 1988
Characterization of rat skeletal muscle sarcolemmal insulin receptors and a sarcolemmal insulin binding inhibitor
RH Whitson, GK Grimditch, E Sternlicht, SA Kaplan, RJ Barnard and K Itakura
Department of Molecular Genetics, Beckman Research Institute of the City of Hope, Duarte, California 91010.
When insulin receptors of rat skeletal muscle sarcolemmal vesicles were
solubilized with Triton X-100, the specific binding of 125I-labeled insulin
increased by more than 10-fold over that seen in the intact vesicles.
Partial purification of the skeletal muscle insulin receptors on wheat germ
agglutinin affinity columns increased the total insulin binding activity by
7-fold and reduced the Kd for insulin binding from 1.92 to 0.20 nM,
suggesting that an inhibitor of insulin binding was removed by this
purification step. This was confirmed when the unbound fractions of the
affinity column were dialyzed and reconstituted with the insulin receptors.
The inhibitory activity in the sarcolemmal extract could not be accounted
for by the presence of Triton X-100. The skeletal muscle inhibitor was more
potent in inhibiting insulin binding to skeletal muscle insulin receptors
than to liver or adipose receptors. The inhibitor was very effective in
inhibiting insulin binding to wheat germ agglutinin-purified IM-9
receptors, but had negligible effects on insulin binding to intact IM-9
cells. The properties of the alpha and beta subunits of the skeletal muscle
insulin receptors appear to be the same as those of insulin receptors of
other tissues: cross-linking of 125I-labeled insulin to the receptor
revealed a band of 130,000 daltons, and insulin stimulated the
phosphorylation of bands of 90,000 and 95,000 daltons in the receptor
preparation. The skeletal muscle insulin binding inhibitor elutes from
molecular sieves in a major 160,000-dalton peak and minor 75,000-dalton
peak. The binding inhibitor is not inactivated by heat, by mercaptoethanol,
or by trypsin, pepsin, or proteinase K. Collectively, these data suggest
that the inhibitor may be a small molecule that aggregates with itself,
with larger proteins, or with detergent micelles.
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