| Abstract |
Previously in our lab, a mutant Chinese hamster fibroblast line,
designated A1-j, was clonally selected based on its resistance to a toxic
insulin-diphtheria A-chain conjugate (Leckett and Germinario, 1992).
Subsequent examination revealed that while the parental V-79 strain showed
2-fold insulin-stimulated growth (P<0.01), the mutant exhibited no
significant growth above basal levels (P>0.10). Since the mutant's growth
was similarly affected in response to insulin-like growth factor-I, but
not to 5% fetal bovine serum or unrelated growth factors (i.e., alpha-
thrombin or epidermal growth factor), the mitogenic block appears to be
confined to a pathway shared by insulin and IGF-I. Analysis of thymidine
incorporation data, as well as c-fos and c-jun mRNA expression, placed the
causative mutation early in the cell cycle (i.e., the G1->S boundary).
Measurement of [3H]-2DG uptake along with [14C]-glucose incorporation into
glycogen revealed that insulin stimulation of metabolic pathways, namely
glucose transport and glycogen synthesis is intact in the mutant. Although
A1-j cells appear to possess approximately half of the insulin receptor
complement of the parental cell line (800 vs 1500 receptors per cell as
estimated by Scatchard analysis), ligand binding, internalization,
autophosphorylation, and tyrosine kinase activity appear to be unaffected.
Finally, the A1-j cell line appears to mediate insulin proteolysis which
is significantly more resistant to chloroquine inhibition than the wild-
type V-79 strain. 100microM chloroquine was able to rescue 26.4+- 1.4% of
a prebound cohort of insulin from degradation in V-79 cells versus only
18.4 +- 1.5% of prebound insulin being rescued in the A1-j cell line under
identical conditions (P<0.05). These data are consistent with a mutation
in A1-j resulting in an alteration in the normal endosomal routing of
insulin and/or its receptor.
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