Intracellular insulin-receptor dissociation and segregation in a rat fibroblast cell line transfected with a human insulin receptor gene

Journal Article · Thu May 05 00:00:00 EDT 1988 · J. Biol. Chem.; (United States)

OSTI ID:6771815

Levy, J R; Olefsky, J M

The cellular processing of insulin and insulin receptors was studied using a rat fibroblast cell line that had been transfected with a normal human insulin receptor gene, expressing approximately 500 times the normal number of native fibroblasts insulin receptors. These cells bind and internalize insulin normally. Biochemically assays based on the selective precipitation by polyethylene glycol of intact insulin-receptor complexes but not of free intracellular insulin were developed to study the time course of intracellular insulin-receptor dissociation. Fibroblasts were incubated with radiolabeled insulin at 4/sup 0/C, and internalization of insulin-receptor complexes was initiated by warming the cells to 37/sup 0/C. Within 2 min, 90% of the internalized radioactivity was composed of intact insulin-receptor complexes. The dissociation of insulin from internalized insulin-receptor complexes was markedly inhibited by monensin and chloroquine. Furthermore, chloroquine markedly increased the number of cross-linkable intracellular insulin-receptor complexes, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. These findings suggest that acidification of intracellular vesicles is responsible for insulin-receptor dissociation. Physical segregation of dissociated intracellular insulin from its receptor was monitored. The results are consistent with the view that segregation of insulin and receptor occurs 5-10 min after initiation of dissociation. These studies demonstrate the intracellular itinerary of insulin-receptor complexes, including internalization, dissociation of insulin from the internalized receptor within an acidified compartment, segregation of insulin from the receptor, and subsequent ligand degradation.

• Research Organization: Univ. of California, San Diego, La Jolla (USA)
• OSTI ID: 6771815
• Journal Information: J. Biol. Chem.; (United States), Vol. 263:13
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
RECEPTORS
BIOCHEMICAL REACTION KINETICS
FIBROBLASTS
GENES
INSULIN
IODINE 125
LABELLED COMPOUNDS
LIGANDS
MAN
RATS
ANIMAL CELLS
ANIMALS
BETA DECAY RADIOISOTOPES
CONNECTIVE TISSUE CELLS
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
HORMONES
INTERMEDIATE MASS NUCLEI
IODINE ISOTOPES
ISOTOPES
KINETICS
MAMMALS
MEMBRANE PROTEINS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PEPTIDE HORMONES
PRIMATES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
RODENTS
SOMATIC CELLS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques