Tyrosine kinase-defective insulin receptors undergo insulin-induced microaggregation but do not concentrate in coated pits.
Biologically active colloid-gold complexes were used to compare ligand-induced microaggregation, redistribution, and internalization of insulin receptors on Rat 1 fibroblasts expressing wild type (HIRc) or tyrosine kinase-defective (HIR A/K1018) human insulin receptors. Insulin-like growth factor I (IGF I) and alpha 2-macroglobulin receptors also were compared. On both cell types, all four unoccupied receptor types occurred predominantly as single receptors. Ligand binding caused receptor microaggregation. Microaggregation of wild type or kinase-defective insulin receptors or IGF I receptors was not different. alpha 2-Macroglobulin receptors formed larger microaggregates. Compared to wild type insulin or IGF I receptors, accumulation of kinase-defective insulin receptor microaggregates in endocytic structures was decreased, and the size of microaggregates in coated pits was significantly smaller. As a result, receptor-mediated internalization of gold-insulin by HIR A/K1018 cells was less than 6% of the cell-associated particles compared to approximately 60% of the particles in HIRc cells. On HIR A/K1018 cells, alpha 2-macroglobulin and IGF I were internalized via coated pits demonstrating that those structures were functional. These results suggest that: 1) ATP binding, receptor autophosphorylation, and activation of receptor kinase activity are not required for receptor microaggregation; 2) receptor microaggregation per se is not sufficient to cause ligand-induced receptor-mediated internalization or the biological effects of insulin; and 3) autophosphorylation of the beta-subunit or activation of the receptor kinase activity is required for the insulin-induced concentration of occupied receptors in coated pits.
Smith
R M
RM
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104.
Seely
B L
BL
Shah
N
N
Olefsky
J M
JM
Jarett
L
L
D000818:Animals; D002478:Cells, Cultured; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008854:Microscopy, Electron; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D011940:Receptor Aggregation; D011972:Receptor, Insulin / Q000378:metabolism* / Q000648:ultrastructure; D013696:Temperature
D008024:Ligands; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin