Antibody binding to the juxtamembrane region of the insulin receptor alters receptor affinity.
The effect of three antibodies that interact with distinct regions of the insulin receptor (the alpha subunit (83-7), the juxtamembrane region near tyrosine 960 (960) or the carboxy terminal region of the beta subunit (CT-1) on insulin binding was examined. Detergent-solubilized insulin receptors from IM-9 cells immobilized on Sepharose beads by 960 antisera bound 2-3 times more 125I-insulin tracer (25-60 pM) than receptors immobilized with either 83-7 or CT-1. Pre-incubation of solubilized receptors with either 83-7 or 960 resulted in equivalent depletion (90%) of insulin binding activity from solubilized IM-9 cell extracts, suggesting that both antibodies were in excess and capable of binding a similar population of receptors. Antibody 960, but not CT-1 or 83-7, also increased insulin binding 2 fold to solubilized receptors precipitated with polyethylene glycol. To determine whether the altered binding observed with antibody 960 was due to increased affinity of the receptor for insulin or appearance of more insulin binding sites, binding studies were performed over a wide range of insulin concentrations. Analysis of the resulting binding curves indicated that 960 increased the affinity of the receptor for insulin 3 fold over control (kd = 0.3 nM for 960, and 0.9 nM for 83-7, respectively). The antibody 960 also specifically increased insulin binding to intact, saponin-permeabilized IM-9 cell membranes. These results indicate that binding of 960 antibody to the juxtamembrane region of the insulin receptor alters the affinity of the receptor for insulin. Since tyrosine 960 in the juxtamembrane region has been suggested to play a role in receptor signalling, changes in receptor conformation in this region that are likely to account for the change in affinity may play a role in signal transduction.
Goodman
D W
DW
Department of Pharmacology, University of Virginia, Charlottesville 22908.
Romero
G
G
Isakson
P
P
D000906:Antibodies / Q000276:immunology / Q000378:metabolism*; D001665:Binding Sites; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D006801:Humans; D007118:Immunoassay; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011972:Receptor, Insulin / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology
D000906:Antibodies; D007328:Insulin; D008565:Membrane Proteins; D011972:Receptor, Insulin