Recently, a mutation substituting Leu for Ser323 in the α-subunit of the human insulin receptor has been identified in an insulin-resistant patient. The Leu323 mutation leads to a severe impairment in insulin binding without significantly altering the processing or cell surface expression of the receptor. In order to study how αβ half-receptors interact to form the insulin-binding site, we cotransfected NIH-3T3 cells with two insulin receptor cDNA constructs: a truncated insulin receptor lacking the C-terminal 43 amino acids (Δ43) and the full-length Leu323 mutant receptor. A clonal cell line from cotransfected cells expresses a hybrid receptor consisting of a Leu323 half-receptor and a Δ43 half- receptor. We demonstrate that the Leu323-Δ43 hybrid receptor binds insulin with high affinity. Furthermore, by cross-linking 125I-insulin to immobilized hybrid receptors, we show that only the αβ(Δ) half of the hybrid receptor binds insulin. Since the isolated half-insulin receptor has low affinity for insulin, this suggests that the addition of even a non- binding α-subunit can result in high affinity binding to the holoreceptor (αα(mut)β(Δ)β). Both β and β(Δ)-subunits of the Leu323-Δ43 hybrid receptor are phosphorylated in vivo and in vitro in an insulin-dependent manner, suggesting an intramolecular transphosphorylation mechanism and that the presence of the Leu323 mutant receptor that lacks an intrinsic high affinity binding site does not prevent the associated β-subunit from functioning either as a tyrosine kinase or as a phosphate acceptor in the hybrid insulin receptor molecule (αα(mut)β(Δ)β). Furthermore, we show that the hybrid receptor can phosphorylate insulin receptor substrate-1 (IRS- 1) in response to insulin and can be coimmunoprecipitated together with IRS- 1 by anti-IRS-1 antibody.
|Original language||English (US)|
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|State||Published - May 27 1994|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology