Analysis of in vitro interactions of protein tyrosine phosphatase 1B with insulin receptors

One strategy to treat the insulin resistance that is central to type II dia betes mellitus may be to maintain insulin receptors (IR) in the active (tyr osine phosphorylated) form. Because protein tyrosine phosphatase 1B (PTP1B) binds and subsequently dephosphorylates IR, inhibitors of PTP1B-IR binding are potential insulin 'sensitizers.' A Scintillation Proximity Assay (SPA) was developed to characterize and quantitate PTP1B-1R binding. Human IR we re solubilized and captured on wheat germ agglutinin (WGA)-coated SPA beads . Subsequent binding of human. catalytically inactive [S-35] PTP1B Cys(215) /Ser (PTP1B(C215S)) to the lectin-anchored IR results in scintillation from the SPA beads that call be quantitated. Binding of PTP1B to IR was PH- and divalent cation-sensitive. Ca2+ and Mn2+, but not Mg2+, dramatically atten uated the loss of PTP1B-1R binding observed when pH was raised from 6.2 to 7.8. PTP1B binding to IR from insulin-stimulated cells was much greater tha n to IR from unstimulated cells and was inhibited by either an antiphosphot yrosine: antibody or treatment of IR with alkaline phosphatase, suggesting that tyrosine phosphorylation of IR is required for PTP1B binding. Phosphop eptides modeled lifter various: IR phosphotyrosine domains each only partia lly inhibited PTP1B-IR binding, indicating that multiple domains of IR are likely involved in binding PTP1B. However. competitive displacement of [S-3 5]PTP1B(C215S) by PTP1B(C215S) fitted best to a single binding site with a K-d in the range 100-1000 nM, depending upon pH and divalent cations. PNU-2 00898, a potent and selective inhibitor of PTP1B whose orientation in the a ctive site of PTP1B has been solved, competitively inhibited catalysis and PTP1B IR binding with equal potency. The results of this novel assay for PT P1B-IR binding suggest that PTP1B binds preferentially to tyrosine phosphor ylated IR through its active site and that binding may be susceptible to th erapeutic disruption by small molecules. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.