TYROSINE 462 OF THE MEMBRANE-PROXIMAL F′-G′ LOOP OF MURINE Mpl IS NOT ESSENTIAL FOR HIGH-AFFINITY BINDING OF THROMBOPOIETIN

Abstract

The ligand binding site of Mpl, the thrombopoietin (Tpo) receptor, has not been determined. Tyr462of murine Mpl corresponds to Tyr421of the common β chain of the human IL-3, IL-5 and GM-CSF receptors. Tyr421has been identified as essential for high-affinity ligand binding. To determine whether Tyr462is similarly required for Tpo binding, wild-type murine Mpl (Mpl-WT) or mutant receptors containing an alanine (Y462A) or lysine (Y462K) in place of Tyr462were expressed in BaF3 cells. In proliferation studies, the Y462A mutation had no effect on Tpo-induced growth. In contrast, the Y462K mutation led to an attenuated proliferative response to Tpo. In single-point binding studies, both Mpl-WT and Y462A cells were able to bind [125I]Tpo in a specific manner. In contrast, there was a marked reduction in binding of [125I]Tpo by Y462K cells. Mpl-WT cells bound Tpo with a Kdof approximately 330pM, while Y462A cells bound Tpo with a Kdof approximately 268pM. The binding affinity of Y462K cells was below that quantifiable by Scatchard analysis. This study suggests that unlike the corresponding Tyr421of the common human β chain, Tyr462of murine Mpl is not required for high-affinity ligand binding, although it may be located in proximity to the ligand binding site.