Y429, Y431, and Y479 mediate ligand-induced EpoR internalization. (A) Wild-type EpoR, but not F8, is degraded (left) on Epo stimulation. Wild-type EpoR, but not F8, is internalized on Epo stimulation as detected by surface IP (right). (B) F8 does not colocalize with clathrin or EEA1 on stimulation (original magnification ×40; Leica TCS SP5). (C) Epo-induced internalization of HA-F8 is dramatically reduced in TER119⁻ erythroid progenitor cells from murine fetal livers. Surface expression of HA-EpoR or HA-F8 was measured 60 minutes after Epo stimulation. (D) Y429, Y431, or Y479 mediates ligand-induced EpoR internalization. Surface IP was performed on γ2A cells stably expressing HA-EpoR constructs with individual cytosolic tyrosine. (E,F) Y429, Y431, or Y479, but not other tyrosines, is sufficient for ligand-induced EpoR internalization. Ligand-induced receptor internalization was measured by flow cytometry in γ2A cells. (G) Replacing Y429, Y431, or Y479 individually on the wild-type EpoR (F429, F431, and F479) or replacing both Y429 and Y431 (Y2F) did not affect receptor internalization by flow cytometry, but simultaneously mutating all 3 tyrosines (Y3F) significantly reduced Epo-induced receptor internalization. *P = .025 (unpaired t test) versus control.