Membrane Raft Localization Is Critical for Erythropoietin Receptor Signaling.

Abstract 2506

Poster Board II-483

Erythropoietin (EPO) is the principal regulator of erythroid progenitor proliferation, differentiation, and survival. Upon ligand engagement, the EPO-receptor (R) homodimerizes to activate the tyrosine kinases, Janus kinase-2 (JAK-2) and Lyn, which in turn phosphorylate the signal transducer and activator of transcription (STAT)-5. Although recent investigations have identified key negative regulators of the EPO-R signal, little is known about the membrane localization and dynamic control of receptor signal fidelity. Here we show a critical role for membrane raft microdomains in the creation of signaling platforms that are essential for signal integrity. Using UT-7 cells, we showed that recombinant human EPO (rhuEPO) stimulation rapidly induced raft formation and aggregation. Confocal microscopy quantitation of the raft ganglioside GM-1 fluorescence showed that raft aggregates increased from a mean of 4.3 ± 1.4 (SE) per cell to 25.6 ± 3.2 aggregates per cell after cytokine stimulation (p≤0.001), accompanied by a greater than 3-fold increase in cluster size (mean stimulated to untreated aggregate size ratio: 3.33 ± 0.11, p≤0.001). Isolation and immunoblot analysis of detergent insoluble raft fractions showed that the EPO-R translocated to lipid rafts after EPO stimulation of UT-7 cells. Confocal microscopy confirmed translocation of the EPO-R into membrane rafts in EPO-stimulated UT-7 cells and normal erythroid bursts. Receptor recruitment into rafts was accompanied by Jak2, Lyn and STAT5 loading into membrane raft fractions upon EPO stimulation. Treatment with methyl-β-cyclodextrin (MBCD) to deplete raft cholesterol and disrupt raft integrity extinguished EPO-induced STAT5 phosphorylation in UT-7 cells and human bone marrow progenitors. Similarly, membrane cholesterol-sequestration by nystatin markedly reduced EPO induced STAT5 phosphorylation in UT-7 cells. MBCD pretreatment of these cells prior to stimulation with phorbol 12-myristate 13-acetate (PMA) did not alter mitogen-activated protein kinase (MAPK) phosphorylation, indicating preservation of non-receptor, non-raft signal integrity despite depletion of cholesterol rich microdomains. These data establish a critical role for membrane raft microdomains in the recruitment and physical assembly of the EPO-R and its signaling intermediates into discrete platforms necessary to optimize signal integrity