YRRL motifs in the cytoplasmic domain of the thrombopoietin receptor regulate receptor internalization and degradation

Thrombopoietin (Tpo), acting through the c-MpI receptor, promotes the survival and proliferation of hematopoietic stem and progenitor cells and drives megakaryocyte differentiation. The proproliferation and survival signals activated by Tpo must therefore be tightly regulated to prevent uncontrolled cell growth. In this work, we determined the mechanisms that control Tpo-stimulated c-MpI internalization and defined the processes leading to its degradation. Stimulation of BaF-MpI cells with Tpo leads to rapid, clathrindependent endocytosis of the receptor. Using small interfering RNA (siRNA), we found that inhibition of adaptor protein 2 (AP2), which mediates endocytosis of transmembrane proteins, strongly attenuates Tpo-stimulated c-MpI internalization. AP2 interacts with YXXΦ motifs and we identified 2 such motifs in c-MpI (Y₈RRL and Y₇₈RRL) and investigated Tpostimulated internalization of receptors bearing point mutations at these sites. After Tpo stimulation, internalization was greatly reduced in c-MpI Y₇₈F and c-MpI Y₈₊₇₈F. and these cell lines also exhibited increased proliferation and increased strength and duration of Jak2, STAT5, AKT, and ERK1/2 activation in response to Tpo. We also found that the Y ₈RRL motif regulates Tpo-stimulated lysosomal degradation of c-Mpl. Our data establishes that c-Mpl cytoplasmic YRRL motifs are responsible for both Tpo-mediated internalization via interactions with AP2 and lysosomal targeting after endocytosis.