Submitted January 15, 2008
Accepted April 29, 2008
YRRL motifs in the cytoplasmic domain of the thrombopoietin receptor regulate receptor internalization and degradation
Ian S Hitchcock*, Maximus M Chen, Jennifer R King, and Kenneth Kaushansky
Department of Medicine, University of California San Diego, La Jolla, CA, United States
* Corresponding author; email: ihitchcock{at}ucsd.edu.
Thrombopoietin (Tpo), acting through the c-Mpl receptor, promotes the survival and proliferation of hematopoietic stem and progenitor cells and drives megakaryocyte differentiation. The pro-proliferation 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-Mpl internalization and defined the processes leading to its degradation. Stimulation of BaF-Mpl cells with Tpo leads to rapid, clathrin-dependent endocytosis of the receptor. Using small interfering (si)RNA we found that inhibition of adaptor protein 2 (AP2), which mediates endocytosis of transmembrane proteins, strongly attenuates Tpo-stimulated c-Mpl internalization. AP2 interacts with YXX
motifs and we identified 2 such motifs in c-Mpl (Y8RRL and Y78RRL) and investigated Tpo-stimulated internalization of receptors bearing point mutations at these sites. After Tpo stimulation, internalization was greatly reduced in c-Mpl Y78F and c-Mpl 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 Y8RRL 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 following endocytosis.
