Key result
Mutation of c-Mpl cytoplasmic YRRL motifs greatly reduced Tpo-stimulated receptor internalization and lysosomal degradation, leading to increased cell proliferation and prolonged signaling.
Why the study?
Do YRRL motifs in the cytoplasmic domain of the c-Mpl receptor regulate Tpo-stimulated receptor internalization and degradation in BaF-Mpl cells?
Do YRRL motifs in the cytoplasmic domain of the c-Mpl receptor regulate Tpo-stimulated receptor internalization and degradation in BaF-Mpl cells?
Answers are grounded in this paper and cite their sources.
The study identifies specific YRRL motifs in the c-Mpl receptor that are essential for its AP2-mediated internalization and lysosomal degradation, preventing uncontrolled cell proliferation.
Thrombopoietin (Tpo), acting through the c-Mpl 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-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 RNA (siRNA), 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 YXXPhi motifs and we identified 2 such motifs in c-Mpl (Y(8)RRL and Y(78)RRL) and investigated Tpo-stimulated internalization of receptors bearing point mutations at these sites. After Tpo stimulation, internalization was greatly reduced in c-Mpl Y(78)F and c-Mpl Y(8+78)F, 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(8)RRL 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.
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Hitchcock et al. (2008) studied this question. Mutation of YRRL motifs in c-Mpl vs. Wild-type c-Mpl was evaluated on c-Mpl internalization and degradation. Mutation of c-Mpl cytoplasmic YRRL motifs greatly reduced Tpo-stimulated receptor internalization and lysosomal degradation, leading to increased cell proliferation and prolonged signaling.
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