Document Type

Dissertation

Date of Degree

Fall 2010

Degree Name

PhD (Doctor of Philosophy)

Degree In

Pharmacology

First Advisor

Frederick W. Quelle

Abstract

Erythropietin (Epo), acting through its receptor (EpoR), is an essential hemotopietic growth factor that regulates the proliferation, differentiation, and survival of erythroid progenitor cells. Perturbations of Epo/EpoR function cause myeloproliferative disease, such as erythrocytosis, or myelodeficient disease, such as anemia. Therefore, defining the mechanisms by which Epo/EpoR control proliferation and differentiation of erythroid cell lineages attracts interest. Ubiquitin-dependent internalization and degradation is a common regulatory mechanisms affecting signaling from a variety of receptors. Although EpoR has been found to be ubiquitinated, the function of EpoR ubiquitination in the regulation of Epo signaling remains unclear. Therefore, the primary goal of this study was to define the role of EpoR ubiquitination in regulating Epo signaling activities and erythroid cell growth. We showed that EpoR was ubiquitinated in response to ligand stimulation, and that loss of EpoR ubiquitination reduced signaling activity and biological responses to low dosages of Epo. We also identified two EpoR lysines that were the primary targets for ubiquitination, and showed that either ubiquitination site supported the enhanced activities of wild-type-EpoR. Ubiquitination of EpoR was also associated with a change in the endocytic pathway mediating internalization of EpoR. Specifically, constitutive internalization of non-ubiquitinated EpoR was found to depend on dynamin activity, while internalization of ubiquitinated EpoR was dynamin-independent but could be inhibited by disrupting lipid raft microdomains in the plasma membrane. Interestingly, inhibiting internalization of ubiquitinated EpoR (by disrupting lipid rafts) specifically reduced signaling from ubiquitinated receptors without affecting signaling from non-ubiquitinated receptors. Conversely, reducing internalization of non-ubiquitinated EpoR (by inhibiting dynamin) reduced its signaling activity without affecting signaling from ubiquitinated receptors. This strong correlation between EpoR internalization and signaling activity suggests a novel regulatory mechanism in which internalization of EpoR facilitates its signaling activity. In this regard, Epo-induced ubiquitination of EpoR promotes more efficient internalization of ligand-activated receptor and may contribute to enhanced responsiveness to low concentrations of Epo.

Keywords

Endocytosis, Erythropoietin, Erythropoietin Receptor, Internalization, Signal Transduction, Ubiquitin

Pages

xiii, 111 pages

Bibliography

Includes bibliographical references (pages 101-111).

Comments

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Copyright

Copyright 2010 Daisuke Mayuzumi

Included in

Pharmacology Commons

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