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Document Type

Dissertation - University Access Only

Award Date

2013

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry and Biochemistry

First Advisor

Adam D. Hoppe

Abstract

Macrophage colony stimulating factor receptor (MCSFR a.k.a CSF-1R), is a receptor tyrosine kinase (RTK) that is essential for the growth of myeloid stem cells, monocytes and macrophages. Proper control of signaling through the MCSFR is critical for proper macrophage development and function. Deregulation of this signaling pathway is associated with numerous inflammatory diseases as well as myelogenous leukemias. Clearance of RTKs from the plasma membrane is an important mechanism for regulating the signal amplitude, however, the exact mechanisms remain elusive. To better define these mechanisms, we characterized MCSFR internalization, endocytic trafficking, and signaling regulation by new fluorescent microscopy and conventional biochemistry methods. In this work we discovered a novel cellular mechanism regulating MCSFR signaling. Specifically, we showed that the MCSFR is internalized by clathrin-mediated endocytosis (CME). Coincident with this internalization, the MCSFR stimulates the formation of macropinosomes. These large vacuoles form at the plasma membrane, but are devoid of the receptor. Following these two steps, the Rab5 positive early endosomes containing MCSFR fuse with nascent macropinosomes and the MCSFR is subsequently transported into the lumen of the macropinsomes and degraded. Pharmacological disruption of macropinocytosis impairs receptor degradation. Entry into endocytic pathway is mediated by the covalent addition of the protein ubiquitin to the MCSFR by the ubiquitin-ligases Cbl and Cbl-b in the hematopoietic stem cell (HSC) compartment. These mice succumb to lethal myeloproliferative disease by 4 months of age, reminiscent of human juvenile leukemia. By analyzing macrophages from these mice we have shown that Cbl and Cbl-b share overlapping functions in controlling MCSFR internalization, traffic to macropinosomes, degradation and ultimately cell growth. This work lays the foundation for uncovering the mechanisms of MCSFR signal regulation via endocytic traffic and creates a new paradigm for the role of macropinosomes in modulating signaling.

Library of Congress Subject Headings

Colony-stimulating factors (Physiology)
Macrophages.
Cellular signal transduction.

Description

Includes bibliographical references (pages 101-109).

Format

application/pdf

Number of Pages

120

Publisher

South Dakota State University

Rights

In Copyright - Non-Commercial Use Permitted
http://rightsstatements.org/vocab/InC-NC/1.0/

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