Biomedical Engineering Reference
In-Depth Information
Chapter 18
Approaches to Analyze the Role of Rab GTPases
in Endocytic Traffi cking of Epidermal Growth Factor
Receptor (EGFR)
Christelle En Lin Chua , Angeline Yim Kiat Lim , and Bor Luen Tang
Abstract
The epidermal growth factor receptor (EGFR), a member of the erythroblastic leukemia viral oncogene
homologue (ErbB) receptor tyrosine kinase family, plays key mitogenic signaling roles in development,
cellular, and tissue physiology, as well as a myriad of malignancies. EGFR signaling occurs concurrently
with ligand-receptor binding and subsequent endocytosis, and its signaling strength and engagement of
different downstream signaling components are modulated by its endocytic traffi cking itinerary.
Understanding the factors and mechanisms that modulate ligand-bound EGFR's endocytic traffi cking is
therefore important for deciphering its role in pathophysiological processes. Endocytic traffi cking of EGFR
is regulated by a bunch of Rab small GTPases associated with the endocytic pathway. In this chapter, we
describe a suite of relatively standard protocols in dissecting the role of a particular Rab protein in EGFR
endocytic traffi cking steps/stages. The approach constitutes a combination of genetic/molecular manipu-
lations, followed by confocal imaging and a range of biochemical analyses. We shall mainly focus on
Rab31 in our illustrations, but the approaches would be equally applicable to any Rab and its associated
regulators/effectors.
Key words
Confocal microscopy, Density gradient sedimentation, Epidermal growth factor receptor
(EGFR), Rab, Small interfering (si)RNA
1
Introduction
Upon ligand binding, EGFR dimerization stimulates its intrinsic
tyrosine kinase activity, resulting in autophosphorylation of sev-
eral tyrosine residues in its intracellular C-terminal domain. These
phosphorylated tyrosine residues attract signaling molecules with
phosphotyrosine-binding SH2 domains, resulting in the activa-
tion of mitogen-activated protein kinase (MAPK) and other mito-
genic pathways [
1
,
2
]. Receptor cross talk also allows EGFR to be
transactivated by other signaling receptors [
3
], and activated
