Biomedical Engineering Reference
In-Depth Information
EGFR could also phosphorylate tyrosine residues of other
molecules in close proximity. Ligand binding triggers receptor-
mediated endocytosis of the complex, ultimately culminating in
the degradation of both receptor and ligand in the lysosome [
2
,
4
].
For EGFR and several other growth factor receptors such as
tropomyosin receptor kinases (Trks), signaling is not limited to
the plasma membrane and does not stop upon internalization. In
fact, signaling continues in the endosomes, and the strength and
duration of EGFR signaling are therefore dependent on, and thus
modulated by, its endocytic traffi cking itinerary [
4
,
5
].
Like the secretary pathway, endocytic traffi cking is regulated
by a suite of small GTPases of the Rab subfamily [
6
,
7
], which is
part of the Ras superfamily of GTPases. The human genome con-
tains over 60 different Rab and Rab-like genes [
6
]. While their
general working principles are known [
8
], the exact role and mech-
anisms of function of many of these are not yet well understood. In
general, Rabs are activated by guanosine triphosphate (GTP)-
guanosine diphosphate (GDP) exchange mediated by guanine
nucleotide exchange factors (GEFs) and are inactivated by GTPase-
activating proteins (GAPs). In their GTP-bound active form, Rabs
engage downstream effector proteins, which include tethering fac-
tors, membrane fusion factors, and motor proteins [
9
]. Rab pro-
teins that are known to be important for EGFR's endocytic traffi c
include the early endosomal Rab5 [
10
], the late endosomal Rab7
[
11
], and Rab11 of the recycling endosome [
12
]. We have recently
shown that Rab31, a Rab that is phylogenetically related to Rab5
and Rab22, has a specifi c role in mediating EGFR traffi cking from
the early to the late endosome [
13
]. Furthermore, Rab31 appears
to associate with EGFR and its effector EEA1 in a putative traffi ck-
ing complex and is dependent on GAPex-5 for its function [
13
].
In the ensuing paragraphs, we shall describe some relatively
standard protocols to functionally dissect the role of a particular
Rab in EGFR endocytic traffi cking steps/stages, using Rab31 for
our illustrations. Rab activities and levels are amenable to some
fairly standard genetic and pharmacological manipulations.
Constitutively active (possible but not always) and dominant nega-
tive mutants could be generated based on analogous mutations in
key conserved residues in other Rabs and other members of the
Ras superfamily. Findings made with these mutants could be com-
plemented by silencing and rescue experiments. Identifi cation of
molecular partners and functional interactions could be achieved
by affi nity pulldowns or co-immunoprecipitation. The conse-
quence of these manipulations could be monitored, often with at
least semiquantitative readouts by confocal fl uorescence micros-
copy imaging in conjunction with a range of common biochemical
analytical methods.
