Biology Reference
In-Depth Information
factors (
Lemmon & Schlessinger, 2010
). The majority of RTK activation depends
upon protein-protein interactions and formation of homodimer or higher-order olig-
omeric states (
Lemmon & Schlessinger, 2010
).
Ligand binding and dimerization initiate activation of the tyrosine kinase
domain, resulting in autophosphorylation of regulatory tyrosine residues.
Phosphorylated residues serve as binding sites for several enzymes (e.g.,
PLCgamma1) or adaptor proteins (e.g., Grb2 or Shc) through Src homology
2 (SH2) domains. Resultant RTK activation proceeds through four main
signaling pathways: mitogen-activated protein kinase (MAPK) cascades, p85/
p110 phosphatidylinositol-3 kinase (PI3-K) lipid kinase family, signal transducers
and activator of transcription (STAT) family, and the phospholipase Cgamma1
(PLCgamma1) pathway.
G protein-coupled receptors (GPCRs) are the most prominent group of cell sur-
face receptors and the principal targets for the majority of therapeutic drugs (
Heilker,
Wolff, Tautermann, & Bieler, 2009
). As indicated by their name, GPCRs exert their
cell signaling actions by activating different heterotrimeric G proteins (
Gilman,
1987
). However, GPCRs are also known to signal via alternative mechanisms, some
of
which involve the adaptor proteins beta arrestin 1 and 2 (
Beaulieu & Caron, 2005;
Ferguson, 2001; Luttrell & Gesty-Palmer, 2010
). Several lines of evidence have re-
cently suggested the interconnection between RTKs activation and GPCR signaling
(
Natarajan & Berk, 2006
).
RTK transactivation refers to a process by which activation of a GPCR in turn
activates an RTK (
Schlessinger, 2000
). RTK transactivation by GPCRs was first
reported in the late 1990s and could be implicated in neurophysiology, cancer,
and cardiac function and dysfunction (
Dicker & Rozengurt, 1980
). To date, the ma-
jority of literature has reported qualitative characterization to describe RTK-GPCR
heterologous activation. Specifically, the bulk of research directed to study transac-
tivation has been limited to biochemical assays relying on measuring downstream
targets, which, in contrast to early events like receptor dimerization, provide limited
information on the dynamics and spatial localization of the transactivation process
(
Eguchi et al., 1998; Rajagopal, Chen, Lee, & Chao, 2004
). Currently, there is a great
need for more quantitative characterization of this relationship that would allow for
understanding mechanisms and relative importance of RTK transactivation leading
to biological outcomes.
The investigation of biological processes at the subcellular level is typically done
using a myriad of microscopy techniques. Confocal laser scanning microscope
(CLSM) has become a centerpiece in cell biology laboratories and remains a popular
tool to study many biological questions, albeit with an optical spatial resolution that
is limited by the diffraction of light. In this chapter, we present a method to study and
quantify RTK cell surface density, dimerization, and internalization that integrates
the analysis of standard CLSM images together with the well-established transgene
expression of
fluorescent protein labels and/or
immunofluorescence labeling
approaches in native or fixed cells or tissue.
