Biology Reference
In-Depth Information
heteroreceptor complexes, GPCRs can activate RTK in the absence of added growth
factor through the use of RTK signaling molecules. This integrative phenomenon is
reciprocal and can place also RTK signaling downstream of GPCR. Formation of
either stable or transient complexes by these two important classes of membrane re-
ceptors is involved in regulating all aspects of receptor function, from ligand binding
to signal transduction, trafficking, desensitization, and downregulation among
others. Functional phenomena can be modulated with conformation-specific inhib-
itors that stabilize defined GPCR states to abrogate both GPCR agonist- and growth
factor-stimulated cell responses or by means of small interfering heteroreceptor com-
plex interface peptides. The bioluminescence resonance energy transfer (BRET)
technology has emerged as a powerful method to study the structure of heterorecep-
tor complexes closely associated with the study of receptor-receptor interactions in
such complexes. In this chapter, we provide an overview of different BRET
2
assays
that can be used to study the structure of GPCR-RTK heteroreceptor complexes and
their functions. Various experimental designs for optimization of these experiments
are also described.
INTRODUCTION
It is now several years since a general dogma established that growth-promoting ac-
tivity of many G protein-coupled receptor (GPCR) ligands involves activation of re-
ceptor tyrosine kinases (RTKs) and their downstream signaling cascades (
Luttrell,
Daaka, & Lefkowitz, 1999
). Such observations led to the emergence of the “trans-
activation” concept, which refers to the activation of RTK signaling pathways by
GPCR ligands; see (
Fuxe et al., 2007
). However, over the past few years, this concept
appears to have become increasingly complex since pharmacological, biochemical,
and biophysical studies provided evidence for an engagement of GPCR signaling
molecules (e.g., heterotrimeric G proteins and arrestins) in signal transduction gen-
erated by various RTK subtypes, which reveals a bidirectional cross communication
between RTKs and GPCRs (
Dalle et al., 2002; Lin, Daaka, & Lefkowitz, 1998; Pyne
& Pyne, 2011
).
Perhaps, an even more interesting concept relates to the apparent ability of dif-
ferent GPCRs to associate with RTKs and forming large receptor complexes
(GPCR-RTK heteroreceptor complexes) (
Borroto-Escuela, Romero-Fernandez,
Mudo, et al., 2012; Flajolet et al., 2008; Fuxe et al., 2010
). Such a higher molecular
organization brings new alternatives in term of physiological functions for each of
these two receptor families, but it also increases the possibilities regarding therapeu-
tic targeting (
Alderton et al., 2001; Delcourt, Bockaert, &Marin, 2007
; Pyne & Pyne,
2011;
Waters et al., 2006
). Biochemical and/or pharmacological properties reported
for GPCR-RTK heteroreceptor complexes are often distinct from those of the cor-
responding protomers (
Borroto-Escuela, Romero-Fernandez, Mudo, et al., 2012;
Delcourt et al., 2007
; Pyne & Pyne, 2011;
Waters et al., 2006
). Thus,
