G Protein-Coupled Receptor Transactivation: From Molecules to Mice - Methods in Cell Biology - page 422

model GPCR. Using transactivation as a mode of assessing receptor dimerization, we
describe our cellular system and functional assays for assessment of transactivation
in vitro and detail our strategy for generating a mouse model to assess GPCR trans-
activation in vivo .
INTRODUCTION
G protein-coupled receptors (GPCRs) act as key core communicators of extracellular
signals within a wide variety of physiological systems. The question of how diversity
and specificity is achieved when many receptor signaling pathways converge
on common downstream pathways is highly pertinent for this superfamily of signal-
ing receptors. This is driven by our more current understanding of the increasing
complexity of these receptor systems and the problems that can impede effective-
ness of current therapeutic ligands and unwanted side effects ( Smith, Bennett, &
Milligan, 2011 ).
One mechanism that has emerged in studies over the past 15 years that influences
rece ptor signal specificity and diversity is the organization of GPCRs in a multitude
of complexes, existing not only as monomers but also as dimers and higher-order
oligomers that function to diversify receptor functionality. Formation of receptor
homodimers and heterodimers can impact all aspects of GPCR biology, includ-
ing alterations in pharmacology, plasma membrane expression, signal transduc-
tion, and receptor trafficking (reviewed by Lohse, 2010 ). However, GPCR
di/oligomerization has been a highly debated issue ( Chabre & le Maire, 2005;
Fotiadis et al., 2006; Milligan, 2013 ), and while there is extensive evidence for re-
ceptor di/oligomerization in vitro , the functional significance of this phenomenon in
vivo is still uncertain. The clearest evidence for the physiological role of GPCR
dimerization was first obtained for class C GPCRs, such as GABA B , taste (T1R1-3),
metabotropic glutamate, and Ca 2 þ -sensing receptors ( Kaupmann et al., 1998; Pin
et al., 2004 ). Although a key question is to understand the requirement and in vivo
significance of GPCR dimerization, such data on the large class A/rhodopsin-like
GPCRs, in particular homodimerization, have not been forthcoming. From now on,
the term“dimerization”will be used, although it refers to both dimers and higher-order
oligomers.
Transactivation, also termed functional complementation or intermolecular
cooperativity, has been employed to study the requirement of GPCR dimerization
on receptor signaling. Receptor transactivation involves coexpression of two distinct
mutants that on their own are nonfunctional but when coexpressed can “rescue” the
functional activity of that receptor ( Fig. 23.1 ), with the binding of ligand to one
receptor protomer within the dimer, which communicates with neighboring protomer
to propagate signal. The phenomenon of transactivation has been demonstrated for
several GPCRs, including GABA B , D2 dopamine, opioid, thyrotropin-releasing hor-
mone, thyroid-stimulating hormone, follicle-stimulating hormone, and luteinizing
hormone/chorionic gonadotropin receptors (LHCGRs), using and employing distinct