Although many GPCR heteromers have been identified
Although many GPCR heteromers have been identified using heterologous cell lines, only very few fit all three criteria. This is mainly due to the difficulties to study these structures in native tissues because of the lack of sensitive and selective tools, not only capable of detecting in vivo evidence of these endogenous heteromers, but also of demonstrating that they are close enough to interact (Gomes, Sierra, & Devi, 2016). Therefore, it was agreed that the fulfillment of two out of three of the criteria is sufficient for the acceptance of a GPCR heteromer (Jonas & Hanyaloglu, 2017). The most widely used methods to detect heteromers are resonance energy transfer based techniques, which involve either fluorescence or bioluminescence resonance energy transfer (FRET or BRET) between a donor and an acceptor molecule fused to two proteins within the heteromer complex, such as BRET saturation assays (Mercier, Salahpour, Angers, Breit, & Bouvier, 2002) or the GPCR heteromer identification technology (Mustafa & Pfleger, 2011). Another extensively used technique is the proximity ligation assay, which is an immunochemical based technique that allows the detection of molecular interactions between two endogenous or transfected proteins. If these proteins are separated <17nm, heteromers will be observed as red dots in a confocal microscope (Gomes, Sierra, et al., 2016; Söderberg et al., 2008; Taura, Fernández-Dueñas, & Ciruela, 2015). Alternative strategies to target GPCR heteromers are the generation of selective compounds (bivalent, multifunctional or small molecule ligands), which exhibit higher efficacy in cells expressing both receptors and in tissues from wild type animals, as compared with cell expressing individual receptors or tissues from animals lacking the individual receptors (Akgün et al., 2013; Gomes, Gupta, & Devi, 2013; Molero et al., 2015; Nimczick & Decker, 2015; Peterson et al., 2017; Qian, Vasudevan, et al., 2018; Qian, Wouters, et al., 2018). Likewise, the use of membrane-permeable peptides that target the dimerization interface or heteromer-selective naloxone hydrochloride that can recognize an epitope in the heteromer but not in the individual protomers, have been emerged as useful tools to detect the presence of GPCR heteromers in vivo (Gupta et al., 2010; He et al., 2011; Jacobs et al., 2018; Moreno et al., 2017; Navarro et al., 2015; Rivera-Oliver et al., 2018; Viñals et al., 2015). Finally, it is important to note the existence of single-molecule techniques, which are ideally designed to understand the dynamic signaling and conformational complexity of GPCRs (Tian, Fürstenberg, & Huber, 2017). Their application can provide tools to directly visualize individual receptors forming homomers and heteromers and how GPCRs can move and interact in living cells; in addition, they are useful to study the effects in their mobility and interactions caused by different ligands that can alter this dynamics, stabilizing preexisting dimers prolonging their lifetime (Calebiro & Sungkaworn, 2018; Jonas, Huhtaniemi, & Hanyaloglu, 2016; Scarselli et al., 2016; Tabor et al., 2016). The class C GABAB receptor is the first known GPCR that was demonstrated to require heterodimerization for functioning. In fact, it is called an obligatory heterodimer (or heteromeric receptor; Ferré et al., 2009) composed by two subunits, GBR1 and GBR2, that have complementary functions in signal transmission (Kniazeff et al., 2011; Moller et al., 2017). While the extracellular domain of GBR1 is responsible for ligand recognition, the transmembrane domain of GBR2 is required for G-protein activation. In addition, GBR2 facilitates the cell surface expression of GBR1 through coiled-coil interactions in the cytoplasmic region. This heteromer has been crystallized bound to different agonists and antagonists (Geng, Bush, Mosyak, Wang, & Fan, 2013). On the other hand, mGluRs, another members of family C GPCRs, can form non-obligatory heterodimers (or receptor heteromers; Ferré et al., 2009), being the mGlu2-4 the most studied pair due to its physiological interest; concretely, they are involved in control synaptic activity at the level of the cortico-striatal terminals in the striatum (Yin et al., 2014) and at the level of lateral performant path terminals in the dendate gyrus (Moreno-Delgado et al., 2017). Recently, Liu et al. (2017) reported an oriented asymmetry in the activation of this heterodimer that can be controlled with allosteric modulators.