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Over the past decade, G protein-coupled receptors were assumed to exist primarily as monomeric polypeptides. This perspective was based on the monophasic binding properties of most antagonistic ligands for individual GPCRs. However, increasing evidence has shown that GPCRs exist as dimeric and/or multimeric structures and can be either homomeric or heteromeric (Milligan 2009). The experimental evidence for the existence of GPCRs as dimers and higher order structures includes dimeric bands on SDS-PAGE, co-operativity in ligand binding studies, co-immunoprecipitation of differentially epitope-tagged but co-expressed forms of the same GPCR and co-trafficking of two receptor subunits following single subunit stimulation. Furthermore, use of tissue-specific knockout animal models and biophysical means such as resonance energy transfer techniques have been employed in a number of GPCR dimerization studies (Milligan, 2004; Milligan and Bouvier, 2006). Elucidation of the quaternary structure of GPCRs also increases our understanding of GPCR dimers or multimers. From a pharmacological and therapeutic viewpoint, this structural understanding provides opportunities for the discovery of physiologically relevant GPCR dimers, which is likely to highlight novel drug targets for diseases such as schizophrenia (Panetta and Greenwood, 2008).
The most convincing demonstration that GPCRs exist as dimers includes a number of studies by Devi (2001), Salahpour et al. (2000) and Galvez et al. (2001) which show that dimerization is important for trafficking and function in the case of -aminobutyric acid (GABAB) receptor. GABAB receptors have been shown to consist of an obligate heterodimer of GABABR-1 and GABABR-2 subunits. Each subunit is a member of the GPCR family C. GABABR-1 contains the orthosteric site and a C-terminal endoplasmic reticulum (ER) retention signal. When GABABR-1 is expressed alone it is trapped in the ER within the cell; the polypeptide is not transported to the cell surface and therefore cannot activate G protein signalling. When the GABABR-2 is expressed alone it is translocated to the plasma membrane, but cannot bind GABA or activate G proteins. When both subunits are co-expressed in the same cell, the receptor subunits interact through a coiled-coil domain in their cytoplasmic carboxyl tails and are translocated to the plasma membrane (fig. 1). GABA binds specifically to the GABABR-1 subunit and G¡i proteins are activated by the GABABR-2 subunit. Allosteric interactions between GABABR-1 and GABABR-2 subunits are required for cell surface delivery and optimal GABAB receptor G-protein signalling (Galvez et al., 2001; Milligan and Bouvier, 2006).
Pierce, L.P. Premont, R.T & Lefkowitz, R.J (2002) Seven-Transmembrane Receptors. Nature Rev. 3: 639-650.
Figure 1. Both GABABR-1 and GABABR-2 subunits are required to generate a functional receptor. The GABABR-1 subunit contains an ER retention signal and when co-expressed alone, it is retained in the ER. Whereas, when GABABR-2 is expressed alone it is translocated to the plasma membrane, but cannot bind GABA or activate G proteins. When both subunits are co-expressed in the same cell, the receptor subunits interact through a coiled-coil domain in their cytoplasmic carboxyl tails and are then translocated to the plasma membrane.
Taste receptors that recognize sugars and amino acids, also members of GPCR family C receptors, have been shown to require heterodimerization to produce a functional receptor. Double label fluorescent in situ hybridization (FISH) (fig. 2) was used to monitor co-expressed taste receptor 1, member 2 (TIR2) and taste receptor 1, member 3 (TIR3) receptor subunits in circumvallate, foliate and palate taste buds by Nelson et al. (2001). Monitoring of expression of native and epitope-tagged rat receptors was done in human embryonic kidney (HEK-293) cells using T1R2 and T1R3 specific antibodies. In addition T1Rs were expressed with G¡16-Gz chimera and G¡15- together these two G protein subunits efficiently couple Gs, Gi and Gq to phospholipase C¢. In this functional assay, receptor activation leads to increase in intracellular calcium [Ca2+]i, which was monitored at the single cell level using the FURA-2 Ca2+ indicator dye. Expression of either T1 receptor subunit alone did not trigger [Ca2+]i change in response to any of the tastants in the assay study, and the response to sweet tastants such as sucrose was dependent on the presence of both T1R2 and T1R3 subunits. These findings suggest that sweet responses require heterodimerization of T1R2 and T1R3 subunits for receptor function. It has also been demonstrated that umami (amino acid) responses require co-expression of the TIR1 and TIR3 receptor subunits using a similar experimental approach (Nelson et al., 2001; Nelson et al., 2002). Additional evidence that heterodimerization is required for a functional T1R was demonstrated by co-immunoprecipitations of differentially epitope-tagged T1R subunits, and by co-expression of a dominant negative T1R. Cotransfection of wild-type T1R2 and T1R3 with a C-terminal truncated T1R2 polypeptide nearly abolished the T1R2 and T1R3 responses (>85% reduction) (Nelson et al., 2001).
Nelson, G. et al. (2001) Mammalian sweet taste receptors. Cell 106: 381-390.
Figure 2. Double-label fluorescent in situ hybridization was used to investigate the overlap in cellular expression of T1Rs. Two-channel fluorescent images (1-2 m optical sections) are overlaid on difference interference contrast images. (a) Illustrates co-expression of T1R1 (red) and T1R3 (green) in fungiform papillae. At least 90% of the cells expressing T1R1 also express T1R3. (b) Illustrates co-expression of T1R2 (green) and T1R3 (red) in circumvallate papillae. Every T1R2 positive cell expresses T1R3.
Co-immunoprecipitation studies remain the most practical means to identify heterodimeric interactions between GPCRs in native tissues and thus, provide relevant information in support of the existence of GPCR dimers/oligomers at the cell surface (Milligan and Bouvier, 2006). McVey et al. (2001) subsequently used combinations of co-immunoprecipitation approaches and two distinct forms of living cell-based fluorescence resonance energy transfer (FRET) studies to confirm the capacity of the ¤-opioid (DOP) receptor to form a homo-multimeric structure at the cell surface. FRET studies utilise two widely used fluorescent partners with altered spectral characteristics- cyan fluorescent protein (CFP) as energy donor and yellow fluorescent protein (YFP) as energy acceptor. FRET can be used to monitor protein-protein interactions of dimers/oligomers in GPCR quaternary structure in intact living cells both in cell populations and single cells (Milligan and Bouvier, 2006). Recent studies have employed this technique to demonstrate that agonist-bound protomer of the leukotriene B4 BLT1 receptor produces a conformational change in the partner subunit of the homodimer (Mesnier and Baneres, 2004). However, the drawback with this technique is that agonist-induced alteration in dimeric or higher oligomeric structures based only on FRET may reflect minute conformational changes in the GPCR that are amplified by the high sensitivity of FRET responses to distances and orientation. Thus, it does not allow one to distinguish easily between dimers or higher oligomers in GPCR quaternary structures (Milligan and Bouvier, 2006).
Figure 3. The paracrystalline organization of rhodopsin dimers in the native disc membrane visualised using atomic-force microscopy (taken from: Fotiadis et al. (2003) Atomic-force microscopy: rhodopsin dimers in native disc membranes. Nature 421:127-128).
Furthermore, recent studies employing atomic force microscopy to visualise the arrangement of rhodopsin in murine rod outer segment disc membranes have indicated that rhodopsin is organised in a series of parallel arrays of dimers and higher oligomers as shown in fig. 3 (Fotiadis et al., 2004). However, Whorton et al. (2008) demonstrated that incorporation of monomeric rhodopsin into reconstituted high-density lipoprotein phospholipid bilayer vesicles, together with G¡t (transducin) protein, demonstrated the capacity of the receptor monomer to activate G protein transducin.
Taken from: Receptor Diversity (GPCRs) Handout 2008: BS3054 Lecture 3- Prof. J. Challiss. 421127a-f2
The concept that GPCRs internalize as dimers has been used to attempt to explore the selectivity of GPCR heterodimerization. For example, co-expression of the ¡1a-adrenoceptor with the ¡1b-adrenoceptors resulted in the co-internalization of these two GPCRs in response to ¡1a-selective agonist oxymetazoline. However, this agonist did not mediate co-internalization of either NK1 tachykinin receptor or the CCR5 chemokine receptor when they were co-expressed with ¡1a-adrenoceptor (stanasila et al., 2003). Such observation suggests that ¡1a- and ¡1b-adrenoceptors form a high-affinity heterodimer but those of the other dimer pairs do not. In an identical approach, the individual selective ligands for NK1 and MOP opioid receptors were able to induce co-trafficking of both co-expressed GPCRs from the plasma membrane to the endosome (Pfeiffer et al., 2003). Further evidence of GPCR dimerization/oligomerization came to light with the use of time-resolved FRET using anti-epitope tag Igs labelled with FRET acceptor and donors to analyse receptor-receptor interaction. Ramsay and Milligan (2005) used time-resolved FRET pairing in membrane fractions generated by sucrose density sedimentation to detect dimers/multimers of ¡1a-adrenoceptors as measured by [3H] antagonist binding studies. However, despite evidence for GPCR dimerization, the major potential caveats for time-resolved FRET assays relates to the obligatory use of antibodies (Milligan and Bouvier, 2006).
Also, use of a peptide corresponding to transmembrane helix VI of the ¢2-adrenoceptor was used to disrupt GPCR-GPCR interactions. Relative proportions of immunodetected GPCR migration were monitored through SDS-PAGE in positions consistent with monomer and dimer. The proportion of dimer was decreased by this competitive synthetic peptide in a concentration-dependent manner (Milligan, 2004). Decreased proportion of dimer correlated with a decrease in the activity of adenylyl cyclase ligand-mediated signalling (adrenaline) but not forskolin (Milligan, 2004). These findings suggest that GPCR dimerization is important for the function of the receptor and would contribute substantially to effective G protein-mediated signalling. Saturation bioluminescence resonance energy transfer (BRET) experiments have also shown that ¢1-adrenoceptor and ¢2-adrenoceptor can form heterodimers with similar affinity as the corresponding homodimers (Milligan, 2004). In addition, the homo-dimeric ¢2-adrenoceptor was shown to undergo internalization from the cell surface in response to binding of a single agonist to either subunit (Sartania et al., 2007).
Interestingly, a number of studies provide evidence suggesting that dimerization occur initially during biosynthesis and maturation of at least certain GPCRs. Terrillon et al. (2003) demonstrated that immature forms of oxytocin and vasopressin receptors were present as dimers while still resident in the ER. Using already established role of the C-terminal ER retention motif of GABABR-1 in the synthesis and cell surface delivery of functional GABAB receptor, Salahpour et al. (2004) replaced the C-terminal tail of the ¢2-adrenoceptor with the C-terminal tail of GABABR-1 and showed intracellular ER retention of this construct when expressed in HEK-293 cells. When the wild-type ¢2-adrenoceptor was co-expressed with the ER trapped mutant, the wild-type form was also retained intracellularly. The dominant negative effect of the chimeric ¢2-adrenoceptor-GABABR-1 construct supports the idea that receptor-receptor interactions between the engineered ER-retained and wild-type forms of the receptor occurred during receptor biosynthesis and maturation. In addition, the replacement of the C-terminal tail of the chemokine CXCR1 receptor with a distinct 14 amino acid ER retention motif from the ¡2c-adrenoceptor resulted in ER retention of this chimeric construct in HEK-293 cells. This ER-retained construct also limited cell surface translocation of co-expressed wild-type forms of both CXCR1 and CXCR2 (Wilson et al., 2005). This supports other evidence of both CXCR1 homo-dimerization and CXCR1-CXCR2 receptor heterodimerization (Milligan et al., 2005). The selectivity of such receptor interactions was noticed because the presence of the trapped CXCR1 receptor was without effect on the plasma membrane delivery of a co-expressed ¡1a-adrenoceptor (Wilson et al., 2005). Dimerization was shown within the ER by adding a distinct bimolecular fluorescence complementation-competent form of enhanced YFP to this receptor (Milligan, 2009).
Advancing knowledge of the structure of GPCR dimers is of increasing importance to our understanding of the alteration in the pharmacology of individual receptors in a dimer. Dimeric pharmacological properties may be useful in defining physiologically relevant dimers and developing novel drug targets. The opioid receptors provide one of the more compelling examples that illustrate the potential that dimerization has for increasing our ability to develop highly specific drugs. The «-opioid (KOR) receptor has been shown to dimerize with the ¤-opioid (DOR) receptor (Panetta and Greenwood, 2008). The pharmacology of this KOR-DOR heterodimer was also indicated to be different from either of the monomers. Waldhoer et al. (2005) showed that the agonist, 6'-guanidinonaltrindole (6'-GNTI) originally thought to be specific for the KOR receptor is more potent in cells co-expressing the KOR-DOR receptor dimer, therefore acting as a heterodimer-selective agonist. It has also been reported to function as a spinally selective analgesic. In line with these data, many groups have tried to identify contact sites in GPCR dimers as a guide in developing models that would permit rational strategies to drug design. Such example is a dimer-inhibiting TMD-4 (transmembrane domain-4) peptide of the chemokine receptor CXCR4, which shows promise as a therapeutic strategy for several inflammatory conditions (Wang and Norcross, 2008).
In conclusion, it is now generally accepted that GPCRs can certainly exist as dimers and/or higher oligomers, and that such interactions may be a requisite for cell surface delivery and function of some receptor dimers. Dimerization of the GPCRs family C and majority of family A is firmly established as an intrinsic requirement for function to the extent that it is no longer a point of contention (Milligan, 2004). This view is based on the coherent body of evidence and the range of experiment approaches that have been applied to study the quaternary structure of GPCRs (Milligan 2009). Further documentation of the potentially large functional and physiological diversity of GPCR dimers should lead to the development of novel drug targets, for example a dimer-selective ligand 6'-GNTI in the case of KOR-DOR heterodimer, and thus provide ultimate tools to investigate the role of heterodimerization in vivo (Panetta and Greenwood, 2008).
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