The experimental evidence that Adhesion G Protein-Coupled Receptors (aGPCRs) functionally couple to heterotrimeric G proteins has been emerging in incremental steps, but attributing biological significance to their G protein signalling function still presents a major challenge. Here, utilising activated truncated forms of the receptors, we show that ADGRE2/EMR2 and ADGRE5/CD97 are G protein-coupled in a variety of recombinant systems. In a yeast-based assay, where heterologous GPCRs are coupled to chimeric G proteins, EMR2 showed broad G protein-coupling, whereas CD97 coupled more specifically to G α12 , G α13 , G α14 and G αz chimeras. Both receptors induced pertussis-toxin (PTX) insensitive inhibition of cyclic AMP (cAMP) levels in mammalian cells, suggesting coupling to G αz. EMR2 was shown to signal via G α16 , and via a G α16 /G αz chimera, to stimulate IP 1 accumulation. Finally, using an NFAT reporter assay, we identified a polyclonal antibody that activates EMR2 G protein signalling in vitro. Our results highlight the potential for the development of soluble agonists to understand further the biological effects and therapeutic opportunities for ADGRE receptor-mediated G protein signalling. The Adhesion G Protein-Coupled Receptors (aGPCRs) constitute an evolutionarily ancient membrane protein family with emerging roles in many important biological processes (for reviews see 1-5). The receptors each contain a 7-transmembrane (7-TM) domain with phylogeny suggesting ancestry to the 'Family B' (Secretin receptor family; also known as Class B) G Protein-Coupled Receptors (GPCRs). However, aGPCRs are distinguished by their large amino-terminal regions that typically contain multiple modular motifs such as EGF (Epidermal Growth Factor-like), cadherin and immunoglobulin domains as well as novel lineage-specific structures. While these are generally thought to mediate inter-cellular 'adhesion' interactions, various examples suggest separable roles for the extracellular domain (ECD) and 7-TM regions 6,7. This complexity, apart from the sheer size of some of the receptors, underlies some of the challenges of studying aGPCRs. As the known interacting partners of aGPCRs are usually tethered to other cells, their identification and characterisation have been difficult. Of those identified, including CD55 for CD97 8 , transglutaminase II (TGII) for GPR56 9 and chondroitin sulphate B (dermatan sulphate) for EMR2 10 , few had measurable effects on G protein signalling in vitro and it is not clear whether these partners can be considered 'ligands' , as understood for the better characterised Family A (Rhodopsin-like), Family B (Secretin receptor family) or Family C (Metabotropic glutamate family) GPCRs, that modulate G protein signalling pathways in response to the binding of soluble activators. Indeed, only recently has evidence become compelling of aGPCR association with G protein alpha subunits (reviewed in Langenhan et al. 11), changes in second messenger levels 11 , and GTP turnover in membranes from cells expressing a...
FFA2 is a receptor for short-chain fatty acids. Propionate (C3) and 4-chloro-α-(1-methylethyl)-N-2-thiazolyl-benzeneacetamide (4-CMTB), the prototypical synthetic FFA2 agonist, evoke calcium mobilization in neutrophils and inhibit lipolysis in adipocytes via this G-protein-coupled receptor. 4-CMTB contains an N-thiazolylamide motif but no acid group, and 4-CMTB and C3 bind to different sites on FFA2 and show allosteric cooperativity. Recently, FFA2 agonists have been described that contain both N-thiazolylamide and carboxylate groups, reminiscent of bitopic ligands. These are thought to engage the carboxylate-binding site on FFA2, but preliminary evidence suggests they do not bind to the same site as 4-CMTB even though both contain N-thiazolylamide. Here, we describe the characterization of four FFA2 ligands containing both N-thiazolylamide and carboxylate. (R)-3-benzyl-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid (compound 14) exhibits allosteric agonism with 4-CMTB but not C3. Three other compounds agonize FFA2 in [35S]GTPγS-incorporation or cAMP assays but behave as inverse agonists in yeast-based gene-reporter assays, showing orthosteric antagonism of C3 responses but allosteric antagonism of 4-CMTB responses. Thus, the bitopic-like FFA2 ligands engage the orthosteric site but do not compete at the site of 4-CMTB binding on an FFA2 receptor molecule. Compound 14 activates FFA2 on human neutrophils and mouse adipocytes, but appears not to inhibit lipolysis upon treatment of human primary adipocytes in spite of the presence of a functional FFA2 receptor in these cells. Hence, these new ligands may reveal differences in coupling of FFA2 between human and rodent adipose tissues.
The Supplementary Information file that accompanies this Article contains errors in Supplementary Figure S1, where the figure keys are incorrect. The correct Figure S1 appears below as Figure 1.
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