Abstract
Adhesion G protein-coupled receptors (aGPCR) are characterized by a large extracellular region containing a conserved GPCR-autoproteolysis-inducing (GAIN) domain. Despite their relevance to several disease conditions, we do not understand the molecular mechanism by which aGPCRs are physiologically activated. GPR110 (ADGRF1) was recently deorphanized as the functional receptor of N-docosahexaenoylethanolamine (synaptamide), a potent synaptogenic metabolite of docosahexaenoic acid. Thus far, synaptamide is the first and only small-molecule endogenous ligand of an aGPCR. Here, we demonstrate the molecular basis of synaptamide-induced activation of GPR110 in living cells. Using in-cell chemical cross-linking/mass spectrometry, computational modeling and mutagenesis-assisted functional assays, we discover that synaptamide specifically binds to the interface of GPR110 GAIN subdomains through interactions with residues Q511, N512 and Y513, causing an intracellular conformational change near TM6 that triggers downstream signaling. This ligand-induced GAIN-targeted activation mechanism provides a framework for understanding the physiological function of aGPCRs and therapeutic targeting in the GAIN domain.
Highlights
Adhesion G protein-coupled receptors are characterized by a large extracellular region containing a conserved GPCR-autoproteolysis-inducing (GAIN) domain
With the help of computational modeling and site-directed mutagenesis, we demonstrate that the smallmolecule ligand synaptamide binds to the GPR110 GAIN domain and causes an intracellular conformational change in living cells, revealing a previously unknown molecular mechanism of Adhesion G protein-coupled receptors (aGPCR) activation through ligand-GAIN domain interaction
Synaptamide-induced GPR110 activity was confirmed by gene-dose-dependent cAMP production detected in CREluc2P HEK 293 cells, which contain a luciferase gene as the cAMP sensor (Fig. 1b, Supplementary Data 1) as well as phosphorylation of downstream cyclic AMP response element binding protein (CREB)[20] (Fig. 1c, Supplementary Fig. 1)
Summary
Adhesion G protein-coupled receptors (aGPCR) are characterized by a large extracellular region containing a conserved GPCR-autoproteolysis-inducing (GAIN) domain Despite their relevance to several disease conditions, we do not understand the molecular mechanism by which aGPCRs are physiologically activated. Using in-cell chemical cross-linking/ mass spectrometry, computational modeling and mutagenesis-assisted functional assays, we discover that synaptamide binds to the interface of GPR110 GAIN subdomains through interactions with residues Q511, N512 and Y513, causing an intracellular conformational change near TM6 that triggers downstream signaling This ligand-induced GAIN-targeted activation mechanism provides a framework for understanding the physiological function of aGPCRs and therapeutic targeting in the GAIN domain. A mechanism independent of either the autoproteolysis or Stachel agonism has been suggested for activation of aGPCRs in vitro[16,17] and recently in vivo[18] In this model, the binding of a ligand to the ECR is thought to modulate G protein signaling through conformational changes at the 7TM17. A detailed molecular understanding of GPR110 activation remains elusive
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