Adhesion G protein-coupled receptors (aGPCRs) comprise the second largest yet least studied class of the GPCR superfamily. aGPCRs are involved in many developmental processes, immune and synaptic functions, but the mode of their signal transduction is unclear. Here, we show that a short peptide sequence (termed the Stachel sequence) within the ectodomain of two aGPCRs, GPR126 and GPR133, functions as a tethered agonist. Upon structural changes within the receptor ectodomain, this intramolecular agonist is exposed to the 7-transmembrane helix domain, which triggers G-protein activation. Our studies show high specificity of a given Stachel sequence for its receptor. Finally, the function of Gpr126 is abrogated in zebrafish with a mutated Stachel sequence, and signaling is restored in hypomorphic gpr126 zebrafish mutants upon exogenous Stachel peptide application. These findings illuminate a previously unknown mode of aGPCR activation, and can initiate the development of specific ligands for this currently untargeted GPCR family.

Members of the adhesion G protein-coupled receptor (aGPCR) family carry an agonistic sequence within their large ectodomains. Peptides derived from this region, called the sequence, can activate the respective receptor. As the conserved core region of the sequence is highly similar between aGPCRs, the agonist specificity of sequence-derived peptides was tested between family members using cell culture-based second messenger assays. peptides derived from aGPCRs of subfamily VI (GPR110/ADGRF1, GPR116/ADGRF5) and subfamily VIII (GPR64/ADGRG2, GPR126/ADGRG6) are able to activate more than one member of the respective subfamily supporting their evolutionary relationship and defining them as pharmacological receptor subtypes. Extended functional analyses of the sequences and derived peptides revealed agonist promiscuity, not only within, but also between aGPCR subfamilies. For example, the-derived peptide of GPR110 (subfamily VI) can activate GPR64 and GPR126 (both subfamily VIII). Our results indicate that key residues in the sequence are very similar between aGPCRs allowing for agonist promiscuity of several-derived peptides. Therefore, aGPCRs appear to be pharmacologically more closely related than previously thought. Our findings have direct implications for many aGPCR studies, as potential functional overlap has to be considered for and studies. However, it also offers the possibility of a broader use of more potent peptides when the original sequence is less effective.