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The GPR15 receptor is a G protein-coupled receptor (GPCR), which is activated by an endogenous peptide GPR15L(25-81) and a C-terminal peptide fragment GPR15L(71-81). GPR15 signals through the G i/o pathway to decrease intracellular cyclic adenosine 3 0 ,5 0 -monophosphate (cAMP). However, the activation profiles of the GPR15 receptor within G i/o subtypes have not been examined. Moreover, whether the receptor can also couple to G s , G q/11 and G 12/13 is unclear. Here, GPR15L(25-81) and GPR15L(71-81) are used as pharmacological tool compounds to delineate the GPR15 receptor-mediated Gα protein signalling using a G protein activation assay and second messenger assay conducted on living cells. The results show that the GPR15 receptor preferentially couples to G i/o rather than other pathways in both assays. Within the G i/o family, the GPR15 receptor activates all the subtypes (G i1 , G i2 , G i3 , G oA , G oB and G z ). The E max and activation rates of G i1, G i2 , G i3, G oA and G oB are similar, whilst the E max of G z is smaller and the activation rate is significantly slower. The potencies of both peptides toward each G i/o subtype have been determined. Furthermore, the GPR15 receptor signals through G i/o to inhibit cAMP accumulation, which could be blocked by the application of the G i/o inhibitor pertussis toxin.

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The 57‐mer full‐length GPR15L(25‐81) peptide has been identified as the principal endogenous agonist of the G protein‐coupled receptor GPR15. Its main activity resides in the C‐terminal 11‐mer GPR15L(71‐81), which has full efficacy but ~40‐fold lower potency than the full‐length peptide. Here, we systematically investigated the structure–activity relationship of GPR15L(71‐81) by truncations/extensions, alanine‐scanning, and N‐ and C‐terminal capping. The synthesized peptide analogues were tested at GPR15 stably expressed in HEK293A cells using a homogenous time‐resolved Förster resonance energy transfer‐based Gi cAMP functional assay. We show that the C‐terminal α carboxyl group and the residues Leu78, Pro75, Val74, and Trp72 are critical for receptor interaction and contribute significantly to the peptide potency. Furthermore, we tested the ability of GPR15L(71‐81), C‐terminally amidated GPR15L(71‐81), and GPR15L(25‐81) to activate the three GPR15 receptor mutants in a bioluminescence resonance energy transfer‐based G protein activation assay. The results demonstrate that the Lys192 and Glu272 residues in GPR15 are important for the potency of the GPR15L peptide. Overall, our study identifies critical residues in the peptide and receptor sequences for future drug design.