Delineation of the GPR15 receptor‐mediated Gα protein signalling profile in recombinant mammalian cells

Abstract

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.

Structure–activity relationship of GPR15L peptide analogues and investigation of their interaction with the GPR15 receptor

“…3 We and others have found that upon GPR15L stimulation, GPR15 couples to the G i/o family decreasing intracellular 3 0 ,5 0 -cyclic adenosine monophosphate (cAMP) levels, recruits multifunction arrestin proteins and undergoes endocytosis. [3][4][5][6] GPR15 has been reported to be a potential therapeutic target for immune diseases such as colitis, dermatitis and multiple sclerosis. [7][8][9] Therefore, it is critical to understand the pharmacological and physiological functions of GPR15.…”

“…The full‐length GPR15L peptide contains two predicted protease cleavage sites, which leads to four potential endogenous variants, among them, the 11‐mer C‐terminal GPR15L(71‐81) peptide (coded as 01 ), which is the starting point of our study 3 . We and others have found that upon GPR15L stimulation, GPR15 couples to the G i/o family decreasing intracellular 3′,5′‐cyclic adenosine monophosphate (cAMP) levels, recruits multifunction arrestin proteins and undergoes endocytosis 3–6 …”

Molecular recognition of the atypical chemokine-like peptide GPR15L by its cognate receptor GPR15

“…While the full-length ligand demonstrates superior activation potency compared to GPR15L C11 (Supplementary Fig. S4a ), prior research has highlighted the pivotal role of the 11 amino acids located in the C-terminus for receptor recognition and activation 3 , 5 , 15 . We hypothesized that the N-terminal portion might participate in interactions with regions outside the orthosteric pocket of the receptor (pertaining to the interaction between chemokine C-termini and their receptors), potentially influencing ligand activation potency to some extent.…”

“…We hypothesized that the N-terminal portion might participate in interactions with regions outside the orthosteric pocket of the receptor (pertaining to the interaction between chemokine C-termini and their receptors), potentially influencing ligand activation potency to some extent. However, truncating the C-terminal 11 amino acids from full-length GPR15L renders the peptide incapable of activating the receptor 3 , 15 , thereby affirming the essential role of interactions between GPR15L C11 and the orthosteric pocket of GPR15. Nonetheless, further investigations into the full-length structure of GPR15L are warranted to comprehensively elucidate its functional significance.…”

Endocytic proteins mediating GPR15 receptor internalization provide insight into the underlying mechanisms

“…It is a potential therapeutical target for immune and inflammatory diseases such as multiple sclerosis, HIV enteropathy, colitis, and dermatitis [24][25][26][27][28]. When the receptor is stimulated by its recently identified endogenous ligand, GPR15L(25-81) peptide, it activates G i/o proteins and inhibits the downstream second messenger (cAMP) effector adenylyl cyclase [29][30][31][32].…”