Abstract
G protein-coupled receptor 35 (GPR35) is poorly characterized but nevertheless has been revealed to have diverse roles in areas including lower gut inflammation and pain. The development of novel reagents and tools will greatly enhance analysis of GPR35 functions in health and disease. Here, we used mass spectrometry, mutagenesis, and [32P] orthophosphate labeling to identify that all five hydroxy-amino acids in the C-terminal tail of human GPR35a became phosphorylated in response to agonist occupancy of the receptor and that, apart from Ser294, each of these contributed to interactions with arretin-3, which inhibits further G protein-coupled receptor signaling. We found that Ser303 was key to such interactions; the serine corresponding to human GPR35a residue 303 also played a dominant role in arrestin-3 interactions for both mouse and rat GPR35. We also demonstrated that fully phospho-site–deficient mutants of human GPR35a and mouse GPR35 failed to interact effectively with arrestin-3, and the human phospho-deficient variant was not internalized from the surface of cells in response to agonist treatment. Even in cells stably expressing species orthologues of GPR35, a substantial proportion of the expressed protein(s) was determined to be immature. Finally, phospho-site–specific antisera targeting the region encompassing Ser303 in human (Ser301 in mouse) GPR35a identified only the mature forms of GPR35 and provided effective sensors of the activation status of the receptors both in immunoblotting and immunocytochemical studies. Such antisera may be useful tools to evaluate target engagement in drug discovery and target validation programs.
Highlights
Classified as an orphan G protein-coupled receptor (GPCR) [1,2], G protein-coupled receptor 35 (GPR35) has a rich pharmacology where, over time, a host of synthetic and naturally generated compounds have 2 been shown to be capable of activating the receptor with potency ranging from modest to high
Across the GPCR superfamily receptor-arrestin interactions are frequently preceded by agonist-promoted phosphorylation of hydroxy-amino acids in the C-terminal tail of the receptor, the third intracellular loop, or both
In cells in which either the receptor was not induced, or those in which the construct was induced but which were not treated with an appropriate agonist, incorporation of [32P] was negligible for both hGPR35-HA (Figure 1B)
Summary
Classified as an orphan G protein-coupled receptor (GPCR) [1,2], GPR35 has a rich pharmacology where, over time, a host of synthetic and naturally generated compounds have 2 been shown to be capable of activating the receptor with potency ranging from modest to high (see 2,3 for review). A range of approaches have been used to identify agonists and antagonists of GPR35 [2], in the main, efforts to identify novel regulators from small molecule chemical libraries have been based on activator-induced interactions between the receptor and an arrestin isoform [4,5,6,7] This reflects that, no-matter the specific assay format employed, agonist-induced interactions between GPR35 orthologues and arrestin isoforms are generally extremely robust, whereas suitable G proteinbased signalling assays have been more challenging to develop and implement [8]. Given the well-known roles of agonist-induced phosphorylation in arrestin interactions with many GPCRs [9,10,11,12] and the roles of arrestins in receptor desensitization and internalization from the surface of cells [13] it is perhaps surprising that detailed analyses of the sites and mechanisms of agonist-regulated phosphorylation of GPR35, and how this might vary between human and rodent orthologues, has not been reported. Journal Pre-proof pathophysiological roles of GPR35 and the potential to target these productively
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