Profiling of HCAR1 signaling reveals Gα _i/o and Gα _s activation without β-arrestin recruitment and the discovery of an allosteric agonist

Lactate was long considered a byproduct of glycolysis and associated with various harmful effects. However, the role of lactate was expanded with the finding that it also can act as a signaling molecule through the G protein–coupled receptor Hydroxycarboxylic Acid Receptor 1 (HCAR1). The receptor was shown to be primarily expressed in adipocytes but is also expressed in many other tissues and cell types. Activation of HCAR1 can help regulate lipolysis and improve insulin sensitivity, making it a promising target for managing obesity and other metabolic disorders. While HCAR1 activation offers therapeutic benefits for metabolic diseases, it can also promote cancer cell survival and metastasis, necessitating a nuanced approach to avoid unintended tumor growth. However, only a few ligands have been reported for HCAR1, and their signaling pathways remain unexplored. Using enhanced bystander bioluminescence resonance energy transfer (ebBRET) to study G protein activation and β-arrestin recruitment following ligand addition, we were able to identify compounds such as AZ7136, a potent HCAR1 agonist, AZ2114 a partial agonist, and establish GPR81 agonist 1 as an ago-positive allosteric modulator. We also show that HCAR1 preferentially activates the Gα _i/o and Gα _s pathways without recruiting β-arrestins. These findings enhance our understanding of the signaling profile of HCAR1 and the newly characterized ligands could be used as molecular tools to understand more about HCAR1 in metabolic disease.