GPR27 mediates L-lactate-induced Ca ²⁺ and cAMP signalling in 3T3 cells

GPR27/SREB1 is a highly conserved orphan class A G-protein coupled receptor implicated in insulin production, metabolic regulation, tumour biology, neurodegeneration and L-lactate homeostasis, but its immediate second-messenger signalling remains poorly defined. We used single-cell Förster resonance energy transfer nanosensors to monitor cytosolic Ca ²⁺ and cAMP in wild-type 3T3 MEF cells, CRISPR–Cas9 GPR27-knockout cells (GPR27KO) and GPR27-knockout cells transiently re-expressing FLAG-tagged GPR27 (GPR27-rescued). The GPR27 surrogate agonist 8535n (1 µM) increased intracellular Ca ²⁺ in wild-type and rescued cells but not in GPR27-knockout cells and produced no significant cAMP response in wild-type cells. Basal Ca ²⁺ and cAMP levels were unaffected by GPR27 deletion. Extracellular L-lactate (2 mM) induced a GPR27-dependent increase in Ca ²⁺ and cAMP in wild-type and rescued cells, but not in knockout cells, raising the possibility that L-lactate acts as an endogenous ligand or modulator of GPR27.