Human GPR65 Stable Cell Line - HEK293

GPR65 is a prototypical proton-sensing G protein-coupled receptor (GPCR). Highly expressed in various solid tumors, these receptors are emerging as promising therapeutic targets due to their ability to respond to the acidic microenvironment and subsequently modulate tumor signaling and immune responses. Despite their physiological significance, the mechanism by which acidic environments alter the signaling patterns of proton-sensing GPCRs remains unclear. Here, researchers demonstrate that the intracellular trafficking of GPR65-a classic proton-sensing receptor-is completely uncoupled from its signaling process, a characteristic that distinguishes it sharply from other known mammalian GPCRs. GPR65 undergoes endocytosis and localizes to early and late endosomes, where it maintains steady-state signaling regardless of extracellular pH. Although an acidic extracellular environment stimulates receptor signaling at the plasma membrane (PM) in a dose-dependent manner, the generation of a full signaling response requires the participation of endosomal GPR65. Receptor mutants incapable of activating the cAMP signaling pathway exhibit normal trafficking, endocytosis, and endosomal localization. These findings indicate that GPR65 is constitutively active within endosomes and suggest a model in which changes in extracellular pH reshape the spatial landscape of receptor signaling, shifting the primary site of signaling to the cell surface.

Researchers determined the pH-dependent activation characteristics of GPR65 by measuring levels of the second messenger cAMP in GPR65-expressing HEK293 cells. They quantified cAMP levels using the GloSensor biosensor, which exhibits increased luminescence upon binding to cAMP (Figure 1A). Upon exposure to proton concentrations corresponding to a pH range of 6.0 to 8.0, GPR65-expressing cells exhibited an increase in cAMP levels that correlated with proton concentration (Figures 1B and C). This increase in cAMP levels reached saturation at pH 6.4. Notably, cAMP levels rose across both acidic and neutral pH ranges. As a negative control, HEK293 cells not expressing GPR65 showed no such response (Figures 1D and E). Similarly, a GPR65 mutant containing substitutions at three key histidine residues (H10, H14, and H243)-mutations known to abolish the cAMP response-failed to elicit a cAMP response (Figures 1D and E). This GPR65-mediated increase in cAMP mirrors the behavior of the prototypical Gs-coupled GPCR, the β-2 adrenergic receptor (B2AR); the researchers used B2AR as a positive control for cAMP activation (Figures 1D and E). When GPR65-expressing cells were exposed to a high-pH environment (i.e., low proton concentration), GloSensor luminescence intensity rapidly declined to levels below those observed in the vehicle control at pH 7.0 (Figures 1B and C). Both acute and chronic exposure to alkaline pH resulted in a reduction of GPR65-mediated GloSensor luminescence (Figure 1B). Collectively, these data indicate that GPR65 elevates cAMP levels under both acidic and neutral pH conditions.

Figure 1. GPR65 increases cAMP levels at both neutral and acidic pH. (Morales Rodríguez L M, et al., 2023)