Differential Roles of Extracellular Histidine Residues of GPR68 for Proton-Sensing and Allosteric Modulation by Divalent Metal Ions

Abstract

GPR68, an orphan G-protein coupled receptor, senses protons, couples to multiple G-proteins, and is also activated or inhibited by divalent metal ions. It has seven extracellular histidine residues, although it is not clear how these histidine residues play a role in both proton-sensing and metal ion modulation. Here we demonstrate that divalent metal ions are allosteric modulators that can activate or inhibit proton activity in a concentration-and pH-dependent manner. We then show that single histidine mutants have differential and varying degrees of effects on proton-sensing and metal ion modulation. Some histidine residues play dual roles in proton-sensing and metal ion modulation, while others are important in one or the other but not both. Two extracellular disulfide bonds are predicted to constrain histidine residues to be spatially close to each other. Combining histidine mutations leads to reduced proton activity and resistance to metal ion modulation, while breaking the less conserved disulfide bond results in a more severe reduction in proton-sensing over metal modulation. The small-molecule positive allosteric modulators (PAMs) ogerin and lorazepam are not affected by these mutations and remain active at mutants with severely reduced proton activity or are resistant to metal ion modulation. These results suggest GPR68 possesses two independent allosteric modulation systems, one through interaction with divalent metal ions at the extracellular surface and another through small-molecule PAMs in the transmembrane domains. A new GPR68 model is developed to accommodate the findings which could serve as a template for further studies and ligand discovery by virtual ligand docking.

Physiological relevance of proton-activated GPCRs

“…Moreover, conditions that stimulate each receptor, i.e., reduction of extracellular pH for OGR1 and elevation of extracellular calcium for CaSR, inhibit the signaling dependent on the other receptor. Interestingly, another study performed by Huang et al with HEK293T cells showed that extracellular calcium had a small stimulatory effect on OGR1 [38]. The discrepancy might be explained by endogenous CaSR expression in HEK293T [33,133].…”

“…Only a few amino acids are protonated at pH values ranging from 5 to 7.4, such as histidine, aspartic acid, arginine, lysine, and glutamic acid [107]. Research on proton sensing by GPCRs has mostly focused on the role of histidine residues [38,75,100,107]. Several histidine residues are present in the extracellular loops of TDAG8, GPR4, and OGR1, but only few in G2A, which could explain the low pH sensitivity of this receptor [100].…”

“…Indeed, multiple ions and conditions can modulate or stimulate proton-activated GPCRs. Sodium and divalent cations can allosteric modify the activation of GPR4, OGR1, and TDAG8 by H + (although GPR4 and TDAG8 have low sensitivity to divalent cations) [38,107] (Fig. 1).…”

pH and proton-sensitive receptors in brain ischemia

“… 108 Interestingly, another study in HEK293T cells shows that divalent metal ions can enhance GPR68 signaling. 109 In organotypic hippocampal and cortical slices, we found that acidosis induced the activation of PKC, and this effect requires GPR68. 63 These results suggest that, in both organotypic brain slices and cerebellar granule cells, GPR68 activation by acidosis activates the Gq-DAG-PKC axis.…”

pH-Sensing G Protein-Coupled Receptor OGR1 (GPR68) Expression and Activation Increases in Intestinal Inflammation and Fibrosis

“…A family of proton-sensing G-protein-coupled receptors (GPCRs), ovarian cancer G-protein-coupled receptor 1 (OGR1, also known as GPR68), G-protein-coupled receptor 4 (GPR4), and T-cell death-associated gene 8 (TDAG8, also known as GPR65), is activated by protons by differing degrees of sensitivity to pH [14]. Protons bind to histidine residues located at the extracellular region of these transmembrane receptors, activating second messenger signalling pathways, which in turn regulate gene expression [14][15][16][17]. OGR1 is almost silent or inactive at pH 7.8, but fully active at pH ≤ 6.8 [14].…”