Site-Directed Mutagenesis and Time-Resolved Fluorescence Spectroscopy Reveal That Two Hydrogen-Bonded Carboxylic Side Chains Are Essential for GPR68 Proton Sensing and G Protein Binding

GPR68 is a pH-sensing G protein-coupled receptor widely
expressed
throughout the body. It has been implicated in various diseases, including
neurodegeneration, chronic inflammation, and cancer, emphasizing its
role in pathophysiology. The identities of the structural elements
essential for pH sensing have been controversial, as experiments and
sequence analyses have been interpreted to suggest that an extracellular
histidine cluster or, by contrast, an internal carboxylic triad, senses
the extracellular pH. Recent molecular simulations and hydrogen-bond
network analyses suggested instead a unifying mechanism whereby the
extracellular histidine residues and the internal carboxylic triad
couple to each other via a protonation-sensitive hydrogen-bond network
that includes a fourth internal carboxylic group, E1033.34. However, without experimental verification, there remains a gap
in our understanding of the mechanism by which GPR68 is activated
by protons. To this aim, here we have studied 16 GPR68 mutations,
which we selected based on the hydrogen-bond network analyses and
conservation of key residues. We implemented a cell-based homogeneous
time-resolved fluorescence assay to monitor the Gαq/11 and Gαs coupled signaling pathways. We used this
assay to study how each mutation alters the basal activity levels,
half-maximal activation values, and reactivation at lower pH values.
Our data identify E1033.34Q as a gain-of-function mutant
essential for the proton-sensing mechanism of GPR68. We further discovered
that E1494.53Q, a constitutively active mutant, prefers
Gαs coupling over Gαq/11 and that
wild-type GPR68 is more sensitive toward Gαq/11 coupling
over Gαs.