Ultimately, this enabled us to identify residues which change pronation with conformation and pH. Combining our DMS, cryo-EM, MD, and extensive pharmacologic validation (by XP Huang), we were able to devise a comprehensive model for pH activation of GPR68 (12/n)

Well, Darko went above and beyond. He developed a new method which uses coevolutionary information to identify both alternative receptor conformations AND the effect of pH on the conformational ensemble. (11/n)

We followed up with a surface expression screen to determine the which mutations alter receptor expression. This allowed to deconvolve the mutational effects on expression vs activation to uncover GOF and LOF activity specific to activation. (9/n)

The result: a comprehensive map of each mutation's effect on pH activation. (8/n)

We then generated a mutational library of GPR68 using the DIMPLE platform developed in @willowcoyote.bsky.social's lab and screened it using our cAMP FACS assay (7/n)

Using GPR68 as our model, we set out to use mutational scanning to determine the effect of every mutation on proton activation. We developed a new FACS-based method to measure Gs coupled receptor activation (enabled by a new TRE's from @justingenglish.bsky.social ) (6/n)

We needed to ascribe a functional role for each residue in proton activation. Unfortunately, these receptors are littered with an enormous number polar and charged residues which may be implicated. (5/n)

To start, Nick Hoppe (in @amanglik.bsky.social's lab) set out to determine the 3D architecture of each human proton sensor (GPR4, GPR65, and GPR68) using cryo-EM. These provided insights into the architecture and arrangement of putative proton-sensing residues in the active-state (4/n)

We have known for some time that several GPCRs respond to changes in pH. However, unlike small molecules, peptides, and other stimuli, protons are a bit wonky--we can't directly see them with standard approaches. (2/n)