Mutagenesis of GPR139 reveals ways to create gain or loss of function receptors

Abstract

GPR139 is a Gq‐coupled receptor activated by the essential amino acids L‐tryptophan (L‐Trp) and L‐phenylalanine (L‐Phe). We carried out mutagenesis studies of the human GPR139 receptor to identify the critical structural motifs required for GPR139 activation. We applied site‐directed and high throughput random mutagenesis approaches using a double addition normalization strategy to identify novel GPR139 sequences coding receptors that have altered sensitivity to endogenous ligands. This approach resulted in GPR139 clones with gain‐of‐function, reduction‐of‐function or loss‐of‐function mutations. The agonist pharmacology of these mutant receptors was characterized and compared to wild‐type receptor using calcium mobilization, radioligand binding, and protein expression assays. The structure‐activity data were incorporated into a homology model which highlights that many of the gain‐of‐function mutations are either in or immediately adjacent to the purported orthosteric ligand binding site, whereas the loss‐of‐function mutations were largely in the intracellular G‐protein binding area or were disrupters of the helix integrity. There were also some reduction‐of‐function mutations in the orthosteric ligand binding site. These findings may not only facilitate the rational design of novel agonists and antagonists of GPR139, but also may guide the design of transgenic animal models to study the physiological function of GPR139.

Orphan peptide and G protein-coupled receptor signalling in alcohol use disorder

“…Further research into the interaction between GPR139 and the opioid system found that this receptor activation led to anti-opioid activity in C. elegans . This was conserved in rodents, where deletion of GPR139 in mice potentiated opioid-induced inhibition of neuronal firing (Wang, Lee, Shih, et al, 2019). These data were further corroborated by electrophysiological data from GPR139 KO mice medial habenula neurons, where GPR139 receptor signalling was suggested to prevent μ-opioid receptor-mediated neuronal inhibition via G q/11 coupling (Stoveken et al, 2020).…”

Pharmacology and function of the orphan GPR139 G protein‐coupled receptor

“…By combining in silico and in vitro mutagenesis, they identified a common binding pocket consisting of a hydrophobic region deeply buried between F109 3x33 , H187 5x43 and W241 6x48 , and a proximal polar region enclosed by N271 7x38 , R244 6x51 and E108 3x32 (Figure ) . The binding site was confirmed by Wang et al, who also identified a less buried hydrophobic binding site defined by V76 2x73 , F79 2x56 , I104 3x28 , V83 2x60 and I80 2x57 shared by larger GPR139 agonists . They furthermore identified several series of human GPR139 mutations resulting in gain, reduction or loss of function.…”

“…Another set of mutations impact the transmembrane helices structure and thus might change the common ligand binding site or the ability to bind G proteins. The authors speculate that the last two set of mutations have a structural impact on GPR139 function …”