In vivo mapping of a GPCR interactome using knockin mice

Abstract

With over 30% of current medications targeting this family of proteins, G-protein–coupled receptors (GPCRs) remain invaluable therapeutic targets. However, due to their unique physicochemical properties, their low abundance, and the lack of highly specific antibodies, GPCRs are still challenging to study in vivo. To overcome these limitations, we combined here transgenic mouse models and proteomic analyses in order to resolve the interactome of the δ-opioid receptor (DOPr) in its native in vivo environment. Given its analgesic properties and milder undesired effects than most clinically prescribed opioids, DOPr is a promising alternative therapeutic target for chronic pain management. However, the molecular and cellular mechanisms regulating its signaling and trafficking remain poorly characterized. We thus performed liquid chromatography–tandem mass spectrometry (LC-MS/MS) analyses on brain homogenates of our newly generated knockin mouse expressing a FLAG-tagged version of DOPr and revealed several endogenous DOPr interactors involved in protein folding, trafficking, and signal transduction. The interactions with a few identified partners such as VPS41, ARF6, Rabaptin-5, and Rab10 were validated. We report an approach to characterize in vivo interacting proteins of GPCRs, the largest family of membrane receptors with crucial implications in virtually all physiological systems.

HA-MOP knockin mice express the canonical µ-opioid receptor but lack detectable splice variants

“…However, the transgene used a tyrosine hydroxylase promoter (in order to drive expression in LC) whereas our approach is based on the targeted insertion of the tag into the endogenous mOPRM1 gene, which should preserve native expression patterns. This strategy is in line with the recently published FLAG-tagged DOP 21 and HA-tagged DOP 22 receptor mouse models that used targeted recombination. In an effort to solve this problem, we have developed a novel mouse model by fusing the HA-epitope tag sequence to the amino terminus of the MOP.…”

Conformational specificity of opioid receptors is determined by subcellular location irrespective of agonist

“…A lower SNC80 concentration in the Golgi, however, is unlikely to explain the differential recruitment we observed, as miniGsi is more potently recruited to PM DOR than Nb39 ( Figure 1G ). Additionally, DOR-interacting proteins, which regulate DOR trafficking and localize to the Golgi, like the coatomer protein I (COPI) complex and Rab10, could also regulate receptor conformations and effector coupling ( Degrandmaison et al, 2020 ; St-Louis et al, 2017 ; Shiwarski et al, 2019 ).…”

“…1G). Additionally, DOR interacting proteins which regulate DOR trafficking and localize to the Golgi, like the COPI complex and Rab10, could also regulate receptor conformations and effector coupling (67)(68)(69).…”

Conformational specificity of opioid receptors is determined by subcellular location irrespective of agonist

“…Sodium acts as an allosteric modulator of class A GPCRs, and DOR specifically has been crystallized with a coordinated sodium ion, which stabilizes the inactive receptor conformation and is required for receptor activation and signaling ( 66, 67 ), suggesting Golgi sodium concentrations could affect DOR activity. Additionally, DOR interacting proteins, especially those which regulate DOR trafficking and localize to the Golgi, like the COPI complex and Rab10, could also regulate receptor conformations and effector coupling ( 68 – 70 ).…”