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

Abstract

The prevailing model for the variety in drug responses is that they stabilize distinct active states of their G protein-coupled receptor (GPCR) targets, allowing coupling to different effectors. However, whether the same ligand can produce different GPCR active states based on the environment of receptors in cells is a fundamental unanswered question. Here we address this question using live cell imaging of conformational biosensors that read out distinct active conformations of the δ-opioid receptor (DOR), a physiologically relevant GPCR localized to Golgi and the surface in neurons. We show that, although Golgi and surface pools of DOR regulated cAMP, the two pools engaged distinct conformational biosensors in response to the same ligand. Further, DOR recruited arrestin on the plasma membrane but not the Golgi. Our results suggest that the same agonist drives different conformations of a GPCR at different locations, allowing receptor coupling to distinct effectors at different locations.

Structures of the human cholecystokinin 1 (CCK1) receptor bound to Gs and Gq mimetic proteins: insight into mechanisms of G protein selectivity

“…In particular, the efficiency of Gs coupling was markedly augmented by either high cholesterol or absence of the primary (Gq/11) transducers demonstrating that the local environment of the receptor within a cell is also critical for the mode of transducer engagement. Differential transducer engagement has been reported for different cellular compartments, linked to different receptor conformations (46) and our current observations expand the potential mechanisms for such observations. Our work supports a model for G protein interaction where the extent of GPCR conformational change that occurs upon agonist activation is intrinsic to the individual receptor, with alterations to G protein preference by biased agonists or the local receptor environment more likely to be governed by changes to the conformational dynamics within this primary activated state, rather than large changes to backbone conformations (e.g.…”

Expression of mini-G proteins specifically halt cognate GPCR trafficking and intracellular signalling

“…Our results have important consequences for the interpretation of data on GPCR subcellular signalling when using a mini-G-based approach. As mini-G proteins are being used to detect the presence of activated GPCRs at different subcellular compartments, with the interrogation of endocytic signalling being a common application (Crilly et al, 2021;Jimenez-Vargas et al, 2020;Wan et al, 2018), there is clear potential for any mini-G-mediated alteration of receptor trafficking responses to confound these measurements. In view of the present observations, we recommend exhaustive checks on specific receptor internalisation rates if a mini-G is to be utilised in any intracellular signalling assay, and to consider changing to other non-mini-G based bystander methods including the use of nanobodies against G protein:active GPCR pairs, amongst other possibilities, such as, for example, the use of fulllength G protein fusion constructs (Novikoff et al, 2021), or strategies to inhibit GPCR internalisation (Sposini et al, 2017).…”

“…Due to this ability of mini-G proteins to act as "conformational biosensors" for active GPCRs, many researchers have constructed vectors encoding a range of mini-G proteins fused to fluorescent or luminescent tags for in-cell experiments (Carpenter, 2018;Martemyanov and Garcia-Marcos, 2018;Wan et al, 2018). Examples include bioluminescence resonance energy transfer (BRET) and NanoLuc Binary Technology (NanoBiT) complementation assays for quantification of subtype-specific coupling to individual receptors (Wan et al, 2018) and spatiotemporal measurements of subcellularly-localised GPCR signalling (Crilly et al, 2021;Truong et al, 2021).…”