G protein-coupled receptors (GPCRs) are transmembrane proteins that modulate physiology across diverse tissues in response to extracellular signals. GPCR signalling can differ due to variation in the sequence (e.g. polymorphisms) or in the expression of receptors in different tissues. The resulting differences in response are an important source of physiological signalling bias. An underexplored source of such bias is the generation of functionally diverse GPCR isoforms that can have distinct patterns of expression in human tissues. Here, we report the findings from a comprehensive study, integrating data from human tissue-level transcriptomes, GPCR sequences and structures, functional annotations, proteomics, single-cell RNA sequencing, population-wide genetic association studies, and pharmacological experiments. Our results show how a single GPCR gene can diversify into multiple isoforms with distinct structural and signalling properties, and how unique combinations of these isoforms can be expressed in different human tissues, contributing to differences in physiological signalling. Based on their structural changes and expression patterns, some of the detected isoforms may also influence drug response and represent new drug targets with improved tissue selectivity. Our findings highlight the need to move from a canonical to a context-specific view of GPCR signalling, in which one considers how the combinatorial expression of receptor isoforms in a specific system (i.e. a particular cell type, tissue, or organism) collectively impacts receptor signalling. These observations pave the way for understanding the impact of isoform variation on GPCR signalling response and have implications for exploiting such variation as a source of GPCR selectivity in drug development.

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Several GPCRs, including receptors previously thought to signal primarily from the cell surface, have been recently shown to signal from many intracellular compartments. This raises the idea that signaling by any given receptor is spatially encoded in the cell, with distinct sites of signal origin dictating distinct downstream consequences. We will discuss recent developments that address this novel facet of GPCR physiology, focusing on spatial segregation of signaling from the cell surface, endosomes, and the Golgi by receptors relevant to the nervous system.

The delta opioid receptor (DOR), a physiologically relevant prototype for G protein–coupled receptors, is retained in intracellular compartments in neuronal cells. This retention is mediated by a nerve growth factor (NGF)-regulated checkpoint that delays the export of DOR from the trans-Golgi network. How DOR is selectively retained in the Golgi, in the midst of dynamic membrane transport and cargo export, is a fundamental unanswered question. Here we address this by investigating sequence elements on DOR that regulate DOR surface delivery, focusing on the C-terminal tail of DOR that is sufficient for NGF-mediated regulation. By systematic mutational analysis, we define conserved dual bi-arginine (RXR) motifs that are required for NGF- and phosphoinositide-regulated DOR export from intracellular compartments in neuroendocrine cells. These motifs were required to bind the coatomer protein I (COPI) complex, a vesicle coat complex that mediates primarily retrograde cargo traffic in the Golgi. Our results suggest that interactions of DOR with COPI, via atypical COPI motifs on the C-terminal tail, retain DOR in the Golgi. These interactions could provide a point of regulation of DOR export and delivery by extracellular signaling pathways.

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 generates different GPCR active states based on the immediate environment of receptors is not known. Here we address this question using spatially resolved 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 neuronal cells. We show that Golgi and surface pools of DOR both inhibit cAMP, but engage distinct conformational biosensors in response to the same ligand in rat neuroendocrine cells. Further, DOR recruits arrestins on the surface but not the Golgi. Our results suggest that the local environment determines the active states of receptors for any given drug, allowing GPCRs to couple to different effectors at different subcellular locations.