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The human calcium-sensing receptor (CaSR) detects fluctuations in the extracellular Ca 2+ concentration and maintains Ca 2+ homeostasis 1 , 2 . It also mediates diverse cellular processes not associated with Ca 2+ balance 3 – 5 . The functional pleiotropy of CaSR arises in part from its ability to signal through several G-protein subtypes 6 . We determined structures of CaSR in complex with G proteins from three different subfamilies: G q , G i and G s . We found that the homodimeric CaSR of each complex couples to a single G protein through a common mode. This involves the C-terminal helix of each Gα subunit binding to a shallow pocket that is formed in one CaSR subunit by all three intracellular loops (ICL1–ICL3), an extended transmembrane helix 3 and an ordered C-terminal region. G-protein binding expands the transmembrane dimer interface, which is further stabilized by phospholipid. The restraint imposed by the receptor dimer, in combination with ICL2, enables G-protein activation by facilitating conformational transition of Gα. We identified a single Gα residue that determines G q and G s versus G i selectivity. The length and flexibility of ICL2 allows CaSR to bind all three Gα subtypes, thereby conferring capacity for promiscuous G-protein coupling. Structures of the human calcium-sensing receptor can be bound into complex with G proteins from three different Gα subtypes while maintaining G-protein-binding specificity.

G protein-coupled receptors (GPCRs) activate four families of heterotrimeric G proteins, and individual receptors must select a subset of G proteins to produce appropriate cellular responses. Although the precise mechanisms of coupling selectivity are uncertain, the Gα subunit C terminus is widely believed to be the primary determinant recognized by cognate receptors. Here, we directly assess coupling between 14 representative GPCRs and 16 Gα subunits, including one wild-type Gα subunit from each of the four families and 12 chimeras with exchanged C termini. We use a sensitive bioluminescence resonance energy transfer (BRET) assay that provides control over both ligand and nucleotide binding, and allows direct comparison across G protein families. We find that the Gs- and Gq-coupled receptors we studied are relatively promiscuous and always couple to some extent to Gi1 heterotrimers. In contrast, Gi-coupled receptors are more selective. Our results with Gα subunit chimeras show that the Gα C terminus is important for coupling selectivity, but no more so than the Gα subunit core. The relative importance of the Gα subunit core and C terminus is highly variable and, for some receptors, the Gα core is more important for selective coupling than the C terminus. Our results suggest general rules for GPCR-G protein coupling and demonstrate that the critical G protein determinants of selectivity vary widely, even for different receptors that couple to the same G protein.

Despite the crowded nature of the cellular milieu, ligand–GPCR–G protein interactions are traditionally viewed as spatially and temporally isolated events. In contrast, recent reports suggest the spatial and temporal coupling of receptor–effector interactions, with the potential to diversify downstream responses. In this study, we combine protein engineering of GPCR–G protein interactions with affinity sequestration and photo-manipulation of the crucial Gα C terminus, to demonstrate the temporal coupling of cognate and non-cognate G protein interactions through priming of the GPCR conformation. We find that interactions of the Gαs and Gαq C termini with the β 2 -adrenergic receptor (β 2 -AR), targeted at the G-protein-binding site, enhance Gs activation and cyclic AMP levels. β 2 -AR–Gα C termini interactions alter receptor conformation, which persists for ~90 s following Gα C terminus dissociation. Non-cognate G-protein expression levels impact cognate signaling in cells. Our study demonstrates temporal allostery in GPCRs, with implications for the modulation of downstream responses through the canonical G-protein-binding interface. Recent reports suggest the spatial and temporal coupling of receptor-effector interactions, with the potential to diversify downstream responses. Here authors demonstrate the temporal coupling of cognate and non-cognate G protein interactions through priming of GPCR conformation.

The MRGPRX family of receptors (MRGPRX1–4) is a family of mas-related G-protein-coupled receptors that have evolved relatively recently 1 . Of these, MRGPRX2 and MRGPRX4 are key physiological and pathological mediators of itch and related mast cell-mediated hypersensitivity reactions 2 – 5 . MRGPRX2 couples to both G i and G q in mast cells 6 . Here we describe agonist-stabilized structures of MRGPRX2 coupled to G i1 and G q in ternary complexes with the endogenous peptide cortistatin-14 and with a synthetic agonist probe, respectively, and the development of potent antagonist probes for MRGPRX2. We also describe a specific MRGPRX4 agonist and the structure of this agonist in a complex with MRGPRX4 and G q . Together, these findings should accelerate the structure-guided discovery of therapeutic agents for pain, itch and mast cell-mediated hypersensitivity. Structural studies of the itch receptors MRGPRX2 and MRGPRX4 in complex with endogenous and synthetic ligands provide a basis for the development of therapeutic compounds for pain, itch and mast cell-mediated hypersensitivity.

The adhesion G-protein-coupled receptor (GPCR) latrophilin 3 (ADGRL3) has been associated with increased risk of attention deficit hyperactivity disorder (ADHD) and substance use in human genetic studies. Knockdown in multiple species leads to hyperlocomotion and altered dopamine signaling. Thus, ADGRL3 is a potential target for treatment of neuropsychiatric disorders that involve dopamine dysfunction, but its basic signaling properties are poorly understood. Identification of adhesion GPCR signaling partners has been limited by a lack of tools to acutely activate these receptors in living cells. Here, we design a novel acute activation strategy to characterize ADGRL3 signaling by engineering a receptor construct in which we could trigger acute activation enzymatically. Using this assay, we found that ADGRL3 signals through G12/G13 and Gq, with G12/13 the most robustly activated. Gα 12/13 is a new player in ADGRL3 biology, opening up unexplored roles for ADGRL3 in the brain. Our methodological advancements should be broadly useful in adhesion GPCR research. Among the adhesion receptor class of GPCRs, which are understudied, the adhesion receptor ADGRL3 can be activated by its own tethered agonist and couples to G protein G12/13 and somewhat to Gq.

G-protein-coupled receptors comprise the largest family of mammalian transmembrane receptors. They mediate numerous cellular pathways by coupling with downstream signalling transducers, including the hetrotrimeric G proteins G s (stimulatory) and G i (inhibitory) and several arrestin proteins. The structural mechanisms that define how G-protein-coupled receptors selectively couple to a specific type of G protein or arrestin remain unknown. Here, using cryo-electron microscopy, we show that the major interactions between activated rhodopsin and G i are mediated by the C-terminal helix of the G i α-subunit, which is wedged into the cytoplasmic cavity of the transmembrane helix bundle and directly contacts the amino terminus of helix 8 of rhodopsin. Structural comparisons of inactive, G i -bound and arrestin-bound forms of rhodopsin with inactive and G s -bound forms of the β 2 -adrenergic receptor provide a foundation to understand the unique structural signatures that are associated with the recognition of G s , G i and arrestin by activated G-protein-coupled receptors. The cryo-electron microscopy structure of human rhodopsin bound to the inhibitory G i protein-coupled receptor provides insights into ligand–receptor–G-protein interactions.

G protein–coupled receptors (GPCRs) are critical regulators of cellular function acting via heterotrimeric G proteins as their primary transducers with individual GPCRs capable of pleiotropic coupling to multiple G proteins. Structural features governing G protein selectivity and promiscuity are currently unclear. Here, we used cryo-electron microscopy (cryo-EM) to determine structures of the cholecystokinin (CCK) type 1 receptor (CCK1R) bound to the CCK peptide agonist, CCK-8 and 2 distinct transducer proteins, its primary transducer Gq, and the more weakly coupled Gs. As seen with other Gq/11–GPCR complexes, the Gq–α5 helix (αH5) bound to a relatively narrow pocket in the CCK1R core. Surprisingly, the backbone of the CCK1R and volume of the G protein binding pocket were essentially equivalent when Gs was bound, with the Gs αH5 displaying a conformation that arises from “unwinding” of the far carboxyl-terminal residues, compared to canonically Gs coupled receptors. Thus, integrated changes in the conformations of both the receptor and G protein are likely to play critical roles in the promiscuous coupling of individual GPCRs.

Heterotrimeric G proteins serve as membrane-associated signaling hubs, in concert with their cognate G-protein-coupled receptors. Fluorine nuclear magnetic resonance spectroscopy was employed to monitor the conformational equilibria of the human stimulatory G-protein α subunit (G s α) alone, in the intact G s αβ 1 γ 2 heterotrimer or in complex with membrane-embedded human adenosine A 2A receptor (A 2A R). The results reveal a concerted equilibrium that is strongly affected by nucleotide and interactions with the βγ subunit, the lipid bilayer and A 2A R. The α1 helix of G s α exhibits significant intermediate timescale dynamics. The α4β6 loop and α5 helix undergo membrane/receptor interactions and order–disorder transitions respectively, associated with G-protein activation. The αN helix adopts a key functional state that serves as an allosteric conduit between the βγ subunit and receptor, while a significant fraction of the ensemble remains tethered to the membrane and receptor upon activation. Fluorine nuclear magnetic resonance spectroscopy of the stimulatory heterotrimeric G protein reveals a conformational landscape shaped by interactions with nucleotides, the lipid bilayer and a G-protein-coupled receptor.

By restoring glucose-regulated insulin secretion, glucagon-like peptide-1-based (GLP-1-based) therapies are becoming increasingly important in diabetes care. Normally, the incretins GLP-1 and glucose-dependent insulinotropic polypeptide (GIP) jointly maintain normal blood glucose levels by stimulation of insulin secretion in pancreatic β cells. However, the reason why only GLP-1-based drugs are effective in improving insulin secretion after presentation of diabetes has not been resolved. ATP-sensitive K+ (KATP) channels play a crucial role in coupling the systemic metabolic status to β cell electrical activity for insulin secretion. Here, we have shown that persistent membrane depolarization of β cells due to genetic (β cell-specific Kcnj11-/- mice) or pharmacological (long-term exposure to sulfonylureas) inhibition of the KATP channel led to a switch from Gs to Gq in a major amplifying pathway of insulin secretion. The switch determined the relative insulinotropic effectiveness of GLP-1 and GIP, as GLP-1 can activate both Gq and Gs, while GIP only activates Gs. The findings were corroborated in other models of persistent depolarization: a spontaneous diabetic KK-Ay mouse and nondiabetic human and mouse β cells of pancreatic islets chronically treated with high glucose. Thus, a Gs/Gq signaling switch in β cells exposed to chronic hyperglycemia underlies the differential insulinotropic potential of incretins in diabetes.

Stress-related alterations in brain-derived neurotrophic factor (BDNF) expression, a neurotrophin that plays a key role in synaptic plasticity, are believed to contribute to the pathophysiology of depression. Here, we show that in a chronic mild stress (CMS) model of depression the Gαi1 and Gαi3 subunits of heterotrimeric G proteins are down-regulated in the hippocampus, a key limbic structure associated with major depressive disorder. We provide evidence that Gαi1 and Gαi3 (Gαi1/3) are required for the activation of TrkB downstream signaling pathways. In mouse embryonic fibroblasts (MEFs) and CNS neurons, Gαi1/3 knockdown inhibited BDNF-induced tropomyosin-related kinase B (TrkB) endocytosis, adaptor protein activation, and Akt-mTORC1 and Erk-MAPK signaling. Functional studies show that Gαi1 and Gαi3 knockdown decreases the number of dendrites and dendritic spines in hippocampal neurons. In vivo, hippocampal Gαi1/3 knockdown after bilateral microinjection of lentiviral constructs containing Gαi1 and Gαi3 shRNA elicited depressive behaviors. Critically, exogenous expression of Gαi3 in the hippocampus reversed depressive behaviors in CMS mice. Similar results were observed in Gαi1/Gαi3 double-knockout mice, which exhibited severe depressive behaviors. These results demonstrate that heterotrimeric Gαi1 and Gαi3 proteins are essential for TrkB signaling and that disruption of Gαi1 or Gαi3 function could contribute to depressive behaviors.