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Class C G protein-coupled receptors (GPCRs) are known to form stable homodimers or heterodimers critical for function, but the oligomeric status of class A and B receptors, which constitute >90% of all GPCRs, remains hotly debated. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful approach with the potential to reveal valuable insights into GPCR organization but has rarely been used in living cells to study protein systems. Here, we report generally applicable methods for using smFRET to detect and track transmembrane proteins diffusing within the plasma membrane of mammalian cells. We leverage this in-cell smFRET approach to show agonist-induced structural dynamics within individual metabotropic glutamate receptor dimers. We apply these methods to representative class A, B and C receptors, finding evidence for receptor monomers, density-dependent dimers and constitutive dimers, respectively.A suite of generally applicable methods and tools, developed to enable single-molecule FRET-based studies of transmembrane proteins diffusing in the cell membrane of living cells, was used to study the oligomerization and dynamics of GPCRs.

G-protein-coupled receptor (GPCR)-mediated signal transduction is central to human physiology and disease intervention, yet the molecular mechanisms responsible for ligand-dependent signalling responses remain poorly understood. In class A GPCRs, receptor activation and G-protein coupling entail outward movements of transmembrane helix 6 (TM6). Here, using single-molecule fluorescence resonance energy transfer imaging, we examine TM6 movements in the β 2 adrenergic receptor (β 2 AR) upon exposure to orthosteric ligands with different efficacies, in the absence and presence of the G s heterotrimer. We show that partial and full agonists differentially affect TM6 motions to regulate the rate at which GDP-bound β 2 AR–G s complexes are formed and the efficiency of nucleotide exchange leading to G s activation. These data also reveal transient nucleotide-bound β 2 AR–G s species that are distinct from known structures, and provide single-molecule perspectives on the allosteric link between ligand- and nucleotide-binding pockets that shed new light on the G-protein activation mechanism. Single-molecule FRET imaging provides insights into the allosteric link between the ligand-binding and G-protein nucleotide-binding pockets of the β 2 adrenergic receptor (β 2 AR) and improved understanding of the G-protein activation mechanism. Monitoring G-protein activation by a GPCR G-protein-coupled receptor (GPCR)-mediated signal transduction is central to human physiology and disease, and understanding the molecular basis of ligand efficacy downstream of receptor activation is important for therapeutic development. For the GPCR β 2 adrenergic receptor (β 2 AR), receptor activation and coupling to the G protein G s involve outward movements of the receptor transmembrane helix 6 (TM6). Here, Scott Blanchard and colleagues apply single-molecule fluorescence resonance energy transfer (smFRET) imaging methods to directly monitor movements of TM6 in β 2 AR bound to a range of ligands with distinct efficacy profiles. They find that partial and full agonists affect TM6 motions in an efficacy-dependent manner. These motions differentially regulate the rate at which β 2 AR couples with GDP-bound G s and the efficiency of nucleotide exchange leading to G s activation. The work provides single-molecule insight into the allosteric link between the ligand- and G-protein-nucleotide-binding pockets of the receptor and improved understanding of the G-protein activation mechanism.

The autoproteolysis-inducing (GAIN) domain of class B2/adhesion G protein-coupled receptors (aGPCRs) is structurally conserved, and its self-cleavage is central to receptor mechanotransduction and signaling. Yet, the influence of factors beyond the protein fold on GAIN domain autoproteolysis remains unclear. Using ADGRE2/EMR2, a self-cleaved aGPCR, we investigated contributions of the seven-transmembrane (7TM) region to GAIN domain autoproteolysis during receptor maturation and trafficking. Retention Upon Selective Hook (RUSH) assays showed that self-cleavage acts as a checkpoint before endoplasmic reticulum (ER) exit, but not during plasma membrane transport. Stepwise truncations of the 7TM domain revealed that cleavage can occur before or at synthesis of the first transmembrane helix, and is enhanced with formation of the full 7TM domain. Analyses of six additional cleavage-competent aGPCRs demonstrated that ER membrane tethering facilitates GAIN domain processing, supported by proteomic evidence linking cleavage to proximity with the N -glycosylation pathway. These results highlight the interplay between GAIN and 7TM domains, offering mechanistic insights and guiding pharmacological strategies to modulate aGPCR activation and signaling. The GAIN domain of adhesion G protein-coupled receptors, a class of biological mechanosensors, was thought to be sufficient for its self-cleavage. Here, the authors show that the event also depends on the seven-transmembrane region of the receptors.

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.

The compositional heterogeneity of proteoliposome reconstitution can skew the results of ensemble-average measurements of transmembrane protein structure and function. These compositional heterogeneities can be exploited, however, with a single-proteoliposome, high-content screening method. Proteoliposome reconstitution is a standard method to stabilize purified transmembrane proteins in membranes for structural and functional assays. Here we quantified intrareconstitution heterogeneities in single proteoliposomes using fluorescence microscopy. Our results suggest that compositional heterogeneities can severely skew ensemble-average proteoliposome measurements but also enable ultraminiaturized high-content screens. We took advantage of this screening capability to map the oligomerization energy of the β 2 -adrenergic receptor using ∼10 9 -fold less protein than conventional assays.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

G proteins are activated when they associate with G protein-coupled receptors (GPCRs), often in response to agonist-mediated receptor activation. It is generally thought that agonist-induced receptor-G protein association necessarily promotes G protein activation and, conversely, that activated GPCRs do not interact with G proteins that they do not activate. Here we show that GPCRs can form agonist-dependent complexes with G proteins that they do not activate. Using cell-based bioluminescence resonance energy transfer (BRET) and luminescence assays we find that vasopressin V₂ receptors (V₂R) associate with both Gs and G12 heterotrimers when stimulated with the agonist arginine vasopressin (AVP). However, unlike V₂R-Gs complexes, V₂R-G12 complexes are not destabilized by guanine nucleotides and do not promote G12 activation. Activating V₂R does not lead to signaling responses downstream of G12 activation, but instead inhibits basal G12-mediated signaling, presumably by sequestering G12 heterotrimers. Overexpressing G12 inhibits G protein receptor kinase (GRK) and arrestin recruitment to V₂R and receptor internalization. Formyl peptide (FPR1 and FPR2) and Smoothened (Smo) receptors also form complexeswith G12 that are insensitive to nucleotides, suggesting that unproductive GPCR-G12 complexes are not unique to V₂R. These results indicate that agonist-dependent receptor-G protein association does not always lead to G protein activation and may in fact inhibit G protein activation.

G protein-coupled receptors (GPCRs) are targeted by a large fraction of approved drugs and regulate many important cellular processes. Association of GPCRs with heterotrimeric G proteins in response to agonist activation is thought to invariably lead to G protein activation. We find instead that G 12 heterotrimers can associate with agonist-bound receptors in a manner that does not lead to activation. These unproductive agonist–receptor-G protein ternary complexes sequester G 12 heterotrimers and thus inhibit rather than support G 12 signaling. These findings reveal a mechanism whereby agonist activation of GPCRs can inhibit as well as promote G protein signaling. G proteins are activated when they associate with G protein-coupled receptors (GPCRs), often in response to agonist-mediated receptor activation. It is generally thought that agonist-induced receptor-G protein association necessarily promotes G protein activation and, conversely, that activated GPCRs do not interact with G proteins that they do not activate. Here we show that GPCRs can form agonist-dependent complexes with G proteins that they do not activate. Using cell-based bioluminescence resonance energy transfer (BRET) and luminescence assays we find that vasopressin V 2 receptors (V 2 R) associate with both G s and G 12 heterotrimers when stimulated with the agonist arginine vasopressin (AVP). However, unlike V 2 R-G s complexes, V 2 R-G 12 complexes are not destabilized by guanine nucleotides and do not promote G 12 activation. Activating V 2 R does not lead to signaling responses downstream of G 12 activation, but instead inhibits basal G 12 -mediated signaling, presumably by sequestering G 12 heterotrimers. Overexpressing G 12 inhibits G protein receptor kinase (GRK) and arrestin recruitment to V 2 R and receptor internalization. Formyl peptide (FPR1 and FPR2) and Smoothened (Smo) receptors also form complexes with G 12 that are insensitive to nucleotides, suggesting that unproductive GPCR-G 12 complexes are not unique to V 2 R. These results indicate that agonist-dependent receptor-G protein association does not always lead to G protein activation and may in fact inhibit G protein activation.

A 'chemical biology of cellular membranes' must capture the way that mesoscale perturbations tune the biochemical properties of constituent lipid and protein molecules and vice versa. Whereas the classical paradigm focuses on chemical composition, dynamic modulation of the physical shape or curvature of a membrane is emerging as a complementary and synergistic modus operandi for regulating cellular membrane biology.