Explore the vast range of titles available.

G protein-independent, arrestin-dependent signaling is a paradigm that broadens the signaling scope of G protein-coupled receptors (GPCRs) beyond G proteins for numerous biological processes. However, arrestin signaling in the collective absence of functional G proteins has never been demonstrated. Here we achieve a state of “zero functional G” at the cellular level using HEK293 cells depleted by CRISPR/Cas9 technology of the Gs/q/12 families of Gα proteins, along with pertussis toxin-mediated inactivation of Gi/o. Together with HEK293 cells lacking β-arrestins (“zero arrestin”), we systematically dissect G protein- from arrestin-driven signaling outcomes for a broad set of GPCRs. We use biochemical, biophysical, label-free whole-cell biosensing and ERK phosphorylation to identify four salient features for all receptors at “zero functional G”: arrestin recruitment and internalization, but—unexpectedly—complete failure to activate ERK and whole-cell responses. These findings change our understanding of how GPCRs function and in particular of how they activate ERK1/2. Arrestins terminate signaling from GPCRs, but several lines of evidence suggest that they are also able to transduce signals independently of G proteins. Here, the authors systematically ablate G proteins in cell lines, and show that arrestins are unable to act as genuine signal initiators.

G protein-gated inwardly rectifying potassium channel (GIRK) plays a key role in regulating neurotransmission. GIRK is opened by the direct binding of the G protein βγ subunit (Gβγ), which is released from the heterotrimeric G protein (Gαβγ) upon the activation of G protein-coupled receptors (GPCRs). GIRK contributes to precise cellular responses by specifically and efficiently responding to the Gi/o-coupled GPCRs. However, the detailed mechanisms underlying this family-specific and efficient activation are largely unknown. Here, we investigate the structural mechanism underlying the Gi/o family-specific activation of GIRK, by combining cell-based BRET experiments and NMR analyses in a reconstituted membrane environment. We show that the interaction formed by the αA helix of Gαi/o mediates the formation of the Gαi/oβγ-GIRK complex, which is responsible for the family-specific activation of GIRK. We also present a model structure of the Gαi/oβγ-GIRK complex, which provides the molecular basis underlying the specific and efficient regulation of GIRK. Opening of G protein-gated inwardly rectifying potassium channels (GIRK) is coupled to the activation of a GPCR. Here the authors use NMR and cell-based BRET assays to gain insights into the mechanisms underlying family-specific activation and find that pre-formation of the Gαi/oβγ-GIRK complex in the inactive state is responsible for specific GIRK activation and present a structural model for the Gαi/oβγ-GIRK complex.

G-protein-coupled receptors (GPCRs) are the largest superfamily of transmembrane proteins and the targets of over 30% of currently marketed pharmaceuticals. Although several structures have been solved for GPCR–G protein complexes, few are in a lipid membrane environment. Here, we report cryo-EM structures of complexes of neurotensin, neurotensin receptor 1 and Gα i1 β 1 γ 1 in two conformational states, resolved to resolutions of 4.1 and 4.2 Å. The structures, determined in a lipid bilayer without any stabilizing antibodies or nanobodies, reveal an extended network of protein–protein interactions at the GPCR–G protein interface as compared to structures obtained in detergent micelles. The findings show that the lipid membrane modulates the structure and dynamics of complex formation and provide a molecular explanation for the stronger interaction between GPCRs and G proteins in lipid bilayers. We propose an allosteric mechanism for GDP release, providing new insights into the activation of G proteins for downstream signaling. Structures of GPCR neurotensin receptor 1 (NTSR1) in complex with neurotensin and Gα i1 β 1 γ 1 in a lipid bilayer environment and without stabilizing antibodies reveal extensive interactions at the GPCR–G protein interface.

Smoothened (SMO), a class Frizzled G protein-coupled receptor (class F GPCR), transduces the Hedgehog signal across the cell membrane. Sterols can bind to its extracellular cysteine-rich domain (CRD) and to several sites in the seven transmembrane helices (7-TMs) of SMO. However, the mechanism by which sterols regulate SMO via multiple sites is unknown. Here we determined the structures of SMO–G i complexes bound to the synthetic SMO agonist (SAG) and to 24( S ),25-epoxycholesterol (24( S ),25-EC). A novel sterol-binding site in the extracellular extension of TM6 was revealed to connect other sites in 7-TMs and CRD, forming an intramolecular sterol channel from the middle side of 7-TMs to CRD. Additional structures of two gain-of-function variants, SMO D384R and SMO G111C/I496C , showed that blocking the channel at its midpoints allows sterols to occupy the binding sites in 7-TMs, thereby activating SMO. These data indicate that sterol transport through the core of SMO is a major regulator of SMO-mediated signaling. Cryo-EM structural work shows sterols binding at four adjacent locations within the class F GPCR Smoothened (SMO), where the transmembrane core functions as a sterol tunnel in which occupancy activates SMO for downstream Hedgehog signaling.

Heterotrimeric G proteinshave been previously linked to plant defense; however a role for the G[beta][gamma] dimer in defense signaling has not been described to date. Using available Arabidopsis (Arabidopsis thaliana) mutants lacking functional G[alpha] or G[beta] subunits, we show that defense against the necrotrophic pathogens Alternaria brassicicola and Fusarium oxysporum is impaired in G[beta]-deficient mutants while G[alpha]-deficient mutants show slightly increased resistance compared to wild-type Columbia ecotype plants. In contrast, responses to virulent (DC3000) and avirulent (JL1065) strains of Pseudomonas syringae appear to be independent of heterotrimeric G proteins. The induction of a number of defense-related genes in G[beta]-deficient mutants were severely reduced in response to A. brassicicola infection. In addition, G[beta]-deficient mutants exhibit decreased sensitivity to a number of methyl jasmonate-induced responses such as induction of the plant defensin gene PDF1.2, inhibition of root elongation, seed germination, and growth of plants in sublethal concentrations of methyl jasmonate. In all cases, the behavior of the G[alpha]-deficient mutants is coherent with the classic heterotrimeric mechanism of action, indicating that jasmonic acid signaling is influenced by the G[beta][gamma] functional subunit but not by G[alpha]. We hypothesize that G[beta][gamma] acts as a direct or indirect enhancer of the jasmonate signaling pathway in plants.

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 (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.

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.

G protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by mediating a GDP to GTP exchange in the Gα subunit. This leads to dissociation of the heterotrimer into Gα-GTP and Gβγ dimer. The Gα-GTP and Gβγ dimer each regulate a variety of downstream pathways to control various aspects of human physiology. Dysregulated Gβγ-signaling is a central element of various neurological and cancer-related anomalies. However, Gβγ also serves as a negative regulator of Gα that is essential for G protein inactivation, and thus has the potential for numerous side effects when targeted therapeutically. Here we report a llama-derived nanobody (Nb5) that binds tightly to the Gβγ dimer. Nb5 responds to all combinations of β-subtypes and γ-subtypes and competes with other Gβγ-regulatory proteins for a common binding site on the Gβγ dimer. Despite its inhibitory effect on Gβγ-mediated signaling, Nb5 has no effect on Gα q -mediated and Gα s -mediated signaling events in living cells. G protein-coupled receptors (GPCRs) activate and dissociate the G protein heterotrimer into Gα-GTP and Gβγ dimer, which facilitate distinct signalling events. Here authors develop a nanobody, Nb5 that modulates Gβγ-mediated signaling without affecting GTP-bound Gαq and Gαs-mediated signaling events.

Control of organ size and shape by cell proliferation and cell expansion is a fundamental developmental process, but the mechanisms that set the size and shape of determinate organs are largely unknown in plants. Molecular, genetic, cytological and biochemical approaches were used to characterize the roles of the Arabidopsis thaliana G protein γ subunit (AGG3) gene in organ growth. Here, we describe A. thaliana AGG3, which promotes petal growth by increasing the period of cell proliferation. Both the N-terminal region and the C-terminal domains of AGG3 are necessary for the function of AGG3. By contrast, analysis of a series of AGG3 derivatives with deletions in specific domains showed that the deletion of any of these domains cannot completely abolish the function of AGG3. The GFP-AGG3 fusion protein is localized to the plasma membrane. The predicted transmembrane domain plays an important role in the plasma membrane localization of AGG3. Genetic analyses revealed that AGG3 action requires a functional G protein α subunit (GPA1) and G protein β subunit (AGB1). Our findings demonstrate that AGG3, GPA1 and AGB1 act in the same genetic pathway to influence organ size and shape in A. thaliana.