GPCR-mediated ß-arrestin activation deconvoluted with single-molecule precision.

ß-arrestins bind G protein-coupled receptors to terminate G protein signaling and to facilitate other downstream signaling pathways. Using single-molecule fluorescence resonance energy transfer imaging, we show that ß-arrestin is strongly autoinhibited in its basal state. Its engagement with a phosphopeptide mimicking phosphorylated receptor tail efficiently releases the ß-arrestin tail from its N domain to assume distinct conformations. Unexpectedly, we find that ß-arrestin binding to phosphorylated receptor, with a phosphorylation barcode identical to the isolated phosphopeptide, is highly inefficient and that agonist-promoted receptor activation is required for ß-arrestin activation, consistent with the release of a sequestered receptor C tail. These findings, together with focused cellular investigations, reveal that agonism and receptor C-tail release are specific determinants of the rate and efficiency of ß-arrestin activation by phosphorylated receptor. We infer that receptor phosphorylation patterns, in combination with receptor agonism, synergistically establish the strength and specificity with which diverse, downstream ß-arrestin-mediated events are directed.

The role of G protein conformation in receptor-G protein selectivity.

G protein-coupled receptors (GPCRs) selectively activate at least one of the four families of heterotrimeric G proteins, but the mechanism of coupling selectivity remains unclear. Structural studies emphasize structural complementarity of GPCRs and nucleotide-free G proteins, but selectivity is likely to be determined by transient intermediate-state complexes that exist before nucleotide release. Here we study coupling to nucleotide-decoupled G protein variants that can adopt conformations similar to receptor-bound G proteins without releasing nucleotide, and are therefore able to bypass intermediate-state complexes. We find that selectivity is degraded when nucleotide release is not required for GPCR-G protein complex formation, to the extent that most GPCRs interact with most nucleotide-decoupled G proteins. These findings demonstrate the absence of absolute structural incompatibility between noncognate receptor-G protein pairs, and are consistent with the hypothesis that transient intermediate states are partly responsible for coupling selectivity.

An inactive receptor-G protein complex maintains the dynamic range of agonist-induced signaling.

Agonist binding promotes activation of G protein-coupled receptors (GPCRs) and association of active receptors with G protein heterotrimers. The resulting active-state ternary complex is the basis for conventional stimulus-response coupling. Although GPCRs can also associate with G proteins before agonist binding, the impact of such preassociated complexes on agonist-induced signaling is poorly understood. Here we show that preassociation of 5-HT7 serotonin receptors with Gs heterotrimers is necessary for agonist-induced signaling. 5-HT7 receptors in their inactive state associate with Gs, as these complexes are stabilized by inverse agonists and receptor mutations that favor the inactive state. Inactive-state 5-HT7-Gs complexes dissociate in response to agonists, allowing the formation of conventional agonist-5-HT7-Gs ternary complexes and subsequent Gs activation. Inactive-state 5-HT7-Gs complexes are required for the full dynamic range of agonist-induced signaling, as 5-HT7 receptors spontaneously activate Gs variants that cannot form inactive-state complexes. Therefore, agonist-induced signaling in this system involves two distinct receptor-G protein complexes, a conventional ternary complex that activates G proteins and an inverse-coupled binary complex that maintains the inactive state when agonist is not present.

Components of the Gs signaling cascade exhibit distinct changes in mobility and membrane domain localization upon ß2 -adrenergic receptor activation.

The G protein signaling cascade is a key player in cell signaling. Cascade activation leads to a redistribution of its members in various cellular compartments. These changes are likely related to the "second wave" of signaling from endosomes. Here, we set out to determine whether Gs signaling cascade members expressed at very low levels exhibit altered mobility and localize in clathrin-coated structures (CCSs) or caveolae upon activation by ß2 -adrenergic receptors (ß2 AR). Activated ß2 AR showed decreased mobility and sustained accumulation in CCSs but not in caveolae. Arrestin 3 translocated to the plasma membrane after ß2 AR activation and showed very low mobility and pronounced accumulation in CCSs. In contrast, Gαs and Gγ2 exhibited a modest reduction in mobility but no detectable accumulation in or exclusion from CCSs or caveolae. The effector adenylyl cyclase 5 (AC5) showed a slight mobility increase upon ß2 AR stimulation, no redistribution to CCSs, and weak activation-independent accumulation in caveolae. Our findings show an overall decrease in the mobility of most activated Gs signaling cascade members and confirm that ß2 AR and arrestin 3 accumulate in CCSs, while Gαs , Gγ2 and AC5 can transiently enter CCSs and caveolae but do not accumulate in and are not excluded from these domains.

Disruption of Fgf13 causes synaptic excitatory-inhibitory imbalance and genetic epilepsy and febrile seizures plus.

We identified a family in which a translocation between chromosomes X and 14 was associated with cognitive impairment and a complex genetic disorder termed "Genetic Epilepsy and Febrile Seizures Plus" (GEFS(+)). We demonstrate that the breakpoint on the X chromosome disrupted a gene that encodes an auxiliary protein of voltage-gated Na(+) channels, fibroblast growth factor 13 (Fgf13). Female mice in which one Fgf13 allele was deleted exhibited hyperthermia-induced seizures and epilepsy. Anatomic studies revealed expression of Fgf13 mRNA in both excitatory and inhibitory neurons of hippocampus. Electrophysiological recordings revealed decreased inhibitory and increased excitatory synaptic inputs in hippocampal neurons of Fgf13 mutants. We speculate that reduced expression of Fgf13 impairs excitability of inhibitory interneurons, resulting in enhanced excitability within local circuits of hippocampus and the clinical phenotype of epilepsy. These findings reveal a novel cause of this syndrome and underscore the powerful role of FGF13 in control of neuronal excitability.

Visualization of endogenous G proteins on endosomes and other organelles.

Classical G protein-coupled receptor (GPCR) signaling takes place in response to extracellular stimuli and involves receptors and heterotrimeric G proteins located at the plasma membrane. It has recently been established that GPCR signaling can also take place from intracellular membrane compartments, including endosomes that contain internalized receptors and ligands. While the mechanisms of GPCR endocytosis are well understood, it is not clear how internalized receptors are supplied with G proteins. To address this gap we use gene editing, confocal microscopy, and bioluminescence resonance energy transfer to study the distribution and trafficking of endogenous G proteins. We show here that constitutive endocytosis is sufficient to supply newly internalized endocytic vesicles with 20-30% of the G protein density found at the plasma membrane. We find that G proteins are present on early, late, and recycling endosomes, are abundant on lysosomes, but are virtually undetectable on the endoplasmic reticulum, mitochondria, and the medial Golgi apparatus. Receptor activation does not change heterotrimer abundance on endosomes. Our results provide a detailed subcellular map of endogenous G protein distribution, suggest that G proteins may be partially excluded from nascent endocytic vesicles, and are likely to have implications for GPCR signaling from endosomes and other intracellular compartments.

Constitutive G protein coupling profiles of understudied orphan GPCRs.

A large number of GPCRs are potentially valuable drug targets but remain understudied. Many of these lack well-validated activating ligands and are considered "orphan" receptors, and G protein coupling profiles have not been defined for many orphan GPCRs. Here we asked if constitutive receptor activity can be used to determine G protein coupling profiles of orphan GPCRs. We monitored nucleotide-sensitive interactions between 48 understudied orphan GPCRs and five G proteins (240 combinations) using bioluminescence resonance energy transfer (BRET). No receptor ligands were used, but GDP was used as a common G protein ligand to disrupt receptor-G protein complexes. Constitutive BRET between the same receptors and ß-arrestins was also measured. We found sufficient GDP-sensitive BRET to generate G protein coupling profiles for 22 of the 48 receptors we studied. Altogether we identified 48 coupled receptor-G protein pairs, many of which have not been described previously. We conclude that receptor-G protein complexes that form spontaneously in the absence of guanine nucleotides can be used to profile G protein coupling of constitutively-active GPCRs. This approach may prove useful for studying G protein coupling of other GPCRs for which activating ligands are not available.

Structure of formylpeptide receptor 2-Gi complex reveals insights into ligand recognition and signaling.

Formylpeptide receptors (FPRs) as G protein-coupled receptors (GPCRs) can recognize formylpeptides derived from pathogens or host cells to function in host defense and cell clearance. In addition, FPRs, especially FPR2, can also recognize other ligands with a large chemical diversity generated at different stages of inflammation to either promote or resolve inflammation in order to maintain a balanced inflammatory response. The mechanism underlying promiscuous ligand recognition and activation of FPRs is not clear. Here we report a cryo-EM structure of FPR2-Gi signaling complex with a peptide agonist. The structure reveals a widely open extracellular region with an amphiphilic environment for ligand binding. Together with computational docking and simulation, the structure suggests a molecular basis for the recognition of formylpeptides and a potential mechanism of receptor activation, and reveals conserved and divergent features in Gi coupling. Our results provide a basis for understanding the molecular mechanism of the functional promiscuity of FPRs.

Common activation mechanism of class A GPCRs.

Class A G-protein-coupled receptors (GPCRs) influence virtually every aspect of human physiology. Understanding receptor activation mechanism is critical for discovering novel therapeutics since about one-third of all marketed drugs target members of this family. GPCR activation is an allosteric process that couples agonist binding to G-protein recruitment, with the hallmark outward movement of transmembrane helix 6 (TM6). However, what leads to TM6 movement and the key residue level changes of this movement remain less well understood. Here, we report a framework to quantify conformational changes. By analyzing the conformational changes in 234 structures from 45 class A GPCRs, we discovered a common GPCR activation pathway comprising of 34 residue pairs and 35 residues. The pathway unifies previous findings into a common activation mechanism and strings together the scattered key motifs such as CWxP, DRY, Na+ pocket, NPxxY and PIF, thereby directly linking the bottom of ligand-binding pocket with G-protein coupling region. Site-directed mutagenesis experiments support this proposition and reveal that rational mutations of residues in this pathway can be used to obtain receptors that are constitutively active or inactive. The common activation pathway provides the mechanistic interpretation of constitutively activating, inactivating and disease mutations. As a module responsible for activation, the common pathway allows for decoupling of the evolution of the ligand binding site and G-protein-binding region. Such an architecture might have facilitated GPCRs to emerge as a highly successful family of proteins for signal transduction in nature.

A Peptide Uncoupling BDNF Receptor TrkB from Phospholipase Cγ1 Prevents Epilepsy Induced by Status Epilepticus.

The BDNF receptor tyrosine kinase, TrkB, underlies nervous system function in both health and disease. Excessive activation of TrkB caused by status epilepticus promotes development of temporal lobe epilepsy (TLE), revealing TrkB as a therapeutic target for prevention of TLE. To circumvent undesirable consequences of global inhibition of TrkB signaling, we implemented a novel strategy aimed at selective inhibition of the TrkB-activated signaling pathway responsible for TLE. Our studies of a mouse model reveal that phospholipase Cγ1 (PLCγ1) is the dominant signaling effector by which excessive activation of TrkB promotes epilepsy. We designed a novel peptide (pY816) that uncouples TrkB from PLCγ1. Treatment with pY816 following status epilepticus inhibited TLE and prevented anxiety-like disorder yet preserved neuroprotective effects of endogenous TrkB signaling. We provide proof-of-concept evidence for a novel strategy targeting receptor tyrosine signaling and identify a therapeutic with promise for prevention of TLE caused by status epilepticus in humans.