Allosteric modulation of adenosine A1 and cannabinoid 1 receptor signaling by G-peptides.

While allosteric modulation of GPCR signaling has gained prominence to address the need for receptor specificity, efforts have mainly focused on allosteric sites adjacent to the orthosteric ligand-binding pocket and lipophilic molecules that target transmembrane helices. In this study, we examined the allosteric influence of native peptides derived from the C-terminus of the Gα subunit (G-peptides) on signaling from two Gi-coupled receptors, adenosine A1 receptor (A R) and cannabinoid receptor 1 (CB ). We expressed A R and CB fusions with G-peptides derived from Gαs, Gαi, and Gαq in HEK 293 cells using systematic protein affinity strength modulation (SPASM) and monitored the impact on downstream signaling in the cell compared to a construct lacking G-peptides.

Minute-scale persistence of a GPCR conformation state triggered by non-cognate G protein interactions primes signaling.

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.

ER/K linked GPCR-G protein fusions systematically modulate second messenger response in cells.

FRET and BRET approaches are well established for detecting ligand induced GPCR-G protein interactions in cells. Currently, FRET/BRET assays rely on co-expression of GPCR and G protein, and hence depend on the stoichiometry and expression levels of the donor and acceptor probes. On the other hand, GPCR-G protein fusions have been used extensively to understand the selectivity of GPCR signaling pathways.

The DRY motif and the four corners of the cubic ternary complex model.

Recent structural data on GPCRs using a variety of spectroscopic approaches suggest that GPCRs adopt a dynamic conformational landscape, with ligands stabilizing subsets of these states to activate one or more downstream signaling effectors. A key outstanding question posed by this emerging dynamic structural model of GPCRs is what states, active, inactive, or intermediate are captured by the numerous crystal structures of GPCRs complexed with a variety of agonists, partial agonists, and antagonists. In the early nineties the discovery of inverse agonists and constitutive activity led to the idea that the active receptor state (R) is an intrinsic property of the receptor itself rather than of the RG complex, eventually leading to the formulation of the cubic ternary complex model (CTC).

Priming GPCR signaling through the synergistic effect of two G proteins.

Although individual G-protein-coupled receptors (GPCRs) are known to activate one or more G proteins, the GPCR-G-protein interaction is viewed as a bimolecular event involving the formation of a ternary ligand-GPCR-G-protein complex. Here, we present evidence that individual GPCR-G-protein interactions can reinforce each other to enhance signaling through canonical downstream second messengers, a phenomenon we term "GPCR priming." Specifically, we find that the presence of noncognate Gq protein enhances cAMP stimulated by two Gs-coupled receptors, β2-adrenergic receptor (β2-AR) and D dopamine receptor (D-R).

G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay.

Fӧrster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group developed an intra-molecular FRET sensor to detect the interaction between Gα subunits and GPCRs in live cells following agonist stimulation. Here, we detail the protocol for detecting changes in FRET between the β2-adrenergic receptor and the Gαs C-terminus peptide upon treatment with 100 µM isoproterenol hydrochloride as previously characterized(1).

Structural Elements in the Gαs and Gαq C Termini That Mediate Selective G Protein-coupled Receptor (GPCR) Signaling.

Although the importance of the C terminus of the α subunit of the heterotrimeric G protein in G protein-coupled receptor (GPCR)-G protein pairing is well established, the structural basis of selective interactions remains unknown. Here, we combine live cell FRET-based measurements and molecular dynamics simulations of the interaction between the GPCR and a peptide derived from the C terminus of the Gα subunit (Gα peptide) to dissect the molecular mechanisms of G protein selectivity. We observe a direct link between Gα peptide binding and stabilization of the GPCR conformational ensemble.

Dynamic Coupling and Allosteric Networks in the α Subunit of Heterotrimeric G Proteins.

G protein α subunits cycle between active and inactive conformations to regulate a multitude of intracellular signaling cascades. Important structural transitions occurring during this cycle have been characterized from extensive crystallographic studies. However, the link between observed conformations and the allosteric regulation of binding events at distal sites critical for signaling through G proteins remain unclear.

Detection of G protein-selective G protein-coupled receptor (GPCR) conformations in live cells.

Although several recent studies have reported that GPCRs adopt multiple conformations, it remains unclear how subtle conformational changes are translated into divergent downstream responses. In this study, we report on a novel class of FRET-based sensors that can detect the ligand/mutagenic stabilization of GPCR conformations that promote interactions with G proteins in live cells. These sensors rely on the well characterized interaction between a GPCR and the C terminus of a Gα subunit.

Pheromone sensing regulates Caenorhabditis elegans lifespan and stress resistance via the deacetylase SIR-2.1.

Lifespan in Caenorhabditis elegans, Drosophila, and mice is regulated by conserved signaling networks, including the insulin/insulin-like growth factor 1 (IGF-1) signaling cascade and pathways depending on sirtuins, a family of NAD(+)-dependent deacetylases. Small molecules such as resveratrol are of great interest because they increase lifespan in many species in a sirtuin-dependent manner. However, no endogenous small molecules that regulate lifespan via sirtuins have been identified, and the mechanisms underlying sirtuin-dependent longevity are not well understood.

A shortcut to identifying small molecule signals that regulate behavior and development in Caenorhabditis elegans.

Small molecule metabolites play important roles in Caenorhabditis elegans biology, but effective approaches for identifying their chemical structures are lacking. Recent studies revealed that a family of glycosides, the ascarosides, differentially regulate C. elegans development and behavior.

NMR-spectroscopic screening of spider venom reveals sulfated nucleosides as major components for the brown recluse and related species.

Extensive chemical analyses of spider venoms from many species have revealed complex mixtures of biologically active compounds, of which several have provided important leads for drug development. We have recently shown that NMR spectroscopy can be used advantageously for a direct structural characterization of the small-molecule content of such complex mixtures. Here, we report the application of this strategy to a larger-scale analysis of a collection of spider venoms representing >70 species, which, in combination with mass spectrometric analyses, allowed the identification of a wide range of known, and several previously undescribed, small molecules.

A blend of small molecules regulates both mating and development in Caenorhabditis elegans.

In many organisms, population-density sensing and sexual attraction rely on small-molecule-based signalling systems. In the nematode Caenorhabditis elegans, population density is monitored through specific glycosides of the dideoxysugar ascarylose (the 'ascarosides') that promote entry into an alternative larval stage, the non-feeding and highly persistent dauer stage. In addition, adult C.