Dataset Information

Tethered ligands reveal glutamate receptor desensitization depends on subunit occupancy.

ABSTRACT: Cell signaling is often mediated by the binding of multiple ligands to multisubunit receptors. The probabilistic nature and sometimes slow rate of binding encountered with diffusible ligands can impede attempts to determine how the ligand occupancy controls signaling in such protein complexes. We describe a solution to this problem that uses a photoswitched tethered ligand as a 'ligand clamp' to induce rapid and stable binding and unbinding at defined subsets of subunits. We applied the approach to study gating in ionotropic glutamate receptors (iGluRs), ligand-gated ion channels that mediate excitatory neurotransmission and plasticity at glutamatergic synapses in the brain. We probed gating in two kainate-type iGluRs, GluK2 homotetramers and GluK2-GluK5 heterotetramers. Ultrafast (submillisecond) photoswitching of an azobenzene-based ligand on specific subunits provided a real-time measure of gating and revealed that partially occupied receptors can activate without desensitizing. The findings have implications for signaling by locally released and spillover glutamate.

SUBMITTER: Reiner A

PROVIDER: S-EPMC4041372 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Tethered ligands reveal glutamate receptor desensitization depends on subunit occupancy.

Reiner Andreas A Isacoff Ehud Y EY

Nature chemical biology 20140223 4

Cell signaling is often mediated by the binding of multiple ligands to multisubunit receptors. The probabilistic nature and sometimes slow rate of binding encountered with diffusible ligands can impede attempts to determine how the ligand occupancy controls signaling in such protein complexes. We describe a solution to this problem that uses a photoswitched tethered ligand as a 'ligand clamp' to induce rapid and stable binding and unbinding at defined subsets of subunits. We applied the approach ...[more]

PMID: 24561661

Similar Datasets

Project description:MAS is a G protein-coupled receptor (GPCR) implicated in multiple physiological processes. Several physiological peptide ligands such as angiotensin-(1-7), angiotensin fragments and neuropeptide FF (NPFF) are reported to act on MAS. Studies of conventional G protein signaling and receptor desensitization upon stimulation of MAS with the peptide ligands are limited so far. Therefore, we systematically analyzed G protein signals activated by the peptide ligands. MAS-selective non-peptide ligands that were previously shown to activate G proteins were used as controls for comparison on a common cell based assay platform. Activation of MAS by the non-peptide agonist (1) increased intracellular calcium and D-myo-inositol-1-phosphate (IP1) levels which are indicative of the activation of classical G?q-phospholipase C signaling pathways, (2) decreased G?i mediated cAMP levels and (3) stimulated G?12-dependent expression of luciferase reporter. In all these assays, MAS exhibited strong constitutive activity that was inhibited by the non-peptide inverse agonist. Further, in the calcium response assay, MAS was resistant to stimulation by a second dose of the non-peptide agonist after the first activation has waned suggesting functional desensitization. In contrast, activation of MAS by the peptide ligand NPFF initiated a rapid rise in intracellular calcium with very weak IP1 accumulation which is unlike classical G?q-phospholipase C signaling pathway. NPFF only weakly stimulated MAS-mediated activation of G?12 and G?i signaling pathways. Furthermore, unlike non-peptide agonist-activated MAS, NPFF-activated MAS could be readily re-stimulated the second time by the agonists. Functional assays with key ligand binding MAS mutants suggest that NPFF and non-peptide ligands bind to overlapping regions. Angiotensin-(1-7) and other angiotensin fragments weakly potentiated an NPFF-like calcium response at non-physiological concentrations (?100 µM). Overall, our data suggest that peptide ligands induce atypical signaling and functional desensitization of MAS.

Dataset Information

Tethered ligands reveal glutamate receptor desensitization depends on subunit occupancy.

Publications

Tethered ligands reveal glutamate receptor desensitization depends on subunit occupancy.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets