visualizing-cellular-gibberellin-levels-using-nlsgps1-frster

Visualizing Cellular Gibberellin Levels Using the nlsGPS1 F&#246;rster Resonance Energy Transfer (FRET) Biosensor

The phytohormone gibberellin (GA) is a small, mobile signaling molecule that plays a key role in seed germination, cellular elongation, and developmental ...

University of Cambridge

Glycine receptors (GlyRs) are Cys-loop receptors that mediate fast synaptic inhibition in the brain stem and spinal cord. They are involved in the generation of motor rhythm, reflex circuit coordination, and sensory signal processing and therefore represent targets for therapeutic interventions. The extracellular domains (ECDs) of Cys-loop receptors typically contain many aromatic amino acids, but only those in the receptor binding pocket have been extensively studied. Here, we show that many Phe residues in the ECD that are not located in the binding pocket are also involved in GlyR function. We examined these Phe residues by creating several GlyR variants, characterizing these variants with the two-electrode voltage clamp technique in  oocytes, and interpreting changes in receptor parameters by using currently available structural information on the open and closed states of the GlyR. Substitution of six of the eight Phe residues in the ECD with Ala resulted in loss of function or significantly increased the EC and also altered the maximal response to the partial GlyR agonist taurine compared with glycine in those receptor variants that were functional. Substitutions with other amino acids, combined with examination of nearby residues that could potentially interact with these Phe residues, suggested interactions that could be important for GlyR function, and possibly similar interactions could contribute to the function of other members of the Cys-loop receptor family. Overall, our results suggest that many ECD regions are important for GlyR function and that these regions could inform the design of therapeutic agents targeting GlyR activity.

Multiple regions in the extracellular domain of the glycine receptor determine receptor activity.

The extracellular domains (ECDs) of Cys-loop receptors contain many aromatic amino acids, but only relatively few have been well studied. Here we explore the roles of Tyr and Trp residues in the ECD of the glycine receptor and show that four such residues that have not been previously studied (Y24, Y58, W170, and Y197) contribute significantly to the function of the protein. The residues were studied by creating mutant receptors, characterizing them using two-electrode voltage clamp in Xenopus oocytes, and interpreting changes in receptor parameters using structural information about the open and closed states of the receptor. Alanine substitution of all these residues ablates function or increases the glycine EC. There are also a number of changes in the relative maximal responses to taurine, a partial agonist, compared to glycine. Further mutations, in combination with structural information, suggest Y24 contributes to an anion-π interaction with a binding loop D residue, Y58 to an S-π interaction stabilizing the Cys loop, W170 to hydrophobic interactions stabilizing the hydrophobic interior of the subunit, and Y197 to a hydrogen bond linking binding loops B and C. These interactions appear to be broadly conserved in other Cys-loop receptors. Thus, we have identified new regions of the glycine receptor that are important contributors to receptor activation and are likely also to contribute to function in other members of this important protein family.

Identification of Novel Functionally Important Aromatic Residue Interactions in the Extracellular Domain of the Glycine Receptor.

Cys-loop receptors play important roles in fast neuronal signal transmission. Functional receptors are pentamers, with each subunit having an extracellular, transmembrane (TM) and intracellular domain. Each TM domain contains 4 α-helices (M1-M4) joined by loops of varying lengths. Many of the amino acid residues that constitute these α-helices are hydrophobic, and there has been particular interest in aromatic residues, especially those in M4, which have the potential to contribute to the assembly and function of the receptor via a range of interactions with nearby residues.

Article	Year
Multiple regions in the extracellular domain of the glycine receptor determine receptor activity. The Journal of biological chemistry\| PubMed ID: 29941455	2018
Identification of Novel Functionally Important Aromatic Residue Interactions in the Extracellular Domain of the Glycine Receptor. Biochemistry\| PubMed ID: 29947514	2018
The roles of aromatic residues in the glycine receptor transmembrane domain. BMC neuroscience\| PubMed ID: 30189850	2018

Bijun Tang

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