g-protein-selective-gpcr-conformations-measured-using-fret-sensors

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

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group ...

Localized activation of Rho GTPases is essential for multiple cellular functions, including cytokinesis and formation and maintenance of cell-cell junctions. Although MgcRacGAP (Mgc) is required for spatially confined RhoA-GTP at the equatorial cortex of dividing cells, both the target specificity of Mgc's GAP activity and the involvement of phosphorylation of Mgc at Ser-386 are controversial. In addition, Mgc's function at cell-cell junctions remains unclear. Here, using gastrula-stage Xenopus laevis embryos as a model system, we examine Mgc's role in regulating localized RhoA-GTP and Rac1-GTP in the intact vertebrate epithelium. We show that Mgc's GAP activity spatially restricts accumulation of both RhoA-GTP and Rac1-GTP in epithelial cells--RhoA at the cleavage furrow and RhoA and Rac1 at cell-cell junctions. Phosphorylation at Ser-386 does not switch the specificity of Mgc's GAP activity and is not required for successful cytokinesis. Furthermore, Mgc regulates adherens junction but not tight junction structure, and the ability to regulate adherens junctions is dependent on GAP activity and signaling via the RhoA pathway. Together these results indicate that Mgc's GAP activity down-regulates the active populations of RhoA and Rac1 at localized regions of epithelial cells and is necessary for successful cytokinesis and cell-cell junction structure.

MgcRacGAP restricts active RhoA at the cytokinetic furrow and both RhoA and Rac1 at cell-cell junctions in epithelial cells.

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. We find that cognate and non-cognate Gα peptides show deep and shallow binding, respectively, and in distinct orientations within the GPCR. Binding of the cognate Gα peptide stabilizes the agonist-bound GPCR conformational ensemble resulting in favorable binding energy and lower flexibility of the agonist-GPCR pair. We identify three hot spot residues (Gαs/Gαq-Gln-384/Leu-349, Gln-390/Glu-355, and Glu-392/Asn-357) that contribute to selective interactions between the β2-adrenergic receptor (β2-AR)-Gαs and V1A receptor (V1AR)-Gαq The Gαs and Gαq peptides adopt different orientations in β2-AR and V1AR, respectively. The β2-AR/Gαs peptide interface is dominated by electrostatic interactions, whereas the V1AR/Gαq peptide interactions are predominantly hydrophobic. Interestingly, our study reveals a role for both favorable and unfavorable interactions in G protein selection. Residue Glu-355 in Gαq prevents this peptide from interacting strongly with β2-AR. Mutagenesis to the Gαs counterpart (E355Q) imparts a cognate-like interaction. Overall, our study highlights the synergy in molecular dynamics and FRET-based approaches to dissect the structural basis of selective G protein interactions.

Article	Year
MgcRacGAP restricts active RhoA at the cytokinetic furrow and both RhoA and Rac1 at cell-cell junctions in epithelial cells. Molecular biology of the cell\| PubMed ID: 25947135	2015
Structural Elements in the Gαs and Gαq C Termini That Mediate Selective G Protein-coupled Receptor (GPCR) Signaling. The Journal of biological chemistry\| PubMed ID: 27330078	2016

Ansley Semack

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