Authors

Volker Jelinek, Nadja Mösslein, and Moritz Bünemann

Affiliations

Institute of Pharmacology and Clinical Pharmacy, Philipps-University Marburg, Marburg, Germany

Topic

Molecular Biology

Abstract

G protein-coupled receptors (GPCRs) selectively couple to specific heterotrimeric G proteins comprised of four subfamilies in order to induce appropriate physiological responses. However, structural determinants in Gα subunits responsible for selective recognition by approximately 800 human GPCRs have remained elusive. Here, we directly compare the influence of subtype-specific Gα structures on the stability of GPCR-G protein complexes and the activation by two Gq-coupled receptors. We used FRET-assays designed to distinguish multiple Go and Gq-based Gα chimeras in their ability to be selectively bound and activated by muscarinic M3 and histaminic H1 receptors. We identify the N-terminus including the αN/β1-hinge, the β2/β3-loop and the α5 helix of Gα to be key selectivity determinants which differ in their impact on selective binding to GPCRs and subsequent activation depending on the specific receptor. Altogether, these findings provide new insights into the molecular basis of G protein-coupling selectivity even beyond the Gα C-terminus.

DOI: https://dx.doi.org/10.1038%2Fs42003-021-02143-9

Extract

FRET measurements were carried out at room temperature using an inverted microscope (Axiovert 100; Zeiss), equipped with a ×60 oil-immersion objective (PlanApo N ×60/1.45 Oil; Nikon), LED light sources with excitation intensities set to 4% for 440 nm and 10% for 500 nm (pE-100; CoolLED) and a high-performance CCD-camera (Spot Pursuit from Spot Imaging/Diagnostic Instruments).

Product Associated Features

The pE-100 series is a family of single-wavelength fluorescence LED Illumination Systems, and in this case two systems at 440 nm and 500 nm provides highly controllable and precise excitation for CFP and YFP FRET

Product Type

pE-100

Journal

Commun. Biol.

Year of Publication

2021

Country of Publication

Germany