FRET-based monitoring of Adhesion class G protein-coupled receptor activity
G protein-coupled receptors (GPCR) of the Adhesion class are a large group of transmembrane receptor molecules that possesses molecular moieties from adhesion proteins and GPCR at equal shares. It is still unknown, which modality Adhesion-GPCR perceive and how they transduce a signal across the membrane. A prevailing hypothesis suggests that changes in distance between the juxtamembraneously positioned GPCR autoproteolysis-inducing (GAIN) domain of Adhesion-GPCR and their heptahelical transmembrane (7TM) unit through mechanical stimuli regulate metabotropic receptor activity (distance hypothesis). The GAIN-7TM domain tandem is harboured by all 33 known Adhesion-GPCR homologs with one exception and is thus suspected to play a paramount role in this novel mechanosensing principle.
Confirmation of the distance hypothesis requires (i) determination of the interaction between the GAIN and 7TM domains of Adhesion-GPCR, (ii) testing whether proximity between GAIN and 7TM domains quantitatively affects signal output of Adhesion-GPCR, and (iii) physiological receptor activity.
This proposal is motivated by the distance hypothesis and will use a combination of optical and physiological approaches to investigate Adhesion-GPCR function. First, selected Adhesion-GPCR (i.e. Latrophilins, CELSR, GPR56) will be adapted for FRET measurements of GAIN-7TM interactions to determine spatial interdomain changes over time when the receptors are challenged with cognate ligands, mechanical stimulation and a combination of both. Resulting changes in intramolecular receptor distances can subsequently be correlated with cellular readouts in vitro and in vivo using established assays for Drosophila melanogaster.
Second, Adhesion-GPCR are involved in a wide scope of human pathologies, where their disruption results in grave organ dysfunction. We will use Adhesion-GPCR FRET sensors to screen a panel of human mutations that map to the extracellular portions of the receptors (including GAIN and 7TM domains) and investigate their impact on distance changes between both domains. Third, we will assess whether relaxation of mechanical tension within the extracellular domain of Adhesion-GPCR results in changes of physiological activity in vivo. This will be tested by the introduction of spacer domains of different lengths between adhesion folds and the GAIN domain. For this set of experiments we will concentrate on Latrophilin/dCIRL, an Adhesion-GPCR in Drosophila, whose function in mechanosensation was recently determined by my group. The effect of stepwise tensile loading and relaxation of Latrophilin/dCIRL will be evaluated through in vivo assays of mechanical qualities.
Obtaining experimental evidence to substantiate the distance hypothesis will fundamentally contribute a mechanistic concept of Adhesion-GPCR signaling. This will lay the foundations to understand the common functional denominator among Adhesion-GPCR, which is still at large, and to manipulate Adhesion-GPCR function.
- University of Leipzig
Tobias Langenhan will coordinate and supervise the project together with other members of the team.
- University of Leipzig
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