Allosteric modulation of GPCR conformational ensembles and signaling selectivity by the cellular environment – DYNALAND

G protein coupled receptors (GPCRs) are the largest family of membrane receptors for hormones and neurotransmitters. As such, they are also major targets for new therapeutics for a broad spectrum of indications including cancer, inflammatory, cardiovascular, respiratory and gastrointestinal diseases. Upon ligand binding, GPCRs trigger diverse signals through the selective coupling to multiple intracellular partners (G proteins, GRKs, arrestins). This functional versatility is further modulated by the receptor cellular environment, in particular the membrane lipids and accessory proteins that both act as endogenous allosteric modulators of signaling. However, the molecular mechanisms underlying this allosteric control are still obscure. The current hypothesis states that ?GPCRs sample? ?a? ?continuum? ?of? ?conformations?, each? tuned ?to? ?variable? ?levels toward the different signaling proteins. The equilibria between these states, and therefore their relative population, would be shifted by the different components in the receptor environment, resulting in a modulation of signaling and, ultimately, of the biological response. In this context, the goal of this proposal is to get an experimental demonstration of this paradigm by deciphering how the membrane composition and accessory proteins impact on the conformational and signaling landscape of a typical GPCR, the ghrelin receptor GHSR. In addition to being a crucial pharmacological target, this receptor is particularly appropriate for analyzing signaling selectivity and its allosteric control. Indeed, GHSR is a pleiotropic receptor that triggers multiple pathways, which in turn regulate many physiological functions (food intake, lipid homeostasis, reward, insulin secretion…). Moreover, allosteric regulation by the lipids in the membrane and an accessory protein, MRAP2, is central to GHSR signaling. In this context, our proposal gathers experts in medicinal chemistry, molecular and cellular pharmacology, membrane protein biochemistry, biophysics and computation to first describe the distribution of states in the GHSR conformational ensemble and the corresponding pharmacological profiles (Task 1). We will then, on this basis, explore how these conformational and functional landscapes are allosterically modulated by the lipids in the membrane (Task 2) and the accessory protein MRAP2 (Task 3). Each of these tasks will rely on the tools we already developed - isolated receptors in lipid nanodiscs, recombinant cell lines - and on a common transdisciplinary approach systematically associating computation (coarse-grained and all atom simulations), biophysics (single molecule fluorescence, solution-state NMR, hydrogen-deuterium exchange mass spectrometry) and cellular pharmacology. Our expertise, the availability of innovative tools, the parallel incorporation of robust still cutting-edge computational, biophysical and cellular methods, together with strong preliminary data, makes this proposal fully feasible. Altogether, DYNALAND should provide a detailed description of the molecular bases underlying GPCR functional selectivity and its allosteric control by the cellular environment as well as it should open new avenues for the design of compounds with fewer adverse side effects because of an increased selectivity toward specific signaling pathways.?????????????????????????????????????????????????????????????????????? In addition, our project will also yield a toolbox of powerful state-of-the-art technologies to be applied to other receptor systems as well.