Allostery and conformational dynamics in GPCR signaling – allosig

The majority of hormones and neurotransmitters communicate information to cells via G protein-coupled receptors (GPCRs). The large number of biological functions they control also makes these membrane receptors one of the most prominent families of pharmacological targets in biomedicine. GPCRs exhibit complex signaling behaviors. Indeed, a single receptor can activate more than one G protein subtype as well as G protein-independent pathways. As a consequence, a given drug can possess distinct intrinsic efficacies towards these different pathways, a concept described as ligand bias. Allostery further increases the complexity of GPCR functioning. Indeed, many substances can act as allosteric modulators of GPCR signaling. These include synthetic ligands, signaling proteins, lipids, or dimerization partners. It has been proposed that the remarkable functional versatility of GPCRs is associated to their intrinsic dynamic properties. In this model, GPCRs are considered as flexible proteins that can visit multiple conformational states linked to distinct functional outcomes. Signaling modulation can then be seen as an alteration of the equilibrium between such states, with the relative amount of the different populations modulated by coupling to orthosteric or allosteric ligands, to intracellular protein partners, by receptor oligomerization, or by alterations in membrane composition. Although major progresses have been made in structural biology of GPCRs, we are nevertheless only beginning to appreciate the role of these dynamics in GPCR signaling. Indeed, a wealth of structural information has been obtained from crystal structures, but these only offer a limited number of static snapshots of a dynamic process. Hence, answering the question of how GPCRs dynamics can control their downstream signaling pathways still requires a variety of additional cutting-edge biophysical methods. In this context, we propose to illuminate how dynamics govern signaling through an integrated, multidisciplinary analysis that will combine innovative tools in biochemistry to state-of-the-art experimental and computational methods. These studies will build upon the experience, reagents (purified receptors in lipid nanodiscs, ligands, signaling proteins) and methods accumulated during the past years by the different members of the consortium. Two different GPCRs, the ghrelin (GHSR) and kappa opioid (KOP) receptors, will be considered. Besides being prototypical class A GPCRs, both are prominent pharmacological targets. GHSR and ghrelin are involved, among others, in the control of growth hormone secretion, of food intake, and of reward-seeking behaviors in alcohol and drug abuse. KOP and its cognate peptide ligand dynorphin are implicated in neuronal pathways associated with addiction, pain, reward, mood, cognition and perception. Specifically, we will decipher the molecular bases of the signaling trigger through an integrated analysis of the structural (NMR), thermodynamic (ITC) and kinetic (fluorescence) features of ligand binding (Task 1). In parallel, we will use solution-state NMR combined to computer simulation analyses to delineate how ligands and allosteric partners – signaling proteins, allosteric ligands, membrane environment, dimerization partners - provoke change in the conformational fluctuations of the receptor (Task 2). The proposed program will therefore provide unprecedented insights into the role of dynamics in major functional processes such as ligand binding, receptor activation, coupling to downstream signaling partners, biased signaling, and allosteric modulation. This is of more than academic interest since stabilization of receptor states is likely the key to modulating GPCR function in drug design.