GPCR conformations · Structural studies of ligand-induced conformational changes in G protein-coupled receptors

Structural studies of ligand-induced conformational changes in G protein-coupled receptors

G protein-coupled receptors (GPCR) are eukaryotic membrane proteins. They are involved in transduction of external signal across the cellular membrane into a cell. Being engaged in different fundamental physiological processes, GPCRs induce intracellular responses to a broad variety of external stimuli, including hormones and neurotransmitters, as well as molecules of smell and taste, pheromones and light. The human genome encodes at least 800 different GPCRs of which 400 are non-sensory receptors with potential medical relevance. According to estimations every third medical drug modulates some kind of GPCR activities. Although being so diverse, all GPCRs have something in common. When activated by the external stimulus, they change their molecular conformation. This change allows them to interact with different protein partners inside a cell and thus initiate diverse signalling pathways. The most prominent GPCR partners are different types of heterotrimeric G proteins. An activated heterotrimeric G protein detaches from the receptor and dissociates into G-alpha and G-beta-gamma subunits, each of which, in turn, modulates cellular signalling pathways and cause a change downstream in the cell. Many GPCRs in different physiological conditions and/or induced by different external signal-carrying molecules (effectors) can interact with more than one type of G proteins and thus initiate different cellular response. In addition to G proteins, GPCRs also interact with and signal via G protein-coupled receptor kinases and beta-arrestins. Chemicals that bind to GPCRs can have different effects. Agonists bind to a receptor and activate certain cellular responses. Neutral antagonists block the action of agonists, while inverse agonists have an opposite effect to agonists. We initiated this project to investigate and understand why different effector molecules induce so drastically different cellular responses and what the underlying molecular mechanism for this difference is. These questions are also important in designing medical drugs which bind to GPCRs. A drug molecule of choice should be specific in targeting only relevant receptor and inducing a certain signalling pathway. We are studying two important therapeutic GPCR targets: human cannabinoid CB2 receptor and human vasopressin V2 receptor. CB2 is a part of the “endocannabinoid system” and is predominantly present in immune and peripheral nervous systems. It is a valuable drug target for treating inflammatory diseases, pain, osteoporosis, atherosclerosis and brain tumours. V2 is present in kidneys and has an important role in maintaining water homeostasis in human organism. It is targeted in treatment of diabetes insipidus and hyponatremia. In order to understand mechanisms by which different effectors modulate activities of CB2 and V2 receptors, we are aiming at determining molecular structures of these receptors with bound effector molecules of different kinds: agonists, antagonists and inverse agonists. This challenging project involves stabilization and crystallization of the receptor molecules and, finally, determination of their structures by X-ray crystallography. Crystallization of GPCRs, and eukaryotic membrane proteins in general, is always a challenging task. Apart from screening of different crystallization conditions and techniques, it usually requires design and extensive screening of modifications in receptor molecules themselves. Potential receptor modifications embrace truncations at N- and C-termini, deletion of flexible parts, thermostabilizing point mutations and insertion of hydrophilic protein domains which should facilitate crystallization. In order to be suitable for crystallization, modified receptors should be more stable than the wild type, less flexible (stabilized in certain conformation), but at the same time at least partially functional and able to express in relatively huge amounts which are necessary in structural studies. In this project we were able to find and express the modified CB2 and V2 receptors which are suitable for crystallization. This is a very important step towards the final goal of the project – determining of the molecular structures of these two receptors and thus understanding the mechanism of their action. The anticipated results are prerequisite for rational design of improved therapeutic drugs specifically affecting the studied receptors, but having no influence on other similar receptors from the same receptor subfamilies.

Data: CORDIS, © European Union