Research project P7/40 (Research action P7)
Introduction
G protein-coupled receptors (GPCR) represent the largest family among membrane receptors. These receptors and their structurally diverse ligands play a major role in all aspects of physiology and pathophysiology, and constitute the targets for about half the active compounds presently used as therapeutic agents. While GPCRs were long thought to function as monomers, it is currently accepted that GPCRs form homo- and/or hetero-complexes and are anchored in membrane regions with specific lipid/(glyco)protein components. These contextual factors modulate signalling and can cause disease-specific receptor dysregulations. Partners of the present programme have played a major role in the characterization of many G protein-coupled receptors and their ligands in different species. The GPCR field is now at an exciting phase of its evolution. Indeed, structural data become available at a rapidly increasing pace, and this will affect deeply all other aspects of the field. Based on the network’s previous experience, and thanks to new partners and external collaborations worldwide, we intend to remain at the forefront of GPCR research by performing world class investigations into novel directions taken by the field. With a new focus onto structural aspects, we will study further both general and specific aspects of GPCRs, with the ultimate goal of improving human health and quality of life.
Structural investigation of GPCR conformational changes
We will determine the crystallographic structure of specific GPCRs in their different functional conformations. For this purpose, we will generate recombinant single chain monoclonal antibodies from Camelidae (nanobodies), able to stabilize specific conformations of the receptors [inactive conformation, active conformation(s), complexes with G proteins, -arrestins or other partners]. These nanobodies will be used as an aid to the purification of correctly folded receptors and to the crystallization of these receptors. The structures will be determined by X-ray crystallography in collaboration with several foreign groups (Brian Kobilka, Stanford, USA; Gebhard Schertler, Paul Scherrer Institute, Switzerland). The crystal structures will be used to model the structural organization of other GPCRs of interest and their interplay with small molecule ligands and signalling proteins (agonists and antagonists, allosteric modulators, G proteins, kinases, -arrestins), aiming to better understand the activation mechanisms of GPCRs in general. We will also pursue the analysis of the functional consequences of GPCR oligomerization in vitro and in animal models, with the help of the structural information obtained.
GPCR signalling pathways
Selected receptors in human, insect species and yeast will be studied for their signalling properties, with a particular focus on the recent concepts of G protein-independent pathways, biased agonism, allosteric regulation within receptor oligomers, and cross-regulation with other sensing/signalling pathways. We will analyse modifications of signalling according to the ligands used, the interaction of the receptor with other partners (among which other GPCRs, GRKs, -arrestins), and the environment of the receptor (lipid composition of the membrane). Energy transfer techniques will be used to study the direct interaction of receptors with specific G protein subunits and other signalling partners, as well as the activation of these proteins. Comparative signalling analyses will be made between mammalian, insect and yeast cells based on structural and functional analogy. In addition, new pathways and/or regulations will be explored and when discovered in one species will be searched in the other species to assess their universal character.
Identification of novel receptors and their ligands
There are still many orphan receptors for which the ligands and functions are not known in the mammalian and insect genomes. We will focus on the characterization of these receptors, through the identification of their ligands, and the subsequent delineation of their functions. Several of such programs have been initiated during the previous phase (identification of ligands or ongoing purification of biological activities), and these programs will be pursued during this phase VII. We will study human receptors for leukocyte chemoattractants (e.g. chemokines with or without posttranslational modifications), neuropeptides, glycoprotein hormone-like proteins and glucose, insect receptors for neuropeptides and peptide/protein hormones, and nutrient-sensing receptors in yeast. Phylogeny and evolutionary clues will be used in this approach.
Functional characterization of specific receptors in physiological processes and diseases
As the major class of signal transducing membrane receptors, GPCRs are expected to have a prominent regulatory function in many biological processes. A number of specific receptors, among which several receptors identified by the partners over the previous years, will be studied in detail in order to determine their role in physiological processes, and their involvement in human diseases. We will consider four areas of crucial importance for future therapeutic applications, namely metabolism, immunity, cancer, and central nervous system disorders. For each of these areas, in which our consortium shares long-standing expertise, this approach will involve in vitro studies, as well as in vivo models of diseases and genetically modified organisms, in combination with advanced post-genomic and phenomic approaches. We will study among other systems, insect and mammalian glycoprotein hormone receptors in development, chemoattractant and nucleotide receptors in immunity and cancer in mammals, neuropeptide receptors in mammals and insects, and nutrient sensing GPCRs in yeasts and mammals. Several partners will contribute in concert to each of these areas. The resulting (patho)physiological data will be complemented with information obtained from the other parts of the program.
The network
The composition of our consortium ensures the continuation of promising activities and interactions initiated in the previous phase and allows for the incorporation of novel and recently emerging themes in GPCR research. In this modified network, we have incorporated a new Belgian partner (P4, Jan Steyaert, VUB), who has played a key role in the recent breakthroughs made in the emerging field of GPCR structural analysis. Indeed, the use of nanobodies generated by this group has been instrumental in the delineation of the first crystal structure of the β2-adrenergic receptor in its active conformation and for the structure of this receptor in complex with its G protein. We expect that these recent developments will lead to an explosion of the GPCR structural field in the years to come, and that our Belgian network will continue to play an essential role in this exciting avenue. We have also incorporated a new foreign partner (INT1, Silvano Sozzani, Brescia, Italy), who is already highly connected to two of the Belgian partners (P1 and P3) in the fields of immunity and cancer. Over the last years, there was also a long awaited convergence between the signalling pathways activated by GPCRs in yeast and in multicellular organisms such as insects and mammals. Besides the conceptual and technological exchanges that have been made between P2 and the other partners over the previous phase, there is now room for a deeper integration of the objectives of partners working on GPCRs in mammals, insects and yeast, in the frame of structure, signalling cascades and role of GPCRs in the metabolism of cells and organisms. The network will therefore reinforce its cohesion around a number of mainstream objectives, and will foster this cohesion by the organization of international meetings and workshops, summer schools and the continuation of student exchange programs.
