Research project P5/30 (Research action P5)
The coordinator and the five associated partners have a strong position in the field of research dealing with the transduction of biological signals. The aim of the present application is to obtain additional support allowing to strengthen their individual programs by joining efforts centered on the study of G protein-coupled receptors (GPCRs) of the rhodopsin-like subfamily. The combined expertise of the various partners covers the entire spectrum of methodologies and concepts required for efficient GPCR research: functional and comparative genomics, proteomics with access to state-of-the-art mass-spectrometry, molecular pharmacology, structure-function studies, molecular modeling, mouse transgenesis. Two of the partners provide additional expertise in the related fields of signal transduction in yeast and molecular virology which will expand the scope of the program and may lead to new methodological developments.
The program will be subdivided in the following work packages (the coordinator and the partners are identified in the text as "P1" and "P2 to P6", respectively. The European partner is identified as "P7").
WP1: Functional genomics and reverse pharmacology of GPCRs.
This part of the study will deal with the characterization of orphan GPCRs and the identification of their natural agonists. It will mainly concentrate on receptor subtypes, as defined from analyses of their primary structures, for which the partners have a previous experience: chemokine-like receptors (P1, P5), vertebrate and invertebrate neuropeptide receptors (P1, P2), putative vertebrate receptors for angiotensinII and related physiologically active fragments (P1,P6), vertebrate and invertebrate receptors belonging to the subfamily of glycoprotein hormone receptors (P1,P2), putative vertebrate and invertebrate receptors serving as glucose sensors (P4, P1, P2) . The studies will include systematic screening of natural (tissue extracts, normal body fluids, inflammatory exudates...) and synthetic (collections of possible ligands, synthetic peptide or compound libraries...) sources of agonists for biological activity, using stably transfected cells expressing the receptors of interest. These cells, of which a series is available, have been engineered to provide a common read-out signal, whatever the regulatory cascade controlled by the receptor. The tissue distribution of the receptors selected for study will be explored by immunohistochemistry and in situ hybridization.
WP2: molecular pharmacology, structure function studies of known receptors.
The molecular steps and players involved in the effects of agonists, antagonists or inverse agonists on a series of receptors will be investigated by a combination of conventional binding studies, activation studies on transfected cells, site-directed mutagenesis and molecular modeling (P7). The receptors selected for such studies will be those for which the partners have a long standing interest: glycoprotein hormone receptors and their invertebrate homologs (P1, P2); insect tachykinin receptors (P2); chemokine receptors (P1, P5), receptors for angiotensinII and physiologically active fragments (P6), sucrose/glucose sensors in yeast (P4).
WP3: study of the biological roles of orphan and known GPCRs by gene invalidation.
Classical KO experiments in the mouse are currently being performed for a limited number of orphan receptors. These experiments will be extended preferentially to those orphans for which the natural agonist will be identified (P1). Depending on the domain, the phenotypes will be studied locally (chemokine-like receptors, P1, P5) or in collaboration with specialized groups abroad (neuropeptide receptors). Spontaneous mutants of Drosophila melanogaster) will be searched for in repositories, that would correspond to KO mutants for receptor genes and their phenotype will be studied in detail (P2).
WP4: study of downstream signaling of GPCRs.
Downstream events generated secondary to GPCR activation and the interacting partner molecules involved will be studied by DNA microarray technology, proteomic and two-hybrid system (P1, P2, P3, P4, P5, P6). The possibility to bridge the frontier between signaling in yeast and higher eukaryotes will be explored by site-directed mutagenesis of yeast receptors, G proteins or RGS proteins (P1, P4).
WP5: study of the role of GPCRs in carcinogenesis and metastasis.
This part of the study will mainly deal with chemokine and chemokine-like receptors. It will involve: (WP5.1) Determination of the expression profile of a series of chemokines and chemokine receptors in spontaneous human tumors and in murine, ovine and bovine tumor cell lines (P1, P3, P5); (WP5.2) study of the role of chemoattractant receptors in chemotaxis of tumors towards their preferred metastasis sites (P1, P3, P5); (WP5.3) study of the angiogenic versus angiostatic activity of chemokines and their variants in an in vivo) model (rabbit cornea micropocket model) (P5).
WP6: development of additional tools for the expression or detection of GPCRs.
The expertise of P4 with signal transduction in yeast will be exploited, in collaboration with the coordinator and P2, in an attempt to develop new biological assays for vertebrate and invertebrate GPCR activation in yeast. The approach will consist in exploring the functional characteristics in yeast of hybrid G alpha proteins made of mammalian G alpha 16 and yeast Gpa2.
