Screening of ligand binding sites of a G-protein coupled receptors by using photocrosslinking
Final Report Abstract
G-protein coupled receptors (GPCRs) mediate many physiological and pathological processes, and represent one of the major targets of modern pharmaceuticals. However, the knowledge of the molecular bases of the ligand-receptor interaction is still quite limited, due to the difficulty of obtaining spectroscopic data for such highly flexible and complex systems constitutively integrated in the cell membrane. For class II GPCRs, information about the binding mode of peptide ligands to the receptors is mostly derived by structure activity relationship studies, studies carried out on receptor chimaeras and expressed receptor domains (especially the N-terminus), and photoaffinity crosslinking studies carried out with ligand analogs containing photoactivatable moieties. As useful as the latter tool is, it has the methodological limitation that most positions of the ligands do not tolerate the introduction of the photoactivatable moieties. The modified ligands either do not bind to the receptor with high affinity anymore, or change their pharmacological behavior (e.g. Gs/Gi selectivity). This limitation prevents comprehensive mapping of the ligand-receptor interaction and prevents comparison of ligands with different pharmacological properties. Recent advances in unnatural amino acid mutagenesis have made possible the site-specific incorporation of a wide variety of unnatural amino acids into proteins as they are synthesized in living cells. This has opened the new possibility of introducing a photocrosslinker into the receptor, rather than into the ligand as it has been done so far. The method of choice to install non canonical moieties into proteins expressed in living cells is to import exogenous orthogonal tRNA /aminoacyl-tRNA synthetase pairs into the cell, which incorporate the desired unnatural amino acid in response to the stop codon. On the contrary, the use of chemically misacylated tRNAs might be fruitful when applied to cell free expression systems, but gives poor perspectives for studies in living systems, particularly when large numbers of cells are involved. In the native environment of the mammalian cell, the photo-activatable amino acid p-azidophenylalanine (Azi) has been genetically incorporated into several positions of the corticotropin releasing factor receptor type 1 (CRF-R1). Good expression yields were achieved through the development of a novel all-in-one expression system, in which three independent expression cassettes, respectively dedicated to the target gene, the suppressor tRNA and the orthogonal synthetase, are incorporated into a unique plasmid. Binding studies have demonstrated that the introduction of the crosslinker did not significantly disturb the high-affinity binding of the antagonist Astressin. On a set of receptor mutants bearing the crosslinker at selected positions chosen according to the established ligand binding model, we have examined the crosslinking pattern of four different peptide ligands: the native agonists CRF, Urocortin 1 and Sauvagine, and the antagonist Astressin. For all ligands, we found a common interaction site in the hinge region between the N-terminal domain and the first transmembrane domain, a result that adds new information in the map of the ligand-receptor interaction, since no spectroscopic data are available for this region. Different crosslinking patterns were observed at the receptor N-terminus and in the loop region, demonstrating that indeed different peptides, although showing the same functional behavior, come into different proximity with different regions of the receptor. For the first time, we have found a crosslinking with the native fully glycosylated receptor N-terminus, which could not be found with the traditional crosslinking approach. However, only Urocortin 1 was crosslinked by the receptor N-terminus, while the other ligands were not. This is a quite surprising result especially for Astressin, which is supposed to have in the N-terminus its major binding determinants. In the loop region, the agonists Urocortin and Sauvagine were crosslinked by Azi installed at the fifth transmembrane domain, in the juxtamembrane region. A weaker crosslinking at this position was observed for agonist CRF, probably coming into tighter contact with other region of the loop domains, while the antagonist Astressin did not show any crosslinking here. Overall our results do not contradict the current binding model for class II GPCRs, but at the same time do not clearly confirm it. From a methodological point of view, we have shown the applicability of the method to a more extensive mapping, which is needed to locate more precisely the peptide ligands at the receptor surface and develop specific binding models for different ligands.
