Development of a scaffold displaying exoloops of RXFP1

Relaxin family peptide receptor 1 (RXFP1), the cognate receptor for relaxin, is a G-protein coupled receptor (GPCR) possessing a unique extracellular region consisting of a domain of 10 leucine rich repeats (LRRs) linked to an N-terminal low density lipoprotein Class A module. Relaxin binds to its receptor primarily by a high affinity interaction with the LRRs. An additional low-affinity interaction has been proposed to occur between relaxin and the the exoloops (ELs) of the transmembrane domain, however the molecular detail of this interaction remains undefined. While site directed mutagenesis and subsequent functional characterisation of these mutants traditionally allows identification of residues contributing to receptor function, in this case results are complicated by the presence of the high affinity binding site in the LRRs. To create a tool to investigate the low-affinity interaction, a protein scaffold system displaying exoloops 1 and 2 from RXFP1 was designed. This was achieved by inserting RXFP1 exoloops 1 and 2 into the native loops of a thermostabilised 6 kDa GB1 protein creating EL1/EL2-GB1. This protein has been expressed and purified in milligram quantities and used in conjunction with biophysical techniques such as NMR to explore relaxin binding to the exoloops of RXFP1

Isoform-Specific Biased Agonism of Histamine H 3 Receptor Agonists s

ABSTRACT The human histamine H 3 receptor (hH 3 R) is subject to extensive gene splicing that gives rise to a large number of functional and nonfunctional isoforms. Despite the general acceptance that G protein-coupled receptors can adopt different ligand-induced conformations that give rise to biased signaling, this has not been studied for the H 3 R; further, it is unknown whether splice variants of the same receptor engender the same or differential biased signaling. Herein, we profiled the pharmacology of histamine receptor agonists at the two most abundant hH 3 R splice variants (hH 3 R 445 and hH 3 R 365 ) across seven signaling endpoints. Both isoforms engender biased signaling, notably for 4-[3-(benzyloxy)propyl]-1H-imidazole (proxyfan) [e.g., strong bias toward phosphorylation of glycogen synthase kinase 3b (GSK3b) via the full-length receptor] and its congener 3-(1H-imidazol-4-yl)propyl-(4-iodophenyl)-methyl ether (iodoproxyfan), which are strongly consistent with the former's designation as a "protean" agonist. The 80 amino acid IL3 deleted isoform hH 3 R 365 is more permissive in its signaling than hH 3 R 445 : 2-(1H-imidazol-5-yl)ethyl imidothiocarbamate (imetit), proxyfan, and iodoproxyfan were all markedly biased away from calcium signaling, and principal component analysis of the full data set revealed divergent profiles for all five agonists. However, most interesting was the identification of differential biased signaling between the two isoforms. Strikingly, hH 3 R 365 was completely unable to stimulate GSK3b phosphorylation, an endpoint robustly activated by the full-length receptor. To the best of our knowledge, this is the first quantitative example of differential biased signaling via isoforms of the same G proteincoupled receptor that are simultaneously expressed in vivo and gives rise to the possibility of selective pharmacological targeting of individual receptor splice variants