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A novel protein engineering approach for investigating GPCR/ligand interactions
Author(s) -
Witteveen Natalie Anne,
Gooley Paul R,
Stone Martin J,
Bathgate Ross A D
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.966.5
Subject(s) - relaxin , g protein coupled receptor , ligand (biochemistry) , chemistry , receptor , peptide , plasma protein binding , protein–protein interaction , biochemistry , biophysics , computational biology , biology
Relaxin family peptide receptors (RXFP) 1 and 2, receptors for relaxin and insulin like peptide‐3 (INSL3) respectively, are G‐protein coupled receptors possessing large N terminal domains consisting of 10 leucine rich repeats (LRRs) and a low density lipoprotein Class A (LDLa) module. Ligand mediated activation requires primary binding to the LRRs as well as a secondary interaction involving the extracellular loops (ELs) of the transmembrane domains. The LDLa is necessary for activation possibly by a specific interaction with the ELs. The project aims to identify residues of RXFP1 and RXFP2 ELs involved in ligand/LDLa binding by mounting them onto a soluble protein scaffold and mapping specific ligand interactions using NMR. We have produced soluble scaffolds containing RXFP1 and RXFP2 EL1 and EL2 and are conducting ligand/LDLa titrations for NMR analysis before residue assignment. Preliminary NMR results show peak changes upon relaxin titration with the RXFP1 scaffold suggesting specific interactions are occurring. Identified interacting residues will be mutated in RXFP1 and functional characterisation will confirm their importance for receptor function. Understanding mechanisms of RXFP1 and RXFP2 activation will aid design of relaxin and INSL3 analogues for therapeutic use. This work is supported by project grant 628427 (to RADB and PRG) from the National Health and Medical Research Council, Australia.