A novel approach for investigating GPCR/ligand interactions: analysis of the secondary binding site within RXFP1
AffiliationDepartment of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute
Document TypePhD thesis
CitationsDiepenhorst, N. (2013). A novel approach for investigating GPCR/ligand interactions: analysis of the secondary binding site within RXFP1. PhD thesis, Florey Institute for Neuroscience and Mental Health, and, Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne.
Access StatusThis item is currently not available from this repository
© 2013 Dr. Natalie Diepenhorst
Relaxin is a peptide hormone which plays a role in mediating a wide variety of physiologies; specifically its positive cardiovascular effects have led to its success in phase three clinical trials for the treatment of acute heart failure. With this therapeutic potential, design of smaller relaxin analogues with oral bioavailability would be beneficial. To achieve this, a sound understanding of the molecular mechanisms of relaxin mediated activation of its G protein-coupled receptor (GPCR) relaxin family peptide receptor 1 (RXFP1) is required. RXFP1 is a class A, rhodopsin like GPCR which possesses a large N-terminal domain consisting of a series of 10 leucine rich repeats and is consequently classified as a leucine rich repeat (LRR) containing GPCR (LGR). Unique to other LGRs and other GPCRs, RXFP1 contains an N-terminal low density lipoprotein class A module (LDLa). Relaxin mediated activation of RXFP1 requires multiple interactions; a well characterised high-affinity interaction at the LRRs positions relaxin for a poorly characterised, lower-affinity interaction within the extracellular loops (ELs) of the transmembrane domain. Additionally, the LDLa module is absolutely required for receptor activation. The current model proposes that RXFP1 exists at the cell surface as a dimer. Relaxin binding the LRRs and ELs of one receptor in the dimer positions the N-terminal LDLa module of the relaxin bound receptor at the ELs of the other receptor in the dimer. Molecular characterisation of interactions at the ELs has been limited due to the inherent difficulties in studying membrane associated GPCRs. This project aimed to confirm interactions between relaxin / LDLa module and the ELs of RXFP1 and then characterise these interactions using NMR. A tool for investigating the interactions at the ELs in isolation of the whole receptor was designed; a soluble protein scaffold system displaying EL1 and EL2 from RXFP1 which were inserted into the native loops of a thermostabilised 6 kDa Streptococcal protein G (GB1) protein creating a soluble scaffold displaying the extracellular loops of RXFP1 (ssRXFP1). EL1 and EL2 were selected as they are the largest loops and there is evidence that they are involved in ligand interactions. Importantly there is a disulphide bond connecting the C-terminal end of EL1 to the middle of EL2 which is known to be important for the structure of EL2 and this was engineered into this construct. This protein was expressed and purified and the formation of the disulphide bond confirmed using mass spectrometry. Interactions with relaxin / LDLa were determined using a streptavidin bead pull down assay where biotinylated relaxin coated streptavidin resin was used to capture ssRXFP1. Interactions were characterised using 15N-HSQC NMR experiments of 15N-ssRXFP1 titrated with relaxin / LDLa. Both the pull down assay and the NMR experiments were able to confirm an interaction between relaxin and ssRXFP1. As a control, GB1 with no loops inserted and ssRXFP1 with the mutations C43S/C81S (ssRXFP1cs) with the disulphide bond mutated out, were unable to interact with relaxin showing the specificity of the interaction and the requirement for the disulphide bond. Truncated ssRXFP1 variants omitting the whole of EL1 or only half of EL1 were also shown to interact with relaxin suggesting that EL2 was the main point of interaction. Unfortunately, 15N-HSQC spectrum assignment was complicated by heterogeneity believed to arise from the predicted cis/trans isomerisation of a proline within EL2 detected by the dual signals arising from the side chain NH of the EL1 tryptophan. Consequently, site directed mutagenesis was applied to the scaffold to further investigate the interaction. Previous site directed mutagenesis of RXFP1 identified the involvement of EL2 F564 in RXFP1 activation. The role of this residue was further investigated using ssRXFP1. Upon mutation to alanine, no interaction with relaxin was detected by NMR or pull down however mutation to tyrosine was able to restore the interaction suggesting the aromatic nature of the residue was required. Corresponding mutations within full length RXFP1 were able to confirm these observations with the tyrosine substitution resulting in restored receptor activity. The scaffold protein was also used to establish whether the LDLa module was able to interact with the ELs of RXFP1. The NMR experiments confirmed an interaction between the LDLa module and ssRXFP1 in addition to the two truncated scaffolds again suggesting EL2 as the main point of interaction. Additionally, the LDLa module was able to compete with biotinylated relaxin for ssRXFP1 binding suggesting the possibility that the LDLa and relaxin binding sites within the loops overlap. This is the first direct experimental evidence that the LDLa module is able to interact with RXFP1 ELs in confirmation of the current model for RXFP1 activation. In this project, a tool for investigating interactions at the ELs of RXFP1 has been developed and successfully used to confirm interactions between the ELs and relaxin and the LDLa module. While further characterisation of these interactions was limited, analysis of mutants and truncations was able to determine the importance of the disulphide bond and an aromatic residue for interactions with relaxin. This data helps to solidify our understanding of the required elements for relaxin mediated RXFP1 activation which is essential for future drug design at RXFP1.
KeywordsGPCR; relaxin; protein engineering; extracellular loops; receptor activation; ligand binding
- Click on "Export Reference in RIS Format" and choose "open with... Endnote".
- Click on "Export Reference in RIS Format". Login to Refworks, go to References => Import References