title: Mapping key regions of the RXFP2 low-density lipoprotein class-A module that are involved in signal activation.
abstract: The peptide hormone INSL3 and its receptor, RXFP2, have co-evolved alongside relaxin and its receptor, RXFP1. Both RXFP1 and RXFP2 are G protein-coupled receptors (GPCRs) containing the hallmark seven transmembrane helices in addition to a distinct ectodomain of leucine-rich repeats (LRRs) and a single low-density lipoprotein class-A (LDLa) module at the N-terminus. RXFP1 and RXFP2 are the only mammalian GPCRs known to contain an LDLa, and its removal does not perturb primary ligand binding to the LRRs; however, signaling is abolished. This presents a general mechanism whereby ligand binding induces a conformational change in the receptor to position the LDLa to elicit a signal response. Although the LDLa interaction site has not been identified, the residues important to the action have been mapped within the RXFP1 LDLa module. In this study, we comprehensively study the RXFP2 LDLa module. We determine its structure using nuclear magnetic resonance (NMR) and concurrently investigate the signaling of an RXFP2 with the LDLa removed (RXFP2-short), confirming that the LDLa is essential to signaling. We then replaced the LDLa with the second ligand binding module from the LDL receptor, LB2, creating the RXFP2-LB2 chimera. Unlike that in the equivalent RXFP1-LB2 chimera, signaling is rescued albeit modestly. Guided by the NMR structure, we dissected regions of the RXFP2 LDLa to identify specific residues that are important to signal activation. We determine that although the module is important to the activation of RXFP2, unlike the RXFP1 receptor, specific residues in the N-terminus of the domain are not involved in signal activation.
authors.affiliation: Department of Biochemistry and Molecular Biology, The Bio21 Molecular Science and Biotechnology Institute, ‡Florey Institute of Neuroscience and Mental Health, and §School of Chemistry, University of Melbourne , Parkville, Victoria 3010, Australia.
authors.forename: Roy C K
authors.initials: RC
authors.lastname: Kong
authors.forename: Ross A D
authors.initials: RA
authors.lastname: Bathgate
authors.forename: Shoni
authors.initials: S
authors.lastname: Bruell
authors.forename: John D
authors.initials: JD
authors.lastname: Wade
authors.forename: Paul R
authors.initials: PR
authors.lastname: Gooley
authors.forename: Emma J
authors.initials: EJ
authors.lastname: Petrie
mesh_terms: D057809:HEK293 Cells; D006801:Humans; D007328:Insulin / Q000235:genetics / Q000378:metabolism; D017270:Lipoprotein(a); D011506:Proteins / Q000235:genetics / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D011973:Receptors, LDL / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D007328:Insulin; C084797:Leydig insulin-like protein; D017270:Lipoprotein(a); D011506:Proteins; C472867:RXFP2 protein, human; D043562:Receptors, G-Protein-Coupled; D011973:Receptors, LDL; D011993:Recombinant Fusion Proteins