title: The different ligand-binding modes of relaxin family peptide receptors RXFP1 and RXFP2.
abstract: Relaxin and insulin-like peptide 3 (INSL3) are peptide hormones with a number of important physiological roles in reproduction, regulation of extracellular matrix turnover, and cardiovascular function. Relaxin and INSL3 mediate their actions through the closely related G-protein coupled receptors, relaxin family peptide receptors 1 and 2 (RXFP1 and RXFP2), respectively. These receptors have large extracellular domains (ECD) that contain high-affinity ligand-binding sites within their 10 leucine-rich repeat (LRR)-containing modules. Although relaxin can bind and activate both RXFP1 and RXFP2, INSL3 can only bind and activate RXFP2. To investigate whether this difference is related to the nature of the high-affinity ECD binding site or to differences in secondary binding sites involving the receptor transmembrane (TM) domain, we created a suite of constructs with RXFP1/2 chimeric ECD attached to single TM helices. We show that by changing as little as one LRR, representing four amino acid substitutions, we were able to engineer a high-affinity INSL3-binding site into the ECD of RXFP1. Molecular modeling of the INSL3-RXFP2 interaction based on extensive experimental data highlights the differences in the binding mechanisms of relaxin and INSL3 to the ECD of their cognate receptors. Interestingly, when the engineered RXFP1/2 ECD were introduced into full-length RXFP1 constructs, INSL3 exhibited only low affinity and efficacy on these receptors. These results highlight critical differences both in the ECD binding and in the coordination of the ECD-binding site with the TM domain, and provide new mechanistic insights into the binding and activation events of RXFP1 and RXFP2 by their native hormone ligands.
authors.affiliation: Florey Neuroscience Institutes and the Department of Biochemistry and Molecular Biology, The University of Melbourne, Melbourne, Victoria 3010, Australia.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Scott
authors.forename: K Johan
authors.initials: KJ
authors.lastname: Rosengren
authors.forename: Ross A D
authors.initials: RA
authors.lastname: Bathgate
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D017124:Conserved Sequence; D000242:Cyclic AMP / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007328:Insulin / Q000378:metabolism; D000090263:Leucine-Rich Repeat Proteins; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000378:metabolism*; D018000:Receptors, Peptide / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012065:Relaxin / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D000596:Amino Acids; D007328:Insulin; D000090263:Leucine-Rich Repeat Proteins; C084797:Leydig insulin-like protein; D008024:Ligands; D011506:Proteins; C417543:RXFP1 protein, human; C472867:RXFP2 protein, human; D043562:Receptors, G-Protein-Coupled; D018000:Receptors, Peptide; D011994:Recombinant Proteins; D012065:Relaxin; D000242:Cyclic AMP