Abstract
Relaxin family peptide 1 (RXFP1) receptor (LGR7) and RXFP2 receptor (LGR8) were recently identified as the receptor targets for H2 relaxin and insulin-like peptide 3 (INSL3), respectively. In this study, we define the pharmacology of these two receptors by using a number of receptor chimeras and relaxin family peptides. We have identified two binding sites on these receptors: one primary, high-affinity site within the ectodomain and a secondary, lower affinity site within the transmembrane region. The primary site was found to dictate receptor binding characteristics, although the lower affinity site also exerts some influence and modulates ligand affinity for the primary site in a manner dependent upon the peptide in question. Not all relaxin peptides were able to bind to the RXFP2 receptor, indicating that the relaxin-RXFP2 receptor interaction is species-specific. INSL3 was found to exhibit characteristics of a partial agonist at the RXFP2 and chimeric RXFP1/2 receptors, with low maximal cAMP responses but high potency in coupling to this pathway. cAMP accumulation studies also revealed that the binding sites couple to cAMP signaling pathways with differing efficiency: the high-affinity site signals with high efficiency, whereas the lower affinity site signals with little to no efficiency. Comparisons between RXFP1, RXFP2, the chimeric receptors, and the truncated receptors revealed that the interaction between receptor sites is critical for optimal ligand binding and signal transduction and that the ectodomain is essential for signaling. Evidence obtained in this study supports a two-stage binding model of receptor activation: binding to the primary site allows a conformational change and interaction with the low-affinity transmembrane site.
Footnotes
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R.B. is an R. D. Wright Fellow of the National Health and Medical Research Council. This work was supported in part by National Health and Medical Research Council Block Grant Reg Key 983001 to the Howard Florey Institute and National Health and Medical Research Council project Grant 300012. C.B. is a Monash University Postgraduate scholar. M.H. is an Australian Postgraduate scholar and recipient of a Monash University Faculty of Medicine, Nursing and Health Sciences Excellence Award.
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doi:10.1124/jpet.104.080655.
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ABBREVIATIONS: INSL, insulin-like peptide; LLR, leucine-rich repeat; GPCR, G protein-coupled receptor; LGR, leucine-rich repeat-containing G protein-coupled receptor; RXFP1, relaxin family peptide receptor; HEK, human embryonic kidney; BSA, bovine serum albumin.
- Received November 14, 2004.
- Accepted January 11, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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