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. 2015 Aug 17:6:128.
doi: 10.3389/fendo.2015.00128. eCollection 2015.

Activation of Relaxin Family Receptor 1 from Different Mammalian Species by Relaxin Peptide and Small-Molecule Agonist ML290

Zaohua Huang  1 , Courtney Myhr  1 , Ross A D Bathgate  2 , Brian A Ho  1 , Amaya Bueno  1 , Xin Hu  3 , Jingbo Xiao  3 , Noel Southall  3 , Elena Barnaeva  3 , Irina U Agoulnik  4 , Juan J Marugan  3 , Marc Ferrer  3 , Alexander I Agoulnik  1
Affiliations

Activation of Relaxin Family Receptor 1 from Different Mammalian Species by Relaxin Peptide and Small-Molecule Agonist ML290

Zaohua Huang et al. Front Endocrinol (Lausanne). .

Abstract

Relaxin peptide (RLN), which signals through the relaxin family peptide 1 (RXFP1) GPCR receptor, has shown therapeutic effects in an acute heart failure clinical trial. We have identified a small-molecule agonist of human RXFP1, ML290; however, it does not activate the mouse receptor. To find a suitable animal model for ML290 testing and to gain mechanistic insights into the interaction of various ligands with RXFP1, we have cloned rhesus macaque, pig, rabbit, and guinea pig RXFP1s and analyzed their activation by RLN and ML290. HEK293T cells expressing macaque or pig RXFP1 responded to relaxin and ML290 treatment as measured by an increase of cAMP production. Guinea pig RXFP1 responded to relaxin but had very low response to ML290 treatment only at highest concentrations used. The rabbit RXFP1 amino acid sequence was the most divergent, with a number of unique substitutions within the ectodomain and the seven-transmembrane domain (7TM). Two splice variants of rabbit RXFP1 derived through alternative splicing of the fourth exon were identified. In contrast to the other species, rabbit RXFP1s were activated by ML290, but not with human, pig, mouse, or rabbit RLNs. Using FLAG-tagged constructs, we have shown that both rabbit RXFP1 variants are expressed on the cell surface. No binding of human Eu-labeled RLN to rabbit RXFP1 was detected, suggesting that in this species, RXFP1 might be non-functional. We used chimeric rabbit-human and guinea pig-human constructs to identify regions important for RLN or ML290 receptor activation. Chimeras with the human ectodomain and rabbit 7TM domain were activated by RLN, whereas substitution of part of the guinea pig 7TM domain with the human sequence only partially restored ML290 activation, confirming the allosteric mode of action for the two ligands. Our data demonstrate that macaque and pig models can be used for ML290 testing.

Keywords: G protein-coupled receptor; RXFP1; receptor structure–function; relaxin; small-molecule allosteric agonist.

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Figures

Figure 1
Figure 1
Alignment of RXFP1 proteins from various species. (A) Amino acid alignment of RXFP1 receptor sequences. The position of the extra amino acid (V98) in rabbit receptor variant R2 is shown above the sequence with an arrow. Functional domains are shown below the sequences. LDLa is low-density lipoprotein class A domain; LRR is leucine-rich repeat, TM1–7 are transmembrane domains; ICL1–3 are intracellular loops of seven-transmembrane domain; ECL1–3 are extracellular loops of seven-transmembrane domain. The highlighted brown box is the third extracellular loop and adjacent amino acids required for ML290 activation of RXFP1. Amino acids conserved in all seven species are in red; amino acids specific for the rabbit sequence are highlighted in yellow. The vertical line at position 324 indicates the fusion site in chimeric rabbit/human receptor (RH-RXFP1). The vertical line at position 502 indicates the fusion site in chimeric guinea pig/human receptor (GH-RXFP1). (B) Evolutionary tree showing the relationship of various RXFP1 proteins. The rabbit sequence is the most diverged.
Figure 2
Figure 2
Alternative splicing of the intron 3 and exon 4 in rabbit RXFP1 genomic DNA. The upper sequence shows the intron (green) and exon (black) boundaries in the genomic DNA. Three additional nucleotides (in red) are added to the mRNA as result of alternative splicing, as shown with the red line. Below is the sequencing chromatogram showing the presence of two sequences after the alternative splicing site. Note an equal size of the peaks in the overlapping sequence, indicating an equal representation of the two variants in the total mRNA pool.
Figure 3
Figure 3
Activation of macaque and pig RXFP1 receptors by RLN and ML290. (A) Porcine RLN-induced cAMP response. (B) ML290-induced cAMP response. HEK293T cells with CRE-luc reporter were transduced with BacMam RXFP1 expression vectors. RLU, relative luciferase units. Data are expressed as mean ± SEM.
Figure 4
Figure 4
Activation of guinea pig and guinea pig–human chimeric receptors by RLN and ML290. (A) Porcine RLN-induced cAMP response. (B) ML290-induced cAMP response. HEK293T cells were transiently transfected with RXFP1 expression vectors and cAMP was determined using HTRF cAMP assay. cAMP activity is expressed as the percentage of 10 μM Forskolin-stimulated response. Data are expressed as mean ± SEM, each point represent triplicate measurements. The experiment was repeated three times. *p < 0.05, **p < 0.01, ***p < 0.001 compared to hRXFP1.
Figure 5
Figure 5
Expression of two rabbit RXFP1 receptors compared to human RXFP1. Shown is the total and cell surface RXFP1 expression in transiently transfected HEK293T cells. Data are expressed as mean ± SEM, each point represent triplicate measurements. The experiment was repeated three times. *p < 0.05 compared to hRXFP1.
Figure 6
Figure 6
Activation of rabbit and rabbit–human chimeric receptors by RLN and ML290. (A) Porcine RLN-induced cAMP response. (B) ML290-induced cAMP response. HEK293T cells were transiently transfected with RXFP1 expression vectors and cAMP was determined using the HTRF cAMP assay. cAMP activity is expressed as the percentage of hRXFP1 cAMP activation by 10 nM of RLN. Data are expressed as mean ± SEM; each point represents triplicate measurements. The experiment was repeated at least three times. ***p < 0.001 compared to hRXFP1.
Figure 7
Figure 7
Activation of rabbit receptors by relaxin peptides from different species. (A) Activation of two rabbit receptor variants (R1- and R2-RXFP1) and hRXFP1with 10 nM of human (hRLN), mouse (mRLN), and porcine (pRLN) relaxin peptides, and by 5 μM of ML290. Treatments of rabbit RXFP1 are statistically significant compared to hRXFP1, ***p < 0.001. (B) Activation of rabbit (R1-), human (h), and guinea pig (G-) RXFP1s with rabbit relaxin SQ10. Conditioned media from HEK293T cells transfected with SQ10 and control empty vector was used for cell stimulation. cAMP activity is expressed as the percentage of 10 μM Forskolin stimulation for each type of cells. Data are expressed as mean ± SEM; each point represents triplicate measurements. The experiment was repeated at least three times. Treatment of R1-RXFP1 is statistically significant (p < 0.0001) compared to hRXFP1 or G-RXFP1.
Figure 8
Figure 8
Rabbit RXFP1 does not bind relaxin peptide. (A) Total and cell surface expression of hRXFP1 and R1-RXFP1. Expression is normalized to the expression of human receptor. ***p < 0.001 compared to hRXFP1. (B) Saturation binding using Eu-labeled H2 RLN. (C) Human RLN-induced cAMP response. (D) ML290-induced cAMP response. cAMP activity is expressed as the percentage of the 5 μM Forskolin-stimulated response for each receptor. Data are expressed as mean ± SEM; each point represents triplicate measurements. The experiment was repeated at least three times.
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References

    1. Sherwood OD. Relaxin’s physiological roles and other diverse actions. Endocr Rev (2004) 25:205–34.10.1210/er.2003-0013 - DOI - PubMed
    1. Halls ML, Bathgate RA, Sutton SW, Dschietzig TB, Summers RJ. International Union of Basic and Clinical Pharmacology. XCV. Recent advances in the understanding of the pharmacology and biological roles of relaxin family peptide receptors 1-4, the receptors for relaxin family peptides. Pharmacol Rev (2015) 67:389–440.10.1124/pr.114.009472 - DOI - PMC - PubMed
    1. Hsu SY, Nakabayashi K, Nishi S, Kumagai J, Kudo M, Sherwood OD, et al. Activation of orphan receptors by the hormone relaxin. Science (2002) 295:671–4.10.1126/science.1065654 - DOI - PubMed
    1. Teerlink JR, Cotter G, Davison BA, Felker GM, Filippatos G, Greenberg BH, et al. Serelaxin, recombinant human relaxin-2, for treatment of acute heart failure (RELAX-AHF): a randomised, placebo-controlled trial. Lancet (2013) 381:29–39.10.1016/S0140-6736(12)61855-8 - DOI - PubMed
    1. Xiao J, Huang Z, Chen CZ, Agoulnik IU, Southall N, Hu X, et al. Identification and optimization of small-molecule agonists of the human relaxin hormone receptor RXFP1. Nat Commun (2013) 4:1953.10.1038/ncomms2953 - DOI - PMC - PubMed