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. 2013 Sep 27;288(39):28138-51.
doi: 10.1074/jbc.M113.499640. Epub 2013 Aug 7.

The relaxin receptor (RXFP1) utilizes hydrophobic moieties on a signaling surface of its N-terminal low density lipoprotein class A module to mediate receptor activation

Roy C K Kong  1 , Emma J Petrie, Biswaranjan Mohanty, Jason Ling, Jeremy C Y Lee, Paul R Gooley, Ross A D Bathgate
Affiliations

The relaxin receptor (RXFP1) utilizes hydrophobic moieties on a signaling surface of its N-terminal low density lipoprotein class A module to mediate receptor activation

Roy C K Kong et al. J Biol Chem. .

Abstract

The peptide hormone relaxin is showing potential as a treatment for acute heart failure. Although it is known that relaxin mediates its actions through the G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), little is known about the molecular mechanisms by which relaxin binding results in receptor activation. Previous studies have highlighted that the unique N-terminal low density lipoprotein class A (LDLa) module of RXFP1 is essential for receptor activation, and it has been hypothesized that this module is the true "ligand" of the receptor that directs the conformational changes necessary for G protein coupling. In this study, we confirmed that an RXFP1 receptor lacking the LDLa module binds ligand normally but cannot signal through any characterized G protein-coupled receptor signaling pathway. Furthermore, we comprehensively examined the contributions of amino acids in the LDLa module to RXFP1 activity using both gain-of-function and loss-of-function mutational analysis together with NMR structural analysis of recombinant LDLa modules. Gain-of-function studies with an inactive RXFP1 chimera containing the LDLa module of the human LDL receptor (LB2) demonstrated two key N-terminal regions of the module that were able to rescue receptor signaling. Loss-of-function mutations of residues in these regions demonstrated that Leu-7, Tyr-9, and Lys-17 all contributed to the ability of the LDLa module to drive receptor activation, and judicious amino acid substitutions suggested this involves hydrophobic interactions. Our results demonstrate that these key residues contribute to interactions driving the active receptor conformation, providing further evidence of a unique mode of G protein-coupled receptor activation.

Keywords: G Protein-coupled Receptors (GPCR); NMR; Peptide Hormones; Peptides; Protein Structure; RXFP1; Relaxin.

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Figures

FIGURE 1.
FIGURE 1.
Boxshade alignment of LDLa module sequences from RXFP1 receptors from various mammalian species in comparison with the sequence of LB2 of the human LDLr. The conserved cysteine residues are highlighted in red, conserved amino acids are highlighted in black, and conservative amino acid substitutions are shaded.
FIGURE 2.
FIGURE 2.
Reporter gene responses upon stimulation of RXFP1 (A) or RXFP1-short (B) stably expressed in HEK293T cells using 100 nm H2 relaxin. Data are -fold change of response from vehicle. Symbols represent means, and vertical bars represent S.E. of triplicate determinations from three independent experiments. GRE, glucocorticoid response element; AP1, activator protein 1; SRE, serum response element; NFAT, nuclear factor of activated T cells; HSE, heat shock element.
FIGURE 3.
FIGURE 3.
H2 relaxin-induced cAMP response of RXFP1-LB2 (A) and G12del-RXFP1-LB2 and S34A-RXFP1-LB2 (B) compared with RXFP1. cAMP activity is expressed as the percentage of the 5 μm forskolin-stimulated response for each receptor and has been normalized for cell surface expression. Symbols represent means and vertical bars (not visible) represent S.E. of triplicate determinations from at least three independent experiments.
FIGURE 4.
FIGURE 4.
H2 relaxin-induced cAMP response of SLGYFP-RXFP1-LB2, NITK-RXFP1-LB2, LLH-RXFP1-LB2, and NGVD-RXFP1-LB2 (A) and SLGYFP-RXFP1-LB2, NITK-RXFP1-LB2, SLGYFP NITK-RXFP1-LB2, and SLGYFP NITK C-term-RXFP1-LB2 (B) compared with RXFP1. cAMP activity is expressed as the percentage of the 5 μm forskolin-stimulated response for each receptor and has been normalized for cell surface expression. Symbols represent means and vertical bars (not visible) represent S.E. of triplicate determinations from at least three independent experiments.
FIGURE 5.
FIGURE 5.
The H2 relaxin-induced cAMP response of L7K-RXFP1 and L7A-RXFP1 (A) and L7K-RXFP1, L7A-RXFP1, L7K/L22K-RXFP1, and L7A/L22A-RXFP1 (B) is shown. C, Y9F-RXFP1, Y9M-RXFP1, and L7K/Y9M-RXFP1 compared with RXFP1. cAMP activity is expressed as the percentage of the 5 μm forskolin-stimulated response for each receptor and has been normalized for cell surface expression. Symbols represent means and vertical bars represent S.E. of triplicate determinations from at least three independent experiments.
FIGURE 6.
FIGURE 6.
H2 relaxin-induced cAMP response of I15A-RXFP1, T16A-RXFP1, K17A-RXFP1, and K17M-RXFP1 (A) and L7K-RXFP1, Y9M-RXFP1, K17A-RXFP1, L7K/Y9M-RXFP1, and L7K/Y9M/K17A-RXFP1 (B) compared with RXFP1. cAMP activity is expressed as the percentage of the 5 μm forskolin-stimulated response for each receptor and has been normalized for cell surface expression. Symbols represent means and vertical bars represent S.E. of triplicate determinations from at least three independent experiments.
FIGURE 7.
FIGURE 7.
1H,15N HSQC of 1 mm13C,15N-labeled SLGYFP NITK-LB2 in 50 mm imidazole, 10 mm CaCl2, 10% D2O, pH 6. 0 and 25 °C acquired at 800 MHz. Assignments of observed peptide and side chain NH resonances are indicated. Numbering is based on the chimeric LB2 module in Protein Data Bank code 2M7P.
FIGURE 8.
FIGURE 8.
A, ensemble of the 20 lowest energy structures of SLYFP NITK-LB2. Structures are represented as an overlay of the backbone structures in MOLMOL that overlay with an r.m.s.d. of 0.35 Å between residues 6 and 42. The side chains from the two “add-back” regions SLYFP and NITK have been represented as lines. B, overlay of SLYFP NITK-LB2 (blue) onto the structure of RXFP1 LDLa (orange) (Protein Data Bank code 2JM4). The side chains of Leu-7, Tyr-9, and Lys-17 overlay with good agreement. The overall r.m.s.d. of the two structures between residues 6 and 42 is 1.68 Å.
FIGURE 9.
FIGURE 9.
Overlay of the mean structure of SLYFP NITK-LB2 (blue), RXFP1 LDLa (orange), and LB2 (pink) represented as a cartoon model. The side chains of Lys-17 and equivalent Arg in LB2 are represented as sticks in addition to Asn-33 (RXFP1) and the equivalent Asn-32 from LB2. The overlay demonstrates that the chimera is more “RXFP1”-like despite the LB2 scaffold.
FIGURE 10.
FIGURE 10.
Proposed surface of the RXFP1 LDLa module involved in receptor activation. Mutagenesis studies confirm the importance of residues Leu-7, Tyr-9, and Lys-17. The positions of Leu-19 and Pro-20 suggest that they could contribute to the surface; however, the conservation of these residues throughout the LDLa module family suggests roles in structural maintenance.
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