Relaxin family peptide receptors--former orphans reunite with their parent ligands to activate multiple signalling pathways

Abstract

The relaxin family peptides, although structurally closely related to insulin, act on a group of four G protein-coupled receptors now known as Relaxin Family Peptide (RXFP) Receptors. The leucine-rich repeat containing RXFP1 and RXFP2 and the small peptide-like RXFP3 and RXFP4 are the physiological targets for relaxin, insulin-like (INSL) peptide 3, relaxin-3 and INSL5, respectively. RXFP1 and RXFP2 have at least two binding sites--a high-affinity site in the leucine-rich repeat region of the ectodomain and a lower-affinity site in an exoloop of the transmembrane region. Although they respond to peptides that are structurally similar, RXFP3 and RXFP4 demonstrate distinct binding properties with relaxin-3 being the only peptide that can recognize these receptors in addition to RXFP1. Activation of RXFP1 or RXFP2 causes increased cAMP and the initial response for both receptors is the resultant of Gs-mediated activation and G(oB)-mediated inhibition of adenylate cyclase. With RXFP1, an additional delayed increase in cAMP involves betagamma subunits released from G(i3). In contrast, RXFP3 and RXFP4 inhibit adenylate cyclase and RXFP3 causes ERK1/2 phosphorylation. Drugs acting at RXFP1 have potential for the treatment of diseases involving tissue fibrosis such as cardiac and renal failure, asthma and scleroderma and may also be useful to facilitate embryo implantation. Activators of RXFP2 may be useful to treat cryptorchidism and infertility and inhibitors have potential as contraceptives. Studies of the distribution and function of RXFP3 suggest that it is a potential target for anti-anxiety and anti-obesity drugs.

Figure 1

Structure of relaxin family peptide receptors (RXFP) 1–4. Snake plot diagram of predicted structure of (a) RXFP1, (b) RXFP2, (c) RXPF3 and (d) RXFP4. All receptors are seven TM spanning GPCRs. Major differences occur within the ectodomain. RXFP1 and RXFP2 have a large ectodomain containing LRRs and an LDL class A module. Contrastingly, RXFP3 and RXFP4 have shorter N-terminal tails than RXFP1 and RXFP2. Predicted N-linked glycosylation sites (Net-Gly 1.0 server) are indicated with Y and residues are colored blue, and predicted phosphorylation sites (Net-Phos 2.0 server) are indicated with ^* and colored in pink.

Figure 2

Alignment of the primary amino-acid sequences of equivalent RXFP1 and RXFP2 from human, dog, rat, mouse and pufferfish. Human, dog, rat and mouse sequences were obtained from NCBI. Pufferfish sequences were obtained by searching the pufferfish genome with the relevant human sequence using BLASTp. ^*Indicates identical amino-acid residues, : shows highly homologous amino-acid residues and . indicates residues with some homology between RXFP1 species receptors or RXFP2 species receptors; indicates identical amino-acid residues, shows highly homologous amino-acid residues and indicates residues with some homology between all receptors.

Figure 3

Alignment of the primary amino-acid sequences of equivalent RXFP3 and RXFP4 from human, dog, chicken, rat, mouse and pufferfish. Human, dog, chicken, rat and mouse sequences were obtained from NCBI. Pufferfish sequences were obtained by searching the pufferfish genome with the relevant human sequence using BLASTp. Multiple pufferfish sequences were found, but only two were selected for alignment based upon highest homology score with the human sequence. ^*Indicates identical amino-acid residues, : shows highly homologous amino-acid residues, and . indicates residues with some homology between RXFP3 species receptors, or RXFP4 species receptors; Indicates identical amino-acid residues, shows highly homologous amino-acid residues, and indicates residues with some homology between all receptors.

Figure 4

Signalling pathways activated by the RXFPs. (a) RXFP1 is activated primarily by relaxin and appears to signal mainly via cAMP. Initially RXFP1 increases cAMP accumulation via Gα_s and negatively modulates this via Gα_oB. These two pathways combined to affect CRE transcription. With time (dotted lines) RXFP1 recruits Gα_i3 which activates the Gβγ-PI3K-PKCζ pathway to further increase cAMP. RXFP1 also appears to activate GRE transcription (and possibly GR) by a currently unknown mechanism. (b) RXFP2 is activated by INSL3 (in addition to some relaxins, although this is species-specific) and both activates and negatively modulates cAMP accumulation by Gα_s and Gα_oB respectively, which leads to effects upon CRE transcription. (c) RXFP3 is activated by relaxin-3 and inhibits forskolin-stimulated cAMP accumulation. This receptor also activated ERK1/2 by a mechanism which requires receptor internalization or movement into lipid-rich signalling platforms, activation of PTX sensitive G proteins, PKC as well as Raf and MEK1/2. PI3K activation and EGF receptor transactivation (if expressed in the cell) are required for full ERK1/2 phosphorylation to occur, however about 50% of ERK1/2 will still be phosphorylated if these entities are blocked with specific inhibitors, (d) RXFP4 is activated by INSL5 and is also coupled to the inhibition of forskolin-stimulated cAMP accumulation.