Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

Relaxin Family Peptide Receptors RXFP3 and RXFP4

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_583

Synonyms

Historical Background: Relaxin Family Peptides and Their Receptors

Relaxin family peptides including the relaxins 1–3, insulin-like peptides (INSL) 3–6, and insulin-like growth factors I and II have a similar architecture to insulin. In the human, three independent genes produce three relaxin peptides, named relaxin-1, relaxin, and the most recently discovered relaxin-3 (Bathgate et al. 2013a; Halls et al. 2015). Relaxin-3 is classified by the presence of the characteristic RxxxRxxI/V relaxin-binding motif in the B-chain but otherwise has relatively low sequence homology to other relaxin peptides. Compared to other relaxins, relaxin-3 is well conserved across species (Wilkinson et al. 2005a; Yegorov et al. 2009), is believed to be the ancestral peptide (Wilkinson et al. 2005a), and in mammals is primarily a neuropeptide (Bathgate et al. 2002; Tanaka et al. 2005; Banerjee et al. 2010; Ma et al. 2007; McGowan et al. 2005; Ganella et al. 2013a, b; Ryan et al. 2013a, b; Smith et al. 2014). INSL5 is widely distributed with high expression in the gastrointestinal tract (Conklin et al. 1999; Mashima et al. 2013) particularly in L cells (Mashima et al. 2013; Thanasupawat et al. 2013) but also in ascending, transverse, and descending colon and proximal rectum, with lower levels in the cecum and distal rectum (Grosse et al. 2014). Low levels of Insl5 mRNA are found in the pancreas, thymus, and eye (Grosse et al. 2014). INSL5 knockout mice display dysfunctional glucose homeostasis (Burnicka-Turek et al. 2012). INSL5 activates RXFP4, but not RXFP1 or RXFP2, with high potency and is a weak antagonist at RXFP3 (Liu et al. 2005a).

The receptors for relaxin-3 and INSL5 are G protein-coupled receptors (GPCRs), where RXFP3 is the relaxin-3 receptor and RXFP4 is the INSL5 receptor. RXFP3 was discovered by probing a human cortical cDNA library (Matsumoto et al. 2000). Although it has high amino acid sequence similarity to the somatostatin and the angiotensin II receptors, it was not activated by these ligands, and therefore it was initially named somatostatin and angiotensin-like peptide receptor (SALPR). The ligand for RXFP3 was later discovered by using the receptor as bait to fish for peptides in extracts derived from various rat tissues (Liu et al. 2003a). Only the brain extracts increased GTPγS binding, leading to the identification of relaxin-3 (Liu et al. 2003a). RXFP4 was subsequently identified by searching the human genome database (Genbank™) with the RXFP3 sequence (Liu et al. 2003b). Since RXFP4 had 43% amino acid sequence identity with RXFP3, it was hypothesized that these receptors may share or have similar ligands (Liu et al. 2003b). Relaxin-3 was also found to activate RXFP4 as well as RXFP3 (Liu et al. 2003b), and INSL5 is a weak antagonist at RXFP3 as well as being the cognate ligand at RXFP4 (Liu et al. 2005a).

Molecular Biology of RXFP3 and RXFP4

Human RXFP3 is located on chromosome 5p15.1–5p14 (Matsumoto et al. 2000), and human RXFP4 is located on chromosome 1q22, and both receptors are coded by a single exon sequence. In the rat RXFP3 gene, there are two potential start codons (ATG) that are not seen in the human or the mouse genes (Wilkinson et al. 2005b). Transcription initiating from the first potential start codon produces rat RXFP3-long, with seven additional residues at the amino terminus. Transcription initiation from the second potential start codon produces rat RXFP3-short, which is equivalent to the human and mouse RXFP3 sequences. The mouse RXFP4 sequence has 74% homology with the human RXFP4 sequence, however, the gene equivalent to RXFP4 in the rat is a pseudogene that does not code a functional protein (Chen et al. 2005), suggesting that the INSL5-RXFP4 system is redundant in rats.

Structural Features and Functional Domains of RXFP3 and RXFP4 Receptors

RXFP3 and RXFP4 are classic rhodopsin family class A GPCRs with small N-terminal domains markedly different from RXFP1 and RXFP2. Unlike RXFP1 where both chains of relaxin-3 are required to activate the receptor, RXFP3 and RXFP4 can bind and be activated by the B-chain alone (Kuei et al. 2007; Hossain et al. 2008). Chimeric RXFP3 and RXFP4 receptors were used to identify potential functional roles for the extracellular domains and TM helices (Bathgate et al. 2013a; Zhu et al. 2008) using the knowledge that relaxin-3 binds to and activates both RXFP3 and RXFP4, while INSL5 only activates RXFP4. Chimeras with swapped N-terminal domains suggested that the N-terminus of RXFP4 is required for INSL5 and possibly relaxin-3 binding. Chimeras of RXFP3 with RXFP4 ECL1 or ECL3 domains demonstrated that these ECLs were not important for INSL5 binding to either receptor (Zhu et al. 2008). However chimeras with swapped ECL2 domains revealed that ECL2 in RXFP3 and RXFP4 are required for ligand binding and possibly receptor activation. Further, insertion of the N-terminus and ECL2 of RXFP4 into RXFP3 produced a chimera with full INSL5 binding, demonstrating that these domains are necessary for INSL5 binding in RXFP4 (Zhu et al. 2008). However, this chimera did not increase GTPγS binding showing that these domains are not sufficient for receptor activation by INSL5.

Extracellular Regions

A recent mapping of the relaxin-3 binding site in RXFP3 (Bathgate et al. 2013b) showed that the first 33 amino acids of the RXFP3 N-terminus are not required for binding. Relaxin-3 appeared to utilize multiple arginine residues to form electrostatic interactions with RXFP3 at glutamic and aspartic acid residues in the ECLs. Mutagenesis suggested that E141 and D145 in ECL1 and E244 in ECL2 were essential for binding. Homology modeling based on the crystal structure of CXCR4 (Wu et al. 2010) was used to dock the solution structure of relaxin-3 (Rosengren et al. 2006a) and showed that three acidic residues, E141, D145, and E244, likely coordinate binding to the three arginines in relaxin-3. This model allows the relaxin-3 C-terminal tail to insert into a classic “GPCR binding pocket” within the TM domains where relaxin-3 R26 can potentially form a salt bridge with RXFP3 E141 (Bathgate et al. 2013b). This model is consistent with the data showing that relaxin-3 residues in the core β-helix are involved in RXFP3 binding, while the relaxin-3 C-terminal RW residues likely drive activation by interactions in the RXFP3 TM core. A slightly different relaxin-3/RXFP3 interaction model has been recently described that is nonetheless still consistent with the activation mode described above (Zhang et al. 2014).

TM Spanning Regions

Chimeras replacing TM3 or TM5 of RXFP4 with those of RXFP3 show decreased affinity and no INSL5 activity (Zhu et al. 2008) (Bathgate et al. 2013a), suggesting that these regions are necessary for INSL5 binding and activation. However, the reverse chimera of RXFP3 with TM3 and TM5 from RXFP4 had increased INSL5 binding affinity but did not activate GTPγS binding (Zhu et al. 2008) demonstrating that TM3 and TM5 alone are not sufficient for INSL5 activation of RXFP3. A chimera of RXFP3 with TM2, TM3, TM5, and ECL2 of RXFP4 displayed similar affinity for both relaxin-3 and INSL5 to wild-type RXFP4 (Zhu et al. 2008), suggesting that all regions influence binding. Relaxin-3 activated GTPγS binding at this chimera similarly to wild-type RXFP4, and while INSL5 showed increased potency, it was still slightly lower than at wild-type RXFP4 (Zhu et al. 2008). This suggests that TM2, TM3, TM5, and ECL2 are all involved in ligand binding and activation of RXFP3 and RXFP4.

Ligands That Bind to RXFP3

Peptide Agonists

Relaxin-3 has a tertiary structure that resembles insulin and other relaxin family peptides (Rosengren et al. 2006b) but interacts with RXFP3 and RXFP4 in a manner different from RXFP1 (Liu et al. 2003a). Replacement of the A-chain with that of INSL5 (R3/I5) or shortening it does not influence RXFP3 binding or activation but reduces activity at RXFP1 (Hossain et al. 2008; Liu et al. 2005b). Deletion of the A-chain disulfide bond results in complete loss of RXFP1 activity but little effect on actions at RXFP3 (Shabanpoor et al. 2012). Further modifications produced a high-affinity, RXFP3-selective, competitive antagonist (analogue 3).

Site-directed mutagenesis of B-chain residues reveal that R8, R16, I5, and F20 are important for relaxin-3 binding to RXFP3 and RXFP4 (Kuei et al. 2007), with R12 also required for binding to RXFP3 but not RXFP4 (Kuei et al. 2007). R26 and W27 in the B-chain are required for activation of RXFP3 (Kuei et al. 2007). Truncation of the B-chain to C22 and addition of an arginine at the N-terminus, when combined with the A-chain of INSL5, led to a high-affinity RXFP3-selective antagonist R3(BΔ23-27)R/I5 (Kuei et al. 2007). A single-chain, easy to synthesize antagonist has since been developed based on the Β-chain of R3(BΔ23-27)R/I5 with the cysteine residues mutated to serine (H3 B1-22R) (Haugaard-Kedstrom et al. 2011).

Biased Agonists

Ligand-directed signaling bias is increasingly common in GPCR pharmacology (Baker and Hill 2007; Kenakin and Miller 2010; Evans et al. 2010). It is now clear that several relaxin peptides interact with RXFP3 to activate distinct signaling profiles through different, although sometimes overlapping, pathways (see below). The original studies suggested that relaxin-3 was selective for RXFP3 in both binding and AC inhibition assays, with no receptor activation by human relaxin or INSL3 (Liu et al. 2003a). However, cross-reactivity with other relaxin peptides was not examined over a wider range of signal transduction pathways, and the sensitivity of inhibitory cAMP assays can be influenced by the degree of activation of AC by forskolin and the time of stimulation (for detailed description see below).

Allosteric Modulators

A novel selective allosteric modulator of RXFP3 has been described (Alvarez-Jaimes et al. 2012). 3-[3,5-Bis(trifluoromethyl)phenyl]-1-(3,4-dichlorobenzyl)-1-[2- (5-methoxy-1H-indol-3-yl)ethyl]urea (135PAM1) is a positive allosteric modulator (PAM) that displays activity with C-terminal amidated relaxin-3 or R3/I5 (probe selectivity). Binding studies conducted in HEK293-RXFP3 cell membranes showed that 135PAM1 does not compete directly with 125I R3/I5(amide) binding at up to 1 μM but at higher concentrations enhances binding consistent with activity as a PAM. As expected the R3/I5(amide) peptide competes for 125I R3/I5(amide) binding in a conventional manner (Alvarez-Jaimes et al. 2012). In HEK293-RXFP3 cells co-expressing Gαqi5, 135PAM1 increased calcium responses to EC20 concentrations of relaxin-3(amide) or R3/I5(amide) but not the free-acid (OH) peptides. Concentration-response curves to the amidated but not the free-acid form of the peptides were shifted in a limiting manner by 135PAM1. Similar specificity of 135PAM1 was also shown in a reporter gene assay that measured inhibition of CRE activity in cells expressing RXFP3. Although it is the only published example, 135PAM1 does identify an allosteric site on RXFP3 that can be modulated by small molecules.

Antagonists

R3(BΔ23-27)R/I5 was derived from R3/I5 by truncation of the relaxin-3 B-chain (Kuei et al. 2007; Hossain et al. 2009). Recombinant production of R3(BΔ23-27)/I5 introduced the extra arginine at the N-terminus to produce a peptide that binds to RXFP3 and RXFP4 with high affinity and is an antagonist. Synthetic R3(BΔ23-27)/I5 (lacking R23) has lower affinity at RXFP3 and retains weak agonist properties (Hossain et al. 2009) highlighting that the extra R23 creates an additional interaction with RXFP3. Administration of R3(BΔ23-27)R/I5 i.c.v. to rats blocks food intake stimulated by R3/I5. However, R3(BΔ23-27)R/I5 has a complex antagonist profile and blocks some but not all pathways activated by RXFP4 and has weak biased agonist properties (see below). Subsequent studies have shown that the intra-A-chain disulfide bond in relaxin-3 is not important for interaction with RXFP3 but is necessary for interaction with RXFP1. Analogues without the A-chain disulfide bond and 10 amino acids removed from the N-terminus of the A-chain remain potent RXFP3 agonists. If in addition, GGSRW is removed from the B-chain and replaced with R, this produces a high-affinity antagonist with similar activity to R3(BΔ23-27)R/I5 (Shabanpoor et al. 2012).

Further modifications have produced a single-chain high-affinity RXFP3 antagonist H3 B1-22R that is far easier to produce than the two chain peptides (Haugaard-Kedstrom et al. 2011), is specific for RXFP3, and has no activity at RXFP4. In rats, the peptide blocks increases in feeding produced by i.c.v. R3/I5 (Haugaard-Kedstrom et al. 2011) and has been used to demonstrate a role for the relaxin-3 system in addiction (Ryan et al. 2013b) and in motivated food seeking and consumption in mice (Smith et al. 2014).

The structure-activity relationships involved in the interaction of relaxin peptides with RXFP3 are now well understood. Unlike RXFP1 and RXFP2, RXFP3 can be activated by peptides comprising only the B-chain, and a number of selective agonists and antagonists have been developed. Both relaxin and some RXFP3 antagonists display biased agonist properties at RXFP3. The utility of a small molecule allosteric modulator in vivo is currently limited by probe selectivity.

Ligands That Bind to RXFP4

Peptides

Insulin-like peptide 5 (INSL5) is the cognate ligand for RXFP4 (Liu et al. 2005a) and is secreted from enteroendocrine L cells and has been shown to regulate insulin secretion and glucose homeostasis (Burnicka-Turek et al. 2012). Most of the characterization of RXFP4 signaling has been carried out with mouse INSL5 because of difficulties associated with synthesis of the human peptide (Belgi et al. 2011). While relaxin-3 binds both RXFP3 and RXFP4 with high affinity, INSL5 is a weak antagonist at RXFP3 (Zhu et al. 2008). Although the relaxin-3 B-chain alone binds to and activates RXFP3, the INSL5 A- and B-chains alone are inactive at RXFP4 (Belgi et al. 2013).

The relaxin-3 B-chain alone is a weak agonist at RXFP4 (Liu et al. 2003a, b). Up to seven B-chain residues of relaxin-3 can be deleted with little effect on activity at RXFP4 (Kuei et al. 2007; Hossain and Wade 2010), whereas the α-helical region of the B-chain is important (Hossain and Wade 2010). R12 and R16 influence binding to both RXFP1 and RXFP3, while only R16 is important for RXFP4 binding (Kuei et al. 2007). A critical residue for RXFP4 and RXFP3 binding is F20, and changes cause a marked loss of affinity (Kuei et al. 2007; Hossain and Wade 2010). The C-terminus of the relaxin-3 B-chain also has a role in activation of RXFP4, and mutations in this region block agonist activity without influencing affinity (Hossain and Wade 2010).

The potential of RXFP4 as a therapeutic target and the difficult synthesis of human INSL5 have led to a search for easily synthesized and selective analogues. Removal of the first eight residues of the relaxin-3 A-chain produces a peptide with similar binding affinity and potency to relaxin-3 at RXFP3 and RXFP4 but no activity at RXFP1 (Shabanpoor et al. 2012). This activity is retained in a peptide where the B-chain is truncated by nine residues (Shabanpoor et al. 2012). These studies suggest that the C-terminus of the B-chain and the interchain disulfide bonds are the major structural features required for activity at RXFP4. Recent studies suggest that unlike the relationship between relaxin-3 and RXFP-3, the INSL5 A-chain is important for interaction with RXFP4. However, simpler analogues have been designed without the intrachain disulfide bond that retain activity but are much easier to synthesize (Patil et al. 2016).

Antagonists

Several RXFP3 antagonists also have antagonist properties at RXFP4 including minimized relaxin-3 analogue 3 (minimized A-chain and truncated B-chain) (Shabanpoor et al. 2012) and R3(BΔ23-27)R/I5 (Kuei et al. 2007). However, the single-chain variant of this peptide H3 B1-22R does not bind to RXFP4 (Haugaard-Kedstrom et al. 2011) again emphasizing the role of the A-chain in RXFP4 binding (Patil et al. 2016).

Much less information is available on RXFP4 compared to the other family members, but this has been accelerating due to the identification of INSL5 as a gut hormone. There is now a good understanding of the mode of interaction of INSL5 with RXFP4 and an increasing range of synthetic peptide agonists and antagonists available.

Signal Transduction Pathways of RXFP3 and RXFP4

RXFP3 Signaling

Canonical Signaling Pathways

RXFP3 is a Gαi/o-coupled receptor as evidenced by PTX-sensitive inhibition of forskolin-stimulated cAMP accumulation by relaxin-3 (Liu et al. 2003a). Activation of RXFP3 is also associated with PI3K- and PKC-dependent phosphorylation of ERK1/2 and other MAPKs (Fig. 1) (van der Westhuizen et al. 2010, 2007). Both human relaxin-3 and its Β-chain compete for 125I-human relaxin-3 binding and inhibit AC activity (Liu et al. 2003a), but subsequent studies show that human relaxin also interacts with human RXFP3 to activate a unique signaling pattern (see below) (Fig. 1) (van der Westhuizen et al. 2010).
Relaxin Family Peptide Receptors RXFP3 and RXFP4, Fig. 1

Relaxin family peptide receptor signaling pathways activated downstream of RXFP3 following activation by relaxin-3 (canonical signaling), relaxin-3 amide in the presence of 135PAM1 (allosteric modulation), or relaxin (biased signaling). RXFP3 inhibits cAMP production by coupling to inhibitory G proteins (Gαi/o) and activates extracellular signal-regulated kinase (ERK)1/2 and either a phosphatidylinositol 3-kinase (PI3K)- or protein kinase C (PKC)-dependent pathway. RXFP3 activation promotes increased gene transcription from nuclear factor (NF)-κB and activator protein (AP)-1 transcription factors. MAPK indicates either ERK, p38 MAPK, or JNK, and TK indicates receptor tyrosine kinase

Studies in CHO-K1 cells transiently transfected with PTX-insensitive (C351I mutation) variants of Gαi/o proteins, and treated with PTX to remove the influence of endogenous Gαi/o proteins, have identified Gαi2 as the major G protein involved in inhibition of cAMP accumulation, whereas in HEK293 cells, Gαi3, GαoB, and GαoA were all involved (Van der Westhuizen 2008). Thus the G proteins involved although broadly similar can vary with the different cell types. Although the response can vary with different peptides, each peptide tends to produce consistent effects within a cell type. Thus, similar effects are observed with human relaxin-3 B-chain peptides or with human relaxin and porcine relaxin in the CHO-K1, HEK293, and SN56 cell backgrounds (Liu et al. 2003a; van der Westhuizen et al. 2010, 2007).

Human relaxin-3 causes a rapid and transient increase in ERK1/2 phosphorylation in CHO-K1 and HEK293 cells stably expressing human RXFP3 (Fig. 1) (van der Westhuizen et al. 2010, 2007) that is inhibited by PTX (van der Westhuizen et al. 2007). Thus coupling of RXFP3 to PTX-sensitive Gαi/o proteins mediates ERK1/2 phosphorylation, in both recombinant and endogenous systems (van der Westhuizen et al. 2007). There are two pathways downstream of Gαi/o involved in RXFP3-mediated ERK1/2 phosphorylation in CHO-K1, HEK293, and SN56 cells. About 50% of the MAPK response is blocked by the PI3K inhibitors LY294002 or Wortmannin, while the remainder is inhibited by general and isoform-selective PKC inhibitors (van der Westhuizen et al. 2007). The ERK1/2 response appears to be involved in central feeding responses in rats (Morikawa et al. 2004; Shen et al. 2004; Sasaguri et al. 2005), suggesting that it is physiologically relevant.

Signaling pathway analysis using reporter genes gives a broader view of the signal transduction mechanisms activated by RXFP3. Since many MAPK signaling pathways (p38 MAPK; Roux and Blenis 2004), JNK (Davis 2000), and ERK1/2 (Price et al. 1996; Whitmarsh and Davis 1996) converge on AP-1 elements to increase gene transcription, AP-1-linked reporter genes and selective inhibitors can provide useful information on signaling in different cellular backgrounds. PTX blocks RXFP3-mediated AP-1 reporter activation in SN56 cells but not in CHO-RXFP3 and HEK-RXFP3 cells (van der Westhuizen et al. 2010) suggesting that AP-1 reporter gene activation was downstream of Gαi/o in the mouse-derived cell line but not in the other two cell types (van der Westhuizen et al. 2010). Human relaxin-3-mediated AP-1 reporter gene activation in CHO-RXFP3, and SN56 cells is completely blocked by the p38 MAPK inhibitor (RWJ67657), whereas MEK (PD98059) or JNK inhibition (SP600125) produced only partial blockade. In contrast, in HEK-RXFP3 cells, JNK inhibition completely blocked human relaxin-3-stimulated AP-1 reporter activation, whereas p38 MAPK or MEK inhibition partially blocked AP-1 activation (van der Westhuizen et al. 2010). This suggests that while all three MAPKs are involved in human relaxin-3-mediated AP-1 activation (Fig. 1), the hierarchy of the different signaling pathways varies with the cell background. Several studies show that MAPK signaling is activated in forced swim tests in rats, where there are dramatic increases in pMEK1/2, pERK1/p2, and pJNK1/2/3 (Shen et al. 2004). Although this is associated with an increase in relaxin-3 mRNA in the nucleus incertus (NI) (Tanaka et al. 2005), direct links between human relaxin-3, RXFP3, MAPK phosphorylation, and stress responses remain to be demonstrated in the brain.

In another reporter gene assay, in CHO-K1 and HEK293 cells transiently expressing human RXFP3, and in SN56 cells endogenously expressing mouse RXFP3, activation of RXFP3 by human relaxin-3 increased NFκB reporter gene activation (van der Westhuizen et al. 2010). Activation of NFκB was blocked by PTX pretreatment (van der Westhuizen et al. 2010) suggesting that the response occurs downstream of Gαi/o. The physiological significance of this pathway remains to be determined.

Ligand-Directed Signaling Bias at RXFP3

Ligand-directed signaling bias has been described for relaxin-3, relaxin (Van Der Westhuizen et al. 2005), and the RXFP3 antagonist R3(BΔ23-27)R/I5 (Kocan et al. 2014). The first indication of a functional relaxin-RXFP3 interaction came from studies using microphysiometry (Van Der Westhuizen et al. 2005) where human relaxin caused a small change in the extracellular acidification rate in CHO-RXFP3 cells. In CHO-K1, HEK293, and SN56 cell backgrounds, subsequent studies demonstrated that human relaxin, porcine relaxin, and human INSL3 caused weak inhibition of forskolin-stimulated cAMP accumulation (van der Westhuizen et al. 2010). Interestingly, the ability of INSL3 to activate RXFP3 appears to be specific for the human but not the mouse receptor (van der Westhuizen et al. 2010). Previous studies failed to report inhibition of cAMP accumulation by either porcine relaxin or human INSL3 (Liu et al. 2003a), but since sensitivity of inhibitory cAMP assays is highly dependent on both the degree of activation of AC by forskolin and the time of stimulation, the differences observed most likely result from different experimental paradigms.

Relaxin and porcine relaxin also caused AP-1 reporter gene activation (Fig. 1) (van der Westhuizen et al. 2010) with an order of potency relaxin > relaxin-3 > porcine relaxin. Again, some AP-1 activation appears to be independent of Gαi/o coupling, as pretreatment with PTX failed to block porcine relaxin-stimulated AP-1 reporter gene activation in CHO-RXFP3 cells. Thus, as for relaxin-3, porcine relaxin can activate AP-1 reporter genes by a Gαi/o-independent mechanism, suggesting ligand-directed signaling bias (van der Westhuizen et al. 2010). In contrast, all of the AP-1 reporter gene responses observed after stimulation of RXFP3 in SN56 cells were blocked by PTX, suggesting that different pathways were involved in mediating the response downstream of the mouse RXFP3 receptor (van der Westhuizen et al. 2010).

The downstream signaling pathways activated by RXFP3 are strongly influenced by the cell background. Thus in CHO-RXFP3 cells, the AP-1 response to relaxin is strongly inhibited by the p38 MAPK inhibitor (RWJ67657) or JNK inhibitor (SP600125), whereas the MEK inhibitor (PD98059) has only a weak effect, implicating p38 MAPK and JNK as the major MAPKs involved in the response to relaxin (Fig. 1). In HEK293 cells, relaxin-stimulated AP-1 reporter activation was decreased by p38 MAPK or MEK inhibition but not by JNK inhibition, suggesting that p38 MAPK and ERK were the major MAPKs involved in these cells. In SN56 cells that endogenously express mouse RXFP3, p38 MAPK, JNK, or MEK inhibition was equally effective in blocking the response to relaxin, suggesting equal contributions (van der Westhuizen et al. 2010). Direct measurement of pERK1/2, p38 MAPK, and pJNK following addition of relaxin family peptides has confirmed the findings of these inhibitor-based studies (Kocan et al. 2014) although the Gαi/o-independent pathway remains to be identified for relaxin-3 and porcine relaxin. Direct MAPK assays in CHO-RXFP3 cells show that relaxin activates p38 MAPK and ERK1/2 with lower efficacy than relaxin-3 but the two peptides have similar efficacy for JNK1/2/3 phosphorylation. Both relaxin and relaxin-3 activation of p38 MAPK, JNK1/2/3, or ERK1/2 involved PTX-sensitive G proteins (van der Westhuizen et al. 2010, 2007; Kocan et al. 2014).

The RXFP3 antagonist R3(BΔ23-27)R/I5 also shows bias and blocked relaxin-3 AP-1 reporter gene activation but not relaxin AP-1 activation or relaxin-3 NFκB activation (Kocan et al. 2014). R3(BΔ23-27)R/I5 itself activated the SRE reporter but did not inhibit activation by relaxin or relaxin-3. R3(BΔ23-27)R/I5 also blocked relaxin-3-stimulated p38MAPK and ERK1/2 phosphorylation, acting as a partial agonist at these pathways. Interestingly, p38MAPK activation by R3(BΔ23-27)R/I5 was G protein independent. BRET studies of interactions between RXFP3 and G proteins showed that relaxin-3-activated RXFP3 interacts with Gαi2, Gαi3, GαoA, and GαoB, whereas relaxin or R3(BΔ23-27)R/I5 promoted interactions only with Gαi2 or GαoB. Only relaxin-3 promoted RXFP3/β-arrestin interactions that were blocked by R3(BΔ23-27)R/I5 (Kocan et al. 2014). This is compelling evidence for ligand-directed signaling bias at RXFP3.

Allosteric Modulation of RXFP3

The RXFP3 allosteric modulator 135PAM1 has only been examined in receptor binding studies and in recombinant systems expressing the chimeric G protein GαqI5 or a CRE reporter gene (Alvarez-Jaimes et al. 2012). 135PAM1 enhanced CRE inhibition and also increased 125I R3/I5NH2 binding (Fig. 1). It is not known what changes in the pattern of signaling if any are observed in systems that naturally express RXFP3, and given that 135PAM1 has poor solubility and displays selectivity for the C-terminal amides that are not naturally occurring, it has limited use experimentally (Alvarez-Jaimes et al. 2012).

RXFP4 Signaling

Canonical Signaling Pathways

RXFP4, like RXFP3, is a Gαi/o-coupled receptor. Stimulation of cells expressing human RXFP4 by INSL5 or relaxin-3 increases GTPγS binding and inhibits forskolin-stimulated cAMP accumulation (Fig. 2) (Liu et al. 2005a, b). Co-expression of RXFP4 with the promiscuous G protein Gα16 in HEK293 cells produces a strong calcium signal in response to INSL5 and human relaxin-3 (Liu et al. 2005a, b) that is not present in normal cells. Recent studies have confirmed that INSL5 acts on RXFP4 to inhibit forskolin-stimulated cAMP accumulation but also to produce increased phosphorylation of ERK1/2, p38MAPK, Akt-Ser473, Akt-Thr308, and S6 ribosomal protein (S6RP) (Fig. 2) (Ang et al. 2016). The cAMP and ERK1/2 responses were abolished by PTX and rescued by transfection of PTX-resistant mutants of G proteins particularly mGαoA, mGαoB, and mGαi2 and to a lesser extent mGαi1 and mGαi3 (Ang et al. 2016). It was also demonstrated that INSL5-activated RXFP4 interacts with GRK2 and β-arrestins and moves toward Rab5a and away from KRas, indicating receptor internalization (Ang et al. 2016). In cells that endogenously express RXFP4, INSL5 treatment inhibited glucose-stimulated insulin secretion and Ca2+ mobilization in MIN6 insulinoma cells and forskolin-stimulated cAMP accumulation in NCI-H716 enteroendocrine cells (Ang et al. 2016).
Relaxin Family Peptide Receptors RXFP3 and RXFP4, Fig. 2

Relaxin family peptide receptor signaling pathways activated downstream of RXFP4 following activation by INSL5 (canonical signaling). RXFP4 inhibits cAMP production by coupling to inhibitory G proteins (Gαi/o) and activates extracellular signal-regulated kinase (ERK)1/2 and p38MAPK through a Ras-dependent pathway. It also activates PI3 kinase and Akt leading to phosphorylation of S6RP. The receptor undergoes phosphorylated by GRK2 leading to recruitment of β-arrestins, desensitization, and internalization

Thus both RXFP3 and RXFP4 are Gαi/o-coupled receptors that show GTPγS binding and inhibition of cAMP accumulation. More extensive studies of RXFP3 and RXFP4 signaling have also revealed coupling to MAP kinases as well as in the case of RXFP3, ligand-directed signaling bias, and allosteric modulation.

Localization of RXFP3 and RXFP4

RXFP3 is highly expressed in human, rat, and mouse brain as determined by RT-PCR (Matsumoto et al. 2000), Northern blotting, and in situ hybridization (Liu et al. 2003a; Boels et al. 2004). In rat brain, high levels of mRNA are found in the olfactory bulb, paraventricular and supraoptic nuclei, and preoptic and posterior areas of the hypothalamus, hippocampus, septum, and amygdala with lower levels in cortex, periaqueductal gray, nucleus incertus, and areas of brainstem (Liu et al. 2003a; Sutton et al. 2004). 125I-R3/I5 has been used to identify RXFP3 binding sites in the cerebral cortex, olfactory bulb, and superior colliculus in the rat (Liu et al. 2005b). Recent studies of the distribution of RXFP3 in mouse brain (Smith et al. 2010) by both in situ hybridization and peptide binding suggest a similar pattern to that in the rat with some differences notably in the substantia innominata and the olivary and posterior pretectal nuclei and in the rat expression in the olfactory bulb, entorhinal cortex, arcuate nucleus, and paraventricular thalamic nucleus being more dominant (Smith et al. 2010). As yet, there are no systematic studies of RXFP3 localization in the human or primate brain. In human peripheral tissues, there is low expression of RXFP3 mRNA in the adrenal gland, testis, salivary gland, and pancreas by RT-PCR (Liu et al. 2003a).

Studies prior to the deorphanization of RXFP4 examined the distribution of the then orphan GPCR GPR100 using Northern blotting in human tissues. Expression was principally in peripheral tissues including the heart, skeletal muscle, salivary gland, bladder, kidney, liver, placenta, stomach, jejunum, thyroid, ovary, and bone marrow with the highest expression in the pancreas (Boels and Schaller 2003). Subsequent studies following deorphanization showed high expression by RT-PCR in the human colon but also in placenta, testis, thymus, prostate, kidney, and brain (Liu et al. 2003b). The expression of RXFP4 in the colon matches the expression of the INSL5 peptide and indicates potential functions of INSL5 as a gut hormone.

Physiological Roles of Relaxin-3

Relaxin-3 and RXFP3 expression is highest in the brain, and evidence supports roles in stress, feeding, and metabolism as well as behavioral activation and arousal. Although relaxin-3 does interact with RXFP1 (Sudo et al. 2003) and RXFP4 (Liu et al. 2003b) and possibly RXFP2 in some species (Scott et al. 2005), there is no evidence that these interactions are important physiologically as the expression pattern of relaxin-3 suggests that it would be unlikely to interact with the other RXFP receptors.

The evidence that suggests that relaxin-3 and RXFP3 are involved in stress includes their co-localization in hypothalamic and extra-hypothalamic regions (Ma et al. 2007; Liu et al. 2005b; Sutton et al. 2004; Smith et al. 2010). RXFP3 is highly expressed in the PVN, as well as in other regions associated with stress and anxiety, such as the bed nucleus of the stria terminalis, lateral septum, periaqueductal gray, and dorsal raphe (Liu et al. 2005b). There is considerable overlap between the relaxin-3 and CRF systems, and the CRF1 receptor is expressed in most relaxin 3-containing neurons in the NI (Tanaka et al. 2005; Sutton et al. 1982). Administration of CRF i.c.v. to rats caused activation of relaxin-3-containing neurons, and neurogenic stressors increased relaxin-3 mRNA in the NI (Tanaka et al. 2005). Relaxin-3 mRNA in the rat NI is increased following a forced swim stress paradigm (Banerjee et al. 2010), and these effects are inhibited by pretreatment with the CRF1 antagonist, antalarmin. Altered relaxin-3 expression in the NI due to stress is unlikely to result from the low levels of CRF in the spinal fluid and most likely comes from CRF neuronal projection(s). Ascending projections from the NI (Olucha-Bordonau et al. 2003) are consistent with RXFP3 receptor autoradiography (Sutton et al. 2004) and with involvement of relaxin-3/RXFP3 in neuropsychiatric disease (Smith et al. 2010).

The expression pattern of RXFP3 also suggests a role for the relaxin-3 system in feeding and metabolism. In rodents, RXFP3 is expressed in the PVN and supraoptic nucleus (SON) (Smith et al. 2010, 2011), and injection of relaxin-3 i.c.v. to rats, either in the early light or early dark phase, transiently increased food intake (McGowan et al. 2005). This effect did not result from increased spontaneous activity or arousal and is mediated by RXFP3 (McGowan et al. 2005). Both acute and chronic relaxin-3 infusion into the PVN increases food intake (McGowan et al. 2006) as does, i.c.v. infusion of R3/I5, an effect blocked by pre-administration of R3(BΔ23-27)R/I5 (Kuei et al. 2007). Administration of R3(BΔ23-27)R/I5 i.c.v. alone has no effect on food intake (Sutton et al. 2009), suggesting that under resting conditions, there is minimal tone in the relaxin-3/RXFP3 system.

In spite of effects on food intake, there is no agreement on effects on body weight since in some studies, chronic i.c.v. relaxin-3 increased body weight (Hida et al. 2006) (Sutton et al. 2009), whereas others show no weight change following i.c.v. or intra-hypothalamic injection of relaxin-3 (McGowan et al. 2005, 2006). In rats, chronic i.c.v. administration of the antagonist R3(BΔ23-27)R/I5 did not affect body weight (Sutton et al. 2009). There is also little evidence to suggest that body weight is influenced by endogenous relaxin-3. Although relaxin-3 knockout mice were initially reported to have lower body weights, this study (Sutton et al. 2009) was performed on a mixed 129:B6 mouse strain before the mice were backcrossed, and the difference was not seen on a C57/Bl6N background. This dependence of phenotype on strain suggests there is compensation for a chronic absence of relaxin-3 by other systems.

Studies investigating the effects of relaxin-3 infusion on feeding also measured blood hormone levels. In rats, chronic infusion of relaxin-3 or R3/I5 i.c.v. increased plasma leptin, insulin (Hida et al. 2006), adiponectin, testosterone, and angiotensinogen and decreased growth hormone levels (Sutton et al. 2009). Administration into the PVN caused increases in leptin in ad libitum fed animals and in TSH in ad libitum and pair fed animals (McGowan et al. 2006). iPVN agonist dosing increased feeding but failed to significantly change other behaviors such as drinking, grooming, burrowing, rearing, general locomotion, apparent sleep, head down, or tremor (McGowan et al. 2005). Similarly, chronic R3(BΔ23-27)R/I5 seemed only to decrease plasma levels of growth hormone (Sutton et al. 2009) while not altering the other hormones measured. These effects occurred in the absence of changes in energy expenditure.

There is relatively little evidence for a direct role for the relaxin-3/RXFP3 system in metabolism. While there is RXFP3 expression in the pancreas and in vitro relaxin-3 suppression of insulin secretion has been reported in isolated tissue (Yamamoto et al. 2009), human plasma relaxin-3 levels are in the low picomolar range and do not vary in the diabetic state (Zhang et al. 2013). The metabolic effects of an RXFP3 agonist may therefore be largely secondary to increased feeding behavior. As noted above, central injection of an RXFP3 agonist in rats increased feeding both acutely (McGowan et al. 2005) and chronically (Sutton et al. 2009; Hida et al. 2006), suggesting a lack of tolerance, and chronic viral expression of an RXFP3 agonist has similar effects (Ganella et al. 2013a, b). In contrast in mice, RXFP3 agonist treatment does not increase feeding (Ganella et al. 2013b), and while an RXFP3 antagonist is ineffective in altering feeding in baseline/sated mice, it blocked motivated feeding behavior (Smith et al. 2014). Thus, R3(B1-22)R/I5 blocked food anticipatory activity after a 4 h food restriction when administered i.c.v. in C57/Bl6J control mice, but not in congenic relaxin-3 knockout mice. Antagonist treatment also significantly reduced feeding in mice trained to expect palatable food at a given time.

There is also evidence that the relaxin-3/RXFP3 system has a role in behavioral activation and arousal. The rodent septohippocampal pathway is heavily innervated by relaxin-3-positive projections from the NI and generates the hippocampal theta rhythm, with oscillations at 4–12 Hz controlled by pacemaker neurons of the medial septum (MS). Theta rhythm is involved in behaviors such as vigilance, exploration, orientation, navigation, locomotor control, and working memory. Electrical stimulation of the NI causes theta rhythm in the hippocampus and lesions of the NI disrupt theta rhythm initiated by stimulation of the reticularis pontine oralis (Nunez et al. 2006). In both anesthetized and conscious rats, RXFP3 modulates neuronal activity in the hippocampus and MS to promote hippocampal theta rhythm (Ma et al. 2009), and blockade of RXFP3 in the MS with the antagonist R3(BΔ23-27)R/I5 dose dependently impairs performance in a paradigm investigating theta rhythm-dependent spatial working memory, effects that are reversed by co-administration of the RXFP3-selective agonist, R3/I5 (Ma et al. 2009).

The relaxin-3/RXFP3 system also influences locomotor activity. While chronic i.c.v. relaxin-3 did not affect locomotor activity in male Wistar rats (Hida et al. 2006), acute injection of the RXFP3 agonist R3/I5 increased locomotor activity, and R3(BΔ23-27)R/I5 had no effect (Sutton et al. 2009). Effects of the RXFP3 agonist on general locomotor activity, while statistically significant in one study (Sutton et al. 2009), seem to be small in magnitude and were not reproduced following iPVN injections (McGowan et al. 2005). Interestingly, female relaxin-3 knockout mice were hypoactive relative to WT littermates in several paradigms including the locomotor cell, large open field, Y maze, and novel object tests (Smith et al. 2009; Hosken et al. 2014). A similar phenotype is displayed in mice receiving the RXFP3 antagonist R3(Β1-22)R/I5 or R3(Β1−22)R (Smith et al. 2014).

Since most relaxin-3-containing neurons of the NI also co-express 5-HT1A receptors and inhibition of 5-HT synthesis for 3 days increased relaxin-3 mRNA in the NI, there appears to be a role for both systems in anxiety and/or depression. Recent studies show anxiolytic and antidepressant effects of i.c.v. RXFP3 agonist dosing in behavioral rat models (Ryan et al. 2013a). These studies emphasize the need for more studies to determine the relationship between 5-HT and relaxin-3/RXFP3 systems.

Recent studies in rats link RXFP3 circuits to ethanol self-administration and reinstatement behaviors (Ryan et al. 2013b). The R3(BΔ23-27)R/I5 or R3(B1-22)R RXFP3 antagonists administered i.c.v. reduced alcohol self-administration without concomitant alterations in feeding, locomotor behaviors, or memory. The effects on alcohol self-administration were specific, as the antagonists had no effect on sucrose self-administration. In addition, RXFP3 antagonism blocked both cue- and stress-induced reinstatement of alcohol seeking. The reduction of alcohol self-administration/reinstatement was linked to the stress-responsive bed nucleus of the stria terminalis (BNST), as direct injection of the RXFP3 agonists into the BNST also reduced alcohol self-administration.

Extensive brain mapping and behavioral studies have established that the relaxin-3 and RXFP3 system is involved in stress, metabolic control, and behavioral activation and arousal. Recent studies suggest that modulation of the system could be useful in the treatment of forms of addiction.

Physiological Roles of INSL5

RXFP4 mRNA is present in a variety of human tissues including the brain, kidney, testis, thymus, placenta, prostate, salivary gland, thyroid, and colon (Mashima et al. 2013; Thanasupawat et al. 2013; Burnicka-Turek et al. 2012; Liu et al. 2005a). Many of these tissues also express INSL5 (Mashima et al. 2013; Thanasupawat et al. 2013; Burnicka-Turek et al. 2012), and both RXFP4 and Insl5 are pseudogenes in rats and dogs (Wilkinson et al. 2005b; Chen et al. 2005). INSL5 was shown to be a high-affinity ligand for RXFP4 (Liu et al. 2005a), and thus the receptor-ligand coevolution, pharmacology, and similarities in expression profiles establish that RXFP4 is the endogenous receptor for INSL5.

Currently available evidence suggests that the INSL5/RXFP4 system has a metabolic role. Like GLP-1, INSL5 is concentrated and released from enteroendocrine L cells (Grosse et al. 2014) into the circulation to act on RXFP4 that in the mouse is expressed in the hypothalamus, pituitary, testis, epididymis, ovary, uterus, pancreas and liver, and also pancreatic islets, anterior pituitary, and Leydig cells in the testis (Burnicka-Turek et al. 2012). In humans, RXFP4 was detected in the heart, placenta, skeletal muscle, and pancreas (Mashima et al. 2013). Insl5 −/− mice have impaired glucose homeostasis, and in mice older than 6 months, glucose levels are significantly greater than in age-matched control littermates. Glucose tolerance tests revealed impaired glucose tolerance but no change in insulin sensitivity in Insl5−/− mice. The knockouts also had reduced pancreatic islet area and a reduced number of β-cells and lower circulating insulin levels. However there was no change in circulating GLP-1 (Burnicka-Turek et al. 2012). Insl5−/− mice also displayed impaired fertility due to a reduction in sperm motility and alterations in the estrus cycle (Burnicka-Turek et al. 2012). Polymorphisms of RXFP4 in humans are associated with a high body mass index (BMI) and show a trend to association with obesity (Munro et al. 2012). Studies in mice show increased food intake in WT mice following i.p. injection of INSL5 that is not seen in RXFP4−/− mice (Grosse et al. 2014). Plasma INSL5 levels increase with fasting or calorie restriction but are lowered by feeding adding weight to the suggestion that INSL5 is an orexigenic hormone released from the enteroendocrine L cells in the gut (Grosse et al. 2014). At present, little is known of the potential mechanisms utilized by INSL5 and RXFP4 to produce these metabolic effects, and a number of apparent anomalies will have to be addressed. For instance, to date all β-islet cell GPCRs that increase insulin secretion are either Gαs or Gαq coupled, and Gαi-coupled receptors actually reduce insulin secretion (Ahren 2009). In accord with this paradigm, recent studies using MIN6 insulinoma cells demonstrated decreased insulin secretion in response to INSL5 (Ang et al. 2016).

Summary

RXFP3 is a Gαi/o-coupled G protein-coupled receptor that activates many signaling pathways in various cell lines and exhibits different but overlapping signaling profiles dependent upon the activating ligand. On stimulation with relaxin-3, RXFP3 inhibits cAMP production and activates MAP kinase signaling pathways. RXFP3 also increases gene transcription from AP-1 and NF-κB promoters, which may be important in regulating feeding and stress responses mediated by relaxin-3 in rats. Ligand-directed signaling bias has also been observed with relaxin and some other related peptides that activate a subset of pathways activated by relaxin-3, but this is unlikely to have physiological significance due to differences in location of peptides and RXFP3. An allosteric modulator has been identified but displays probe dependence that results in activity only when receptors are activated by C-terminal amide peptides that are not naturally occurring. Linking these signaling pathways activated by relaxin-3 to pathophysiology should encourage the design and development of antianxiety and antiobesity therapies that specifically target particular RXFP3 signaling pathways. RXFP4 like RXFP3 is a Gαi/o-coupled receptor that inhibits cAMP production and activates MAP kinases. Recent studies indicate increased phosphorylation of ERK1/2, p38MAPK, Akt, and S6RP. The synthesis and release of INSL5 from enteroendocrine L cells, together with a metabolic phenotype in some animal models, suggest a metabolic role. Extensive studies of RXFP4 will be required to determine its physiological role and its importance in pathophysiology and to assess its potential as a drug target.

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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  • Martina Kocan
    • 1
  • Sheng Yu Ang
    • 2
  • Roger J. Summers
    • 2
  1. 1.Neuropeptides Division, Florey Institute of Neuroscience and Mental Health and Department of Biochemistry and Molecular Biology, University of MelbourneParkvilleAustralia
  2. 2.Drug Discovery Biology, Monash Institute of Pharmaceutical SciencesMonash UniversityParkvilleAustralia