Relaxin receptors and nitric oxide synthases: search for the missing link

Researchers involved in the study of relaxin have welcomed the article by Hsu et al. [1], who eventually identified the receptors for this hormone. In the past, using labeled relaxin, specific binding sites have been found in several target organs and tissues for this hormone [2–4], but the exact molecular and functional nature of the relaxin receptor remained elusive. Relaxin is structurally similar to insulin and insulin-like growth factors (IGFs) [5], hence it seemed logical to assume that its putative receptor should belong to the insulin family of receptors, which are membrane-associated tyrosine kinases. Nonetheless, studies in this direction have been unfruitful and even misleading, as experienced by Dr Ivell and his team. In a recent study they were at a very short step from a crucial discovery, because they found that the relaxin-induced cAMP accumulation in target cells in in vitro culture was prevented by a pharmacologic inhibitor of G protein activation [6]. However, their attention being focused on tyrosine phosphorylation, they did not go deep into this finding. Using a completely different approach, Hsu and coworkers noticed that knock-out mice for the relaxin-like factor/Insl3 (also known as Leydig cell relaxin) had an abnormal testis descent phenotype [7, 8] which was similar to that of mice with a disruption of a G protein-coupled receptor (GPCR) gene [9]. On these grounds, they screened the human and mouse genome for orphan GPCRs and they could smartly demonstrate that relaxin is a cognate ligand for two leucine-rich repeat-containing GPCRs, LGR7 and LGR8, acting through G_s proteins. Activation of adenylate cyclase by G_s proteins can explain why relaxin induces an elevation of cAMP in target cells and tissues [5]. In particular, in the same article cited above [1], Hsu and coworkers demonstrated that LGR7 and LGR8 are capable of mediating the action of relaxin through a cAMP-dependent pathway distinct from that of insulin and IGF family ligands.

In recent years, our own studies and those of other investigators have provided increasing evidence that relaxin can also act on several of its targets by increasing the expression and/or activity of nitric oxide synthase (NOS) isoenzymes, thereby promoting the generation of nitric oxide (NO) [10–16]. Time is ripe for investigating how the newly discovered relaxin GPCRs may account for this. Based on the current literature, there are multiple pathways by which GPCRs can stimulate NO biosynthesis. In endothelial cells, the best-described agonists for the constitutive NOS III isoform, acetylcholine and bradykinin, activate specific membrane-associated GPCRs [17, 18]. Similar effects are exerted by surface estrogen receptors [19]. It has been reported that NOS III can be also activated by direct stimulation of GPCRs with sphingosine 1-phosphate [20]. The classical signaling pathway appears to involve G protein βγ subunits that, by means of phosphoinositide 3-kinase (PI3-K), switch on protein kinase B (Akt), which in turn activates eNOS by phosphorylation at Ser-1179 [20]. Another mechanism, which may either coexist in the same cell or be alternatively operating in different cell types, could involve cAMP and the inducible NOS II isoform. In rat vascular smooth muscle cells, GPCR-activated adenylate cyclase and the consequent rise in cAMP upregulates protein kinase A (PKA) activity [21]. In turn, PKA is able to phosphorilate and inactivate IkB-α, the inhibitor subunit of the transcription factor NF-κB, thus allowing NF-κB to translocate into the nucleus and to promote the expression of NOS II [22]. This latter mechanism may be operating in some relaxin targets, in which induction of NOS II expression and/or high-output, sustained NO generation have been observed [11, 12, 15, 16], NOS II being far more active than NOS III [23]. As summarized in Figure 1, at present there are some clues but no definitive data about the molecular mechanisms by which relaxin, by binding to its GPCRs, may induce the activation of the NO pathway in its targets. Hopefully, future research would clarify this point.

Figure 1

Putative interactions between relaxin receptor signaling and intrinsic NO pathway. AC: adenylate cyclase Akt: protein kinase B Gs: G_s proteins IκB inhibitor subunit of nuclear factor kappa-B LGR7-8: leucine-rich repeat-containing G protein-coupled receptors 7 and 8 NF-κB nuclear factor kappa-B NO: nitric oxide NOS II: inducible NO synthase NOS III: constitutive NO synthase PI3-K: phosphoinositide 3-kinase PKA: protein kinase A RLX: relaxin