The NMR solution structure of the relaxin (RXFP1) receptor lipoprotein receptor class A module and identification of key residues in the N-terminal region of the module that mediate receptor activation

The receptors for the peptide hormones relaxin and insulin-like peptide 3 (INSL3) are the leucine-rich repeat-containing G-protein-coupled receptors LGR7 and LGR8 recently renamed as the relaxin family peptide (RXFP) receptors, RXFP1 and RXFP2, respectively. These receptors differ from other LGRs by the addition of an N-terminal low density lipoprotein receptor class A (LDLa) module and are the only human G-protein-coupled receptors to contain such a domain. Recently it was shown that the LDLa module of the RXFP1 and RXFP2 receptors is essential for ligand-stimulated cAMP signaling. The mechanism by which the LDLa module modulates receptor signaling is unknown; however, it represents a unique paradigm in understanding G-protein-coupled receptor signaling. Here we present the structure of the RXFP1 receptor LDLa module determined by solution NMR spectroscopy. The structure is similar to other LDLa modules but shows small differences in side chain orientations and inter-residue packing. Interchange of the module with the second ligand binding domain of the LDL receptor, LB2, results in a receptor that binds relaxin with full affinity but is unable to signal. Furthermore, we demonstrate via structural studies on mutated LDLa modules and functional studies on mutated full-length receptors that a hydrophobic surface within the N-terminal region of the module is essential for activation of RXFP1 receptor signal in response to relaxin stimulation. This study has highlighted the necessity to understand the structural effects of single amino acid mutations on the LDLa module to fully interpret the effects of these mutations on receptor activity.     

H3 relaxin is a specific ligand for LGR7 and activates the receptor by interacting with both the ectodomain and the exoloop 2

Leucine-rich repeat-containing, G protein-coupled receptors (LGRs) represent a unique subgroup of G protein-coupled receptors with a large ectodomain. Recent studies demonstrated that relaxin activates two orphan LGRs, LGR7 and LGR8, whereas INSL3/Leydig insulin-like peptide specifically activates LGR8. Human relaxin 3 (H3 relaxin) was recently discovered as a novel ligand for relaxin receptors. Here, we demonstrate that H3 relaxin activates LGR7 but not LGR8. Taking advantage of the overlapping specificity of these three ligands for the two related LGRs, chimeric receptors were generated to elucidate the mechanism of ligand activation of LGR7. Chimeric receptor LGR7/8 with the ectodomain from LGR7 but the transmembrane region from LGR8 maintains responsiveness to relaxin but was less responsive to H3 relaxin based on ligand stimulation of cAMP production. The decreased ligand signaling was accompanied by decreases in the ability of H3 relaxin to compete for (33)P-relaxin binding to the chimeric receptor. However, replacement of the exoloop 2, but not exoloop 1 or 3, of LGR7 to the chimeric LGR7/8 restored ligand binding and receptor-mediated cAMP production. These results suggested that activation of LGR7 by H3 relaxin involves specific binding of the ligand to both the ectodomain and the exoloop 2, thus providing a model with which to understand the molecular basis of ligand signaling for this unique subgroup of G protein-coupled receptors.     

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

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.     

Defining the LGR8 residues involved in binding insulin-like peptide 3

The peptide hormone insulin-like peptide 3 (INSL3) is essential for testicular descent and has been implicated in the control of adult fertility in both sexes. The human INSL3 receptor leucine-rich repeat-containing G protein-coupled receptor 8 (LGR8) binds INSL3 and relaxin with high affinity, whereas the relaxin receptor LGR7 only binds relaxin. LGR7 and LGR8 bind their ligands within the 10 leucine-rich repeats (LRRs) that comprise the majority of their ectodomains. To define the primary INSL3 binding site in LGR8, its LRRs were first modeled on the crystal structure of the Nogo receptor (NgR) and the most likely binding surface identified. Multiple sequence alignment of this surface revealed the presence of seven of the nine residues implicated in relaxin binding to LGR7. Replacement of these residues with alanine caused reduced [(125)I]INSL3 binding, and a specific peptide/receptor interaction point was revealed using competition binding assays with mutant INSL3 peptides. This point was used to crudely dock the solution structure of INSL3 onto the LRR model of LGR8, allowing the prediction of the INSL3 Trp-B27 binding site. This prediction was then validated using mutant INSL3 peptide competition binding assays on LGR8 mutants. Our results indicated that LGR8 Asp-227 was crucial for binding INSL3 Arg-B16, whereas LGR8 Phe-131 and Gln-133 were involved in INSL3 Trp-B27 binding. From these two defined interactions, we predicted the complete INSL3/LGR8 primary binding site, including interactions between INSL3 His-B12 and LGR8 Trp-177, INSL3 Val-B19 and LGR8 Ile-179, and INSL3 Arg-B20 with LGR8 Asp-181 and Glu-229.     

Structural Insights into the Unique Modes of Relaxin-Binding and Tethered-Agonist Mediated Activation of RXFP1 and RXFP2

Our poor understanding of the mechanism by which the peptide-hormone H2 relaxin activates its G protein coupled receptor, RXFP1 and the related receptor RXFP2, has hindered progress in its therapeutic development. Both receptors possess large ectodomains, which bind H2 relaxin, and contain an N-terminal LDLa module that is essential for receptor signaling and postulated to be a tethered agonist. Here, we show that a conserved motif (GDxxGWxxxF), C-terminal to the LDLa module, is critical for receptor activity. Importantly, this motif adopts different structures in RXFP1 and RXFP2, suggesting distinct activation mechanisms. For RXFP1, the motif is flexible, weakly associates with the LDLa module, and requires H2 relaxin binding to stabilize an active conformation. Conversely, the GDxxGWxxxF motif in RXFP2 is more closely associated with the LDLa module, forming an essential binding interface for H2 relaxin. These differences in the activation mechanism will aid drug development targeting these receptors.     

T222P mutation of the insulin-like 3 hormone receptor LGR8 is associated with testicular maldescent and hinders receptor expression on the cell surface membrane

Insulin-like 3 (INSL3) hormone plays a crucial role in testicular descent during embryonic development. Genetic ablation of Insl3 or its G protein-coupled receptor (GPCR) Lgr8 causes cryptorchidism in mice. Previously, we identified a nonfunctional T222P mutation of LGR8 in several human patients with testicular maldescent. Using a large population of patients and healthy controls from Italy, we have demonstrated that T222P LGR8 mutation is present only in affected patients (19 T222P/+ of 598 vs. 0/450, P < 0.0001). We have also identified a novel allele of LGR8 (R223K) found in one patient with retractile testes. Both mutations are located in the leucine-rich repeats (LRRs) of GPCR ectodomain. The expression analysis of T222P mutant receptor transfected into 293T cells revealed that the mutation severely compromised GPCR cell membrane expression. The substitution of Thr(222) with the neutral Ser or Ala, or the R223K mutation, did not alter receptor cell membrane expression or ligand-induced cAMP increase. Additional mutations, affecting first leucine in a signature LxxLxLxxN/CxL stretch of LRR (L283F), or the amino acid residues, forming the disulfide bond or coordinating calcium ion in the LDLa module (C71Y and D70Y), also rendered proteins with reduced cell surface expression. The structural alterations of both LRRs and LDLa of the ligand-binding part of LGR8 cause the inability of receptor to express on the cell surface membrane and might be responsible for the abnormal testicular phenotype in patients.     

Investigation of Interactions at the Extracellular Loops of the Relaxin Family Peptide Receptor 1 (RXFP1)

Relaxin, an emerging pharmaceutical treatment for acute heart failure, activates the relaxin family peptide receptor (RXFP1), which is a class A G-protein-coupled receptor. In addition to the classic transmembrane (TM) domain, RXFP1 possesses a large extracellular domain consisting of 10 leucine-rich repeats and an N-terminal low density lipoprotein class A (LDLa) module. Relaxin-mediated activation of RXFP1 requires multiple coordinated interactions between the ligand and various receptor domains including a high affinity interaction involving the leucine-rich repeats and a predicted lower affinity interaction involving the extracellular loops (ELs). The LDLa is essential for signal activation; therefore the ELs/TM may additionally present an interaction site to facilitate this LDLa-mediated signaling. To overcome the many challenges of investigating relaxin and the LDLa module interactions with the ELs, we engineered the EL1 and EL2 loops onto a soluble protein scaffold, mapping specific ligand and loop interactions using nuclear magnetic resonance spectroscopy. Key EL residues were subsequently mutated in RXFP1, and changes in function and relaxin binding were assessed alongside the RXFP1 agonist ML290 to monitor the functional integrity of the TM domain of these mutant receptors. The outcomes of this work make an important contribution to understanding the mechanism of RXFP1 activation and will aid future development of small molecule RXFP1 agonists/antagonists.     

Impaired nipple development and parturition in LGR7 knockout mice

LGR7 is a G-protein coupled receptor with structural homology to the gonadotrophin and thyrotrophin receptors. Recently, LGR7 was deorphanized, and it was shown that relaxin is the ligand for LGR7. To further study the function of this receptor, mice deficient for LGR7 were generated by replacing part of the transmembrane-encoding region with a LacZ reporter cassette. Here we show that LGR7 is expressed in various tissues, including the uterus, heart, brain, and testis. Fertility studies using female LGR7-/- mice showed normal fertility and litter size. However, some females were incapable of delivering their pups, and several pups were found dead. Moreover, all offspring died within 24 to 48 h after delivery because female LGR7-/- mice were unable to feed their offspring due to impaired nipple development. In some male LGR7-/- mice, spermatogenesis was impaired, leading to azoospermia and a reduction in fertility. Interestingly, these phenomena were absent in mutant mice at older ages or in later generations. Taken together, results from LGR7 knockout mice indicate an essential role for the LGR7 receptor in nipple development during pregnancy. Moreover, a defect in parturition was observed, suggesting a role for LGR7 in the process of cervical ripening.     

Identification of the N-linked glycosylation sites of the human relaxin receptor and effect of glycosylation on receptor function

The relaxin receptor, RXFP1, is a member of the leucine-rich repeat-containing G-protein-coupled receptor (LGR) family. These receptors are characterized by a large extracellular ectodomain containing leucine-rich repeats which contain the primary ligand binding site. RXFP1 contains six putative Asn-linked glycosylation sites in the ectodomain at positions Asn-14, Asn-105, Asn-242, Asn-250, Asn-303, and Asn-346, which are highly conserved across species. N-Linked glycosylation is the most common post-translational modification of G-protein-coupled receptors, although its role in modulating receptor function differs. We herein investigate the actual N-linked glycosylation status of RXFP1 and the functional ramifications of these post-translational modifications. Site-directed mutagenesis was utilized to generate single- or multiple-glycosylation site mutants of FLAG-tagged human RXFP1 which were then transiently expressed in HEK-293T cells. Glycosylation status was analyzed by immunoprecipitation and Western blot and receptor function analyzed with an anti-FLAG ELISA, (33)P-H2 relaxin competition binding, and cAMP activity measurement. All of the potential N-glycosylation sites of RXFP1 were utilized in HEK-293T cells, and importantly, disruption of glycosylation at individual or combinations of double and triple sites had little effect on relaxin binding. However, combinations of glycosylation sites were required for cell surface expression and cAMP signaling. In particular, N-glycosylation at Asn-303 of RXFP1 was required for optimal intracellular cAMP signaling. Hence, as is the case for other LGR family members, N-glycosylation is essential for the transport of the receptor to the cell surface. Additionally, it is likely that glycosylation is also essential for the conformational changes required for G-protein coupling and subsequent cAMP signaling.     

Synthetic human insulin 4 does not activate the G-protein-coupled receptors LGR7 or LGR8.

In contrast to the cellular receptors for insulin and insulin-like growth factors that are known to be protein tyrosine kinases, those of both insulin 3 and relaxin have recently been identified as being members of the leucine-rich repeat-containing G-protein coupled receptor (LGR) family, LGR8 and LGR7, respectively. This has prompted an examination into the possibility that they might also be specific for another member of the insulin superfamily, namely, insulin 4. Towards this aim, a two-chain peptide corresponding to the predicted primary structure of insulin 4 was prepared by solid phase synthesis. As conventional aeration and combination of the two S-reduced chains in solution at high pH failed to produce target product, selectively S-protected A- and B-chains were prepared followed by stepwise, individual formation of each of the three disulfides, one intramolecular within the A-chain and two intermolecular. Chemical characterization confirmed the purity and identity of the synthetic insulin 4 analogue. However, secondary structural analysis indicated that the peptide was devoid of tertiary conformation suggesting that the native peptide may well be either significantly longer in length or is similar to insulin-like growth factor I or II in that it is a single chain product. Screening of the synthetic analogue for activation of transfected cells bearing LGR7, and LGR7 splice variant or LGR8 failed to identify a specific interaction. Thus, the in vivo structural identity of insulin 4 and its receptor (if any) as well as its potential function remains unknown.     

Relaxin-3: improved synthesis strategy and demonstration of its high-affinity interaction with the relaxin receptor LGR7 both in vitro and in vivo

Relaxin-3 is a member of the human relaxin peptide family, the gene for which, RLN3, is predominantly expressed in the brain. Mapping studies in the rodent indicate a highly developed network of RLN3, RLN1, and relaxin receptor-expressing cells in the brain, suggesting that relaxin peptides have important functional roles in the central nervous system. A regioselective disulfide-bond synthesis protocol was developed and used for the chemical synthesis of human (H3) relaxin-3. The selectively S-protected A and B chains were combined by stepwise formation of each of the three insulin-like disulfides via aeration, thioloysis, and iodolysis. Judicious positioning of the three sets of S-protecting groups was crucial for acquisition of synthetic H3 relaxin in a good overall yield. The activity of the peptide was tested against relaxin family peptide receptors. Although the highest activity was demonstrated on the human relaxin-3 receptor (GPCR135), the peptide also showed high activity on relaxin receptors (LGR7) from various species and variable activity on the INSL3 receptor (LGR8). Recombinant mouse prorelaxin-3 demonstrated similar activity to H3 relaxin, suggesting that the presence of the C peptide did not influence the conformation of the active site. H3 relaxin was also able to activate native LGR7 receptors. It stimulated increased MMP-2 expression in LGR7-expressing rat ventricular fibroblasts in a dose-dependent manner and, following infusion into the lateral ventricle of the brain, stimulated water drinking in rats, activating LGR7 receptors located in the subfornical organ. Thus, H3 relaxin is able to interact with the relaxin receptor LGR7 both in vitro and in vivo.     

Relaxin stimulates leukocyte adhesion and migration through a relaxin receptor LGR7-dependent mechanism

Leukocytes are critical effectors of inflammation and tumor biology. Chemokine-like factors produced by such inflammatory sites are key mediators of tumor growth that activate leukocytic recruitment and tumor infiltration and suppress immune surveillance. Here we report that the endocrine peptide hormone, relaxin, is a regulator of leukocyte biology with properties important in recruitment to sites of inflammation. This study uses the human monocytic cell line THP-1 and normal human peripheral blood mononuclear cells to define a novel role for relaxin in regulation of leukocyte adhesion and migration. Our studies indicate that relaxin promotes adenylate cyclase activation, substrate adhesion, and migratory capacity of mononuclear leukocytes through a relaxin receptor LGR7-dependent mechanism. Relaxin-stimulated cAMP accumulation was observed to occur primarily in non-adherent cells. Relaxin stimulation results in increased substrate adhesion and increased migratory activity of leukocytes. In addition, relaxin-stimulated substrate adhesion resulted in enhanced chemotaxis to monocyte chemoattractant protein-1. These responses in THP-1 and peripheral blood mononuclear cells are relaxin dose-dependent and proportional to cAMP accumulation. We further demonstrate that LGR7 is critical for mediating these biological responses by use of RNA interference lentiviral short hairpin constructs. In summary, we provide evidence that relaxin is a novel leukocyte stimulatory agent with properties affecting adhesion and chemomigration.     

Regulation of receptor signaling by relaxin A chain motifs: derivation of pan-specific and LGR7-specific human relaxin analogs

Relaxin peptides are important hormones for the regulation of reproductive tissue remodeling and the renal cardiovascular system during pregnancy. Recent studies demonstrated that two of the seven human relaxin family peptides, relaxin H2 (RLN2) and INSL3, signal exclusively through leucine-rich repeat-containing G protein-coupled receptors, LGR7 and LGR8. Although it was well characterized that an RXXXRXXI motif at the RLN2 B chain confers receptor activation activity, it is not clear what roles RLN2 A chain plays in receptor interaction. Analyses of relaxin family genes on syntenic regions of model tetrapods showed that the A chain of RLN2 orthologs exhibited a greater sequence divergence as compared with the receptor-binding domain-containing B chain, foreshadowing a potential role in receptor interactions; hence, defining receptor selectivity in this fast evolving peptide hormone. To test our hypothesis that select residues in the human RLN2 A chain play key roles in receptor interaction, we studied mutant peptides with residue substitution(s) in the A chain. Here, we showed that alanine substitution at the A16 and A17 positions enhances LGR8-activation activity of RLN2, whereas mutation at the A22-23 region (RLN2A22-23) ablates LGR8, but not LGR7, activation activity. In addition, we demonstrated that the functional characteristics of the RLN2A22-23 mutant are mainly attributed to modifications at the PheA23 position. Taken together, our studies indicated that ThrA16, LysA17, and PheA23 constitute part of the receptor-binding interface of human RLN2, and that modification of these residues has led to the generation of novel human RLN2 analogs that would allow selective activation of human LGR7, but not LGR8, in vivo.     

An evolutionary comparison of leucine-rich repeat containing G protein-coupled receptors reveals a novel LGR subtype

Leucine-rich repeat containing G protein-coupled receptors or LGRs are receptors with important functions in development and reproduction. Belonging to this evolutionarily conserved group of receptors are the well-studied glycoprotein hormone receptors and relaxin receptors in mammals, as well as the bursicon receptor, which triggers cuticle hardening and tanning in freshly enclosed insects. In this study, the numerous LGR sequences in different animal phyla are analyzed and compared. Based on these data a phylogenetic tree was generated. This information sheds new light on structural and evolutionary aspects regarding this receptor group. Apart from vertebrates and insects, LGRs are also present in early chordates (Urochordata, Cephalochordata and Hyperoartia) and other arthropods (Arachnida and Branchiopoda) as well as in Mollusca, Echinodermata, Hemichordata, Nematoda, and even in ancient animal life forms, such as Cnidaria and Placozoa. Three distinct types of LGR exist, distinguishable by their number of leucine-rich repeats (LRRs), their type-specific hinge region and the presence or absence of an LDLa motif. Type C LGRs containing only one LDLa (C1 subtype) appear to be present in nearly all animal phyla. We here describe a second subtype, C2, containing multiple LDLa motifs, which was discovered in echinoderms, mollusks and in one insect species (Pediculus humanis corporis). In addition, eight putative LGRs can be predicted from the genome data of the placozoan species Trichoplax adhaerens. They may represent an ancient form of the LGRs, however, more genomic data will be required to confirm this hypothesis.     

Study of molecular mechanisms of the relaxin action on adenylyl cyclase signaling system using synthetic peptides derived from the relaxin receptor LGR7

The peptide hormone relaxin in dose-dependent manner stimulates adenylyl cyclase activity in the rat tissues (brain striatum, heart and skeletal muscles) and the muscle tissues of invertebrates--bivalve mollusk Anodonta cygnea and earthworm Lumbricus terrestris. Adenylyl cyclase stimulating effect of the hormone is most expressed in striatum and heart muscles of rats. For identification of the type ofrelaxin receptors, participating in the realization of this effect of the hormone, the peptides 619-629, 619-629-Lys(Palm) and 615-629 derived from the primary structure of C-terminal region of the third intracellular loop of the relaxin receptor of type 1 (LGR7), were synthesized by us for the first time. It is shown that peptide: 619-629-Lys(Palm) and 615-629 in competitive manner inhibit the stimulation of the adenylyl cyclase by relaxin in brain striatum and heart muscle of rats. At the same time, these peptides do not change stimulating effect of the hormone in the skeletal muscles of rat and in the muscles of invertebrates. Thus, the peptide action on adenylyl cyclase effect of relaxin is tissue- and species-specific. These data, on the one hand, demonstrate participation of receptor LGR7 in realization of adenylyl cyclase stimulating effect of relaxin in striatum and heart muscle of rats and, on the other, give evidence for existence of another adenylyl cyclase signaling mechanisms of relaxin action in the skeletal muscles and the muscle of invertebrates, which do not involve LGR7 receptor. The adenylyl cyclase stimulating effect of relaxin in striatum and heart muscle was decreased in the presence of C-terminal peptides 385-394 of alpha(s)-subunit of mammalian G protein and was blocked by treatment of the membranes with cholera toxin. On the basis of data obtained the following conclusions were made: (i) in striatum and heart muscle the relaxin stimulates adenylyl cyclase through LGR7 receptors functionally coupled with Gs protein, and (ii) the coupling between hormoneactivated relaxin receptor LGR7 and Gs protein is realized via the interaction of C-terminal part of receptor third intracellular loop and C-terminal segment of Gs protein alpha-subunit.     

The A-chain of human relaxin family peptides has distinct roles in the binding and activation of the different relaxin family peptide receptors

The relaxin peptides are a family of hormones that share a structural fold characterized by two chains, A and B, that are cross-braced by three disulfide bonds. Relaxins signal through two different classes of G-protein-coupled receptors (GPCRs), leucine-rich repeat-containing GPCRs LGR7 and LGR8 together with GPCR135 and GPCR142, now referred to as the relaxin family peptide (RXFP) receptors 1-4, respectively. Although key binding residues have been identified in the B-chain of the relaxin peptides, the role of the A-chain in their activity is currently unknown. A recent study showed that INSL3 can be truncated at the N terminus of its A-chain by up to 9 residues without affecting the binding affinity to its receptor RXFP2 while becoming a high affinity antagonist. This suggests that the N terminus of the INSL3 A-chain contains residues essential for RXFP2 activation. In this study, we have synthesized A-chain truncated human relaxin-2 and -3 (H2 and H3) relaxin peptides, characterized their structure by both CD and NMR spectroscopy, and tested their binding and cAMP activities on RXFP1, RXFP2, and RXFP3. In stark contrast to INSL3, A-chain-truncated H2 relaxin peptides lost RXFP1 and RXFP2 binding affinity and concurrently cAMP-stimulatory activity. H3 relaxin A-chain-truncated peptides displayed similar properties on RXFP1, highlighting a similar binding mechanism for H2 and H3 relaxin. In contrast, A-chain-truncated H3 relaxin peptides showed identical activity on RXFP3, highlighting that the B-chain is the sole determinant of the H3 relaxin-RXFP3 interaction. Our results provide new insights into the action of relaxins and demonstrate that the role of the A-chain for relaxin activity is both peptide- and receptor-dependent.     

Relaxin family peptide receptors Rxfp1 and Rxfp2: mapping of the mRNA and protein distribution in the reproductive tract of the male rat

Background  

Relaxin is the endogenous ligand of the G-protein coupled receptor RXFP1, previously known as LGR7. In humans relaxin can also activate, but with lower affinity, the closely related receptor for the insulin-like peptide from Leydig cells, RXFP2, previously known as LGR8. The lack of relaxin impairs male fertility but the precise distribution and the function of relaxin receptors in the male reproductive tract is not known. We investigated the distribution of Rxfp1 and Rxfp2 in the reproductive tract of the male rat and the function of relaxin in the vas deferens, a tissue with high expression of both receptors.     

Phosphoinositide 3-kinase activity is required for biphasic stimulation of cyclic adenosine 3',5'-monophosphate by relaxin

The G protein-coupled receptors LGR7 and LGR8 have recently been identified as the primary receptors for the polypeptide hormone relaxin and relaxin-like factors. RT-PCR confirmed the existence of mRNA for both LGR7 and LRG8 in THP-1 cells. Whole cell treatment of THP-1 cells with relaxin produced a biphasic time course in cAMP accumulation, where the first peak appeared as early as 1-2 min with a second peak at 10-20 min. Selective inhibitors for phosphoinositide 3-kinase (PI3K), such as wortmannin and LY294002, showed a dose-dependent inhibition of relaxin-mediated increases in cAMP, specific for the second peak of the relaxin time course. Adenylyl cyclase activation by relaxin in purified plasma membranes from THP-1 cells was not inhibited by LY294002, consistent with a mechanism involving direct stimulation by a Galphas-coupled relaxin receptor. However, reconstitution of membranes with cytosol from THP-1 cells enhanced adenylyl cyclase activity and restored LY294002 sensitivity. In addition, relaxin increased PI3K activity in THP-1 cells. Neither the effects of relaxin nor the inhibition of relaxin by LY294002 was mediated by the activity of phosphodiesterases. Taken together, we show that PI3K is required for the biphasic stimulation of cAMP by relaxin in THP-1 cells and present a novel signal transduction pathway for the activation of adenylyl cyclase by a G protein-coupled receptor.