Michelle L. Halls

Relaxin Family Peptides and Their Receptors

There are seven relaxin family peptides that are all structurally related to insulin. Relaxin has many roles in female and male reproduction, as a neuropeptide in the central nervous system, as a vasodilator and cardiac stimulant in the cardiovascular system, and as an antifibrotic agent. Insulin-like peptide-3 (INSL3) has clearly defined specialist roles in male and female reproduction, relaxin-3 is primarily a neuropeptide involved in stress and metabolic control, and INSL5 is widely distributed particularly in the gastrointestinal tract. Although they are structurally related to insulin, the relaxin family peptides produce their physiological effects by activating a group of four G protein-coupled receptors (GPCRs), relaxin family peptide receptors 1-4 (RXFP1-4). Relaxin and INSL3 are the cognate ligands for RXFP1 and RXFP2, respectively, that are leucine-rich repeat containing GPCRs. RXFP1 activates a wide spectrum of signaling pathways to generate second messengers that include cAMP and nitric oxide, whereas RXFP2 activates a subset of these pathways. Relaxin-3 and INSL5 are the cognate ligands for RXFP3 and RXFP4 that are closely related to small peptide receptors that when activated inhibit cAMP production and activate MAP kinases. Although there are still many unanswered questions regarding the mode of action of relaxin family peptides, it is clear that they have important physiological roles that could be exploited for therapeutic benefit.

Relaxin Family Peptide Receptors RXFP1 and RXFP2 Modulate cAMP Signaling by Distinct Mechanisms

Two orphan leucine-rich repeat-containing G protein-coupled receptors were recently identified as targets for the relaxin family peptides relaxin and insulin-like peptide (INSL) 3. Human gene 2 relaxin is the cognate ligand for relaxin family peptide receptor (RXFP) 1, whereas INSL3 is the ligand for RXFP2. Constitutively active mutants of both receptors when expressed in human embryonic kidney (HEK) 293T cells signal through Gαs to increase cAMP. However, recent studies using cells that endogenously express the receptors revealed greater complexity: cAMP accumulation after activation of RXFP1 involves a time-dependent biphasic pathway with a delayed phase involving phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC) ζ, whereas the RXFP2 response involves inhibition of adenylate cyclase via pertussis toxin-sensitive G proteins. The aim of this study was to compare and contrast the cAMP signaling pathways used by these two related receptors. In HEK293T cells stably transfected with RXFP1, preliminary studies confirmed the biphasic cAMP response, with an initial Gαs component and a delayed response involving PI3K and PKCζ. This delayed pathway was dependent upon G-βγ subunits derived from Gαi3. An additional inhibitory pathway involving GαoB affecting cAMP accumulation was also identified. In HEK293T cells stably transfected with RXFP2, the cAMP response involved Gαs and was modulated by inhibition mediated by GαoB and release of inhibitory G-βγ subunits. Thus, initially both RXFP1 and RXFP2 couple to Gαs and an inhibitory GαoB pathway. Differences in cAMP accumulation stem from the ability of RXFP1 to recruit coupling to Gαi3, release G-βγ subunits and thus activate a delayed PI3K-PKCζ pathway to further increase cAMP accumulation.

Direct Binding Between Orai1 and AC8 Mediates Dynamic Interplay Between Ca2+ and cAMP Signaling

A signaling complex enables the compartmentalized regulation of cyclic AMP signaling by calcium entering through a specific channel. Bound to Signal in Close Quarters Interplay between the calcium and the cyclic adenosine monophosphate (cAMP) signaling pathways is crucial to numerous physiological events. Although membrane-bound calcium-sensitive adenylyl cyclases (ACs) are sensitive to submicromolar concentrations of calcium in vitro, in cells they are highly selective in responding to store-operated calcium (SOC) entry rather than to calcium released from intracellular stores or entering the cell through ionophores. Here, Willoughby et al. used a combination of live-cell imaging techniques and biochemical approaches to resolve this conundrum and showed that AC8, which is stimulated by calcium-bound calmodulin, forms a direct protein-protein interaction with Orai1, the pore-forming component of the channel that mediates SOC entry. The existence of AC8 in a complex with SOC channels provides a mechanism for the compartmentalized regulation of cAMP signaling by specific subcellular calcium signals. The interplay between calcium ion (Ca2+) and cyclic adenosine monophosphate (cAMP) signaling underlies crucial aspects of cell homeostasis. The membrane-bound Ca2+-regulated adenylyl cyclases (ACs) are pivotal points of this integration. These enzymes display high selectivity for Ca2+ entry arising from the activation of store-operated Ca2+ (SOC) channels, and they have been proposed to functionally colocalize with SOC channels to reinforce crosstalk between the two signaling pathways. Using a multidisciplinary approach, we have identified a direct interaction between the amino termini of Ca2+-stimulated AC8 and Orai1, the pore component of SOC channels. High-resolution biosensors targeted to the AC8 and Orai1 microdomains revealed that this protein-protein interaction is responsible for coordinating subcellular changes in both Ca2+ and cAMP. The demonstration that Orai1 functions as an integral component of a highly organized signaling complex to coordinate Ca2+ and cAMP signals underscores how SOC channels can be recruited to maximize the efficiency of the interplay between these two ubiquitous signaling pathways.

Relaxin inhibits renal myofibroblast differentiation via RXFP1, the nitric oxide pathway, and Smad2

The hormone relaxin inhibits renal myofibroblast differentiation by interfering with TGF‐β1/ Smad2 signaling. However, the pathways involved in the relaxin‐TGF‐β1/Smad2 interaction remain unknown. This study investigated the signaling mechanisms by which human gene‐2 (H2) relaxin regulates myofibroblast differentiation in vitro by examining its effects on mixed populations of fibroblasts and myofibroblasts propagated from injured rat kidneys. Cultures containing ~60–70% myofibroblasts were used to determine which relaxin receptors, G‐proteins, and signaling pathways were involved in the H2 relaxin‐mediated regulation of α‐smooth muscle actin (α‐SMA; a marker of myofibroblast differentiation). H2 relaxin only inhibited α‐SMA immunostaining and collagen concentration in the presence of relaxin family peptide receptor 1 (RXFP1). H2 relaxin also induced a transient rise in cAMP in the presence of Gi/o inhibition, and a sustained increase in extracellular signal‐regulated kinase (ERK)‐1/2 phosphorylation. Furthermore, inhibition of neuronal nitric oxide synthase (nNOS), NO, and cGMP significantly blocked the inhibitory effects of relaxin on α‐SMA and Smad2 phosphorylation, while the NO inhibitor, l‐nitroarginine methyl ester (hydrochloride) (l‐NAME) significantly blocked the inhibitory actions of relaxin on collagen concentration in vivo. These findings suggest that relaxin signals through RXFP1, and a nNOS‐NO‐cGMP‐dependent pathway to inhibit Smad2 phosphorylation and interfere with TGF‐β1‐mediated renal myofibroblast differentiation and collagen production.—Mookerjee, I., Hewitson, T. D., Halls, M. L., Summers, R. J., Mathai, M. L., Bathgate, R. A. D., Tregear, G. W., Samuel, C. S. Relaxin inhibits renal myofibroblast differentiation via RXFP1, the nitric oxide pathway, and Smad2. FASEB J. 23, 1219–1229 (2009)

Relaxin Family Peptide Receptors - former orphans reunite with their parent ligands to activate multiple signalling pathways

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 GoB‐mediated inhibition of adenylate cyclase. With RXFP1, an additional delayed increase in cAMP involves βγ subunits released from Gi3. 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.

Regulation by Ca2+-Signaling Pathways of Adenylyl Cyclases

Interplay between the signaling pathways of the intracellular second messengers, cAMP and Ca(2+), has vital consequences for numerous essential physiological processes. Although cAMP can impact on Ca(2+)-homeostasis at many levels, Ca(2+) either directly, or indirectly (via calmodulin [CaM], CaM-binding proteins, protein kinase C [PKC] or Gβγ subunits) may also regulate cAMP synthesis. Here, we have evaluated the evidence for regulation of adenylyl cyclases (ACs) by Ca(2+)-signaling pathways, with an emphasis on verification of this regulation in a physiological context. The effects of compartmentalization and protein signaling complexes on the regulation of AC activity by Ca(2+)-signaling pathways are also addressed. Major gaps are apparent in the interactions that have been assumed, revealing a need to comprehensively clarify the effects of Ca(2+) signaling on individual ACs, so that the important ramifications of this critical interplay between Ca(2+) and cAMP are fully appreciated.

International Union of Basic and Clinical Pharmacology. XCV. Recent Advances in the Understanding of the Pharmacology and Biological Roles of Relaxin Family Peptide Receptors 1–4, the Receptors for Relaxin Family Peptides

Relaxin, insulin-like peptide 3 (INSL3), relaxin-3, and INSL5 are the cognate ligands for the relaxin family peptide (RXFP) receptors 1–4, respectively. RXFP1 activates pleiotropic signaling pathways including the signalosome protein complex that facilitates high-sensitivity signaling; coupling to Gαs, Gαi, and Gαo proteins; interaction with glucocorticoid receptors; and the formation of hetero-oligomers with distinctive pharmacological properties. In addition to relaxin-related ligands, RXFP1 is activated by Clq-tumor necrosis factor-related protein 8 and by small-molecular-weight agonists, such as ML290 [2-isopropoxy-N-(2-(3-(trifluoromethylsulfonyl)phenylcarbamoyl)phenyl)benzamide], that act allosterically. RXFP2 activates only the Gαs- and Gαo-coupled pathways. Relaxin-3 is primarily a neuropeptide, and its cognate receptor RXFP3 is a target for the treatment of depression, anxiety, and autism. A variety of peptide agonists, antagonists, biased agonists, and an allosteric modulator target RXFP3. Both RXFP3 and the related RXFP4 couple to Gαi/Gαo proteins. INSL5 has the properties of an incretin; it is secreted from the gut and is orexigenic. The expression of RXFP4 in gut, adipose tissue, and β-islets together with compromised glucose tolerance in INSL5 or RXFP4 knockout mice suggests a metabolic role. This review focuses on the many advances in our understanding of RXFP receptors in the last 5 years, their signal transduction mechanisms, the development of novel compounds that target RXFP1–4, the challenges facing the field, and current prospects for new therapeutics.

AKAP79/150 Interacts with AC8 and Regulates Ca2+-dependent cAMP Synthesis in Pancreatic and Neuronal Systems

Protein kinase A anchoring proteins (AKAPs) provide the backbone for targeted multimolecular signaling complexes that serve to localize the activities of cAMP. Evidence is accumulating of direct associations between AKAPs and specific adenylyl cyclase (AC) isoforms to facilitate the actions of protein kinase A on cAMP production. It happens that some of the AC isoforms (AC1 and AC5/6) that bind specific AKAPs are regulated by submicromolar shifts in intracellular Ca2+. However, whether AKAPs play a role in the control of AC activity by Ca2+ is unknown. Using a combination of co-immunoprecipitation and high resolution live cell imaging techniques, we reveal an association of the Ca2+-stimulable AC8 with AKAP79/150 that limits the sensitivity of AC8 to intracellular Ca2+ events. This functional interaction between AKAP79/150 and AC8 was observed in HEK293 cells overexpressing the two signaling molecules. Similar findings were made in pancreatic insulin-secreting cells and cultured hippocampal neurons that endogenously express AKAP79/150 and AC8, which suggests important physiological implications for this protein-protein interaction with respect to Ca2+-stimulated cAMP production.

Relaxin family peptide receptors--from orphans to therapeutic targets.

The relaxin family peptides have distinct expression profiles and physiological functions. Several of them are the cognate ligands for 4 G-protein-coupled relaxin family peptide receptors (RXFPs; formerly LGR7, LGR8, GPCR135, GPCR142). The relaxin/RXFP1 system has roles in reproductive physiology but is also involved in fibrosis, wound healing and responses to infarction. Relaxin has a potential use in congestive heart failure where fibrosis plays an important role in organ failure. The INSL3/RXFP2 system has biological roles in reproductive biology that may have limited therapeutic potential. However, the recently characterized relaxin-3/RXFP3 system is important in stress/anxiety and body composition. RXFP3 receptor antagonists are potentially novel anti-anxiety and anti-obesity drugs.

Sub‐picomolar relaxin signalling by a pre‐assembled RXFP1, AKAP79, AC2, β‐arrestin 2, PDE4D3 complex

Biochemical studies suggest that G‐protein‐coupled receptors (GPCRs) achieve exquisite signalling specificity by forming selective complexes, termed signalosomes. Here, using cAMP biosensors in single cells, we uncover a pre‐assembled, constitutively active GPCR signalosome, that couples the relaxin receptor, relaxin family peptide receptor 1 (RXFP1), to cAMP following receptor stimulation with sub‐picomolar concentrations of peptide. The physiological effects of relaxin, a pleiotropic hormone with therapeutic potential in cancer metastasis and heart failure, are generally attributed to local production of the peptide, that occur in response to sub‐micromolar concentrations. The highly sensitive signalosome identified here provides a regulatory mechanism for the extremely low levels of relaxin that circulate. The signalosome includes requisite Gαs, Gβγ and adenylyl cyclase 2 (AC2); AC2 is functionally coupled to RXFP1 through AKAP79 binding to helix 8 of the receptor; activation of AC2 is tonically opposed by protein kinase A (PKA)‐activated PDE4D3, scaffolded through a β‐arrestin 2 interaction with Ser704 of the receptor C‐terminus. This elaborate, pre‐assembled, ligand‐independent GPCR signalosome represents a new paradigm in GPCR signalling and provides a mechanism for the distal actions of low circulating levels of relaxin.