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Members of the insulin superfamily regulate pleiotropic biological processes through two types of target-specific but structurally conserved peptides, insulin/insulin-like growth factors and relaxin/insulin-like peptides. The latter bind to the human relaxin family peptide receptors (RXFPs). Here, we report three cryo-electron microscopy structures of RXFP4–Gi protein complexes in the presence of the endogenous ligand insulin-like peptide 5 (INSL5) or one of the two small molecule agonists, compound 4 and DC591053. The B chain of INSL5 adopts a single α-helix that penetrates into the orthosteric pocket, while the A chain sits above the orthosteric pocket, revealing a peptide-binding mode previously unknown. Together with mutagenesis and functional analyses, the key determinants responsible for the peptidomimetic agonism and subtype selectivity were identified. Our findings not only provide insights into ligand recognition and subtype selectivity among class A G protein-coupled receptors, but also expand the knowledge of signaling mechanisms in the insulin superfamily.Relaxin family peptide receptor 4 (RXFP4) regulates pleiotropic biological processes. Here, authors report cryo-EM structures revealing the ligand-binding modes and key determinants of peptidomimetic agonism and subtype selectivity [ABSTRACT FROM AUTHOR]

α1A‐ and α1B‐adrenoceptors (ARs) are G protein‐coupled receptors (GPCRs) that are activated by adrenaline and noradrenaline to modulate smooth muscle contraction in the periphery, and neuronal outputs in the central nervous system (CNS). α1A‐ and α1B‐AR are clinically targeted with antagonists for hypertension and benign prostatic hyperplasia and are emerging CNS targets for treating neurodegenerative diseases. The benzodiazepines midazolam, diazepam, and lorazepam are proposed to be positive allosteric modulators (PAMs) of α1‐ARs. Here, using thermostabilized, purified, α1A‐ and α1B‐ARs, we sought to identify the benzodiazepine binding site and modulatory mechanism to inform the design of selective PAMs. However, using a combination of biophysical approaches no evidence was found for direct binding of several benzodiazepines to purified, stabilized α1A‐ and α1B‐ARs. Similarly, in cell‐based assays expressing unmodified α1A‐ and α1B‐ARs, benzodiazepine treatment had no effect on fluorescent ligand binding, agonist‐stimulated Ca2+ release, or G protein activation. In contrast, several benzodiazepines positively modulated phenylephrine stimulation of a cAMP response element pathway by α1A‐ and α1B‐ARs; however, this was shown to be caused by off‐target inhibition of phosphodiesterases, known targets of diazepam. This study highlights how purified, stabilized GPCRs are useful for validating allosteric ligand binding and that care needs to be taken before assigning new targets to benzodiazepines. [ABSTRACT FROM AUTHOR]

Nuclear magnetic resonance (NMR) studies have revealed that fast methyl sidechain dynamics can report on entropically-driven allostery. Yet, NMR applications have been largely limited to the super-microsecond motional regimes of G protein-coupled receptors (GPCRs). We use 13Cε-methionine chemical shift-based global order parameters to test if ligands affect the fast dynamics of a thermostabilized GPCR, neurotensin receptor 1 (NTS 1). We establish that the NTS 1 solution ensemble includes substates with lifetimes on several, discrete timescales. The longest-lived states reflect those captured in agonist- and inverse agonist-bound crystal structures, separated by large energy barriers. We observe that the rapid fluctuations of individual methionine residues, superimposed on these long-lived states, respond collectively with the degree of fast, global dynamics correlating with ligand pharmacology. This approach lends confidence to interpreting spectra in terms of local structure and methyl dihedral angle geometry. The results suggest a role for sub-microsecond dynamics and conformational entropy in GPCR ligand discrimination. [Display omitted] • Methionine residues sense concerted GPCR motions on the sub-μs timescale • GPCR ensembles contain multiple meta-states including the crystalized conformer • The degree of global dynamics correlates with ligand pharmacological efficacy The very fast motions of proteins can dictate function but are largely invisible to static structural techniques. NMR spectroscopy can observe these motions, but technical challenges have limited their characterization for GPCR activation. Bumbak et al. combine available crystallography data, quantum calculations, and NMR to probe the fast global dynamics of receptors. [ABSTRACT FROM AUTHOR]

Studies the differential expression of receptors of the oxytocin-like peptide, mesotocin, in the uterus and ovary of pregnant tammar wallabies. Mesotocin receptor gene expression in wallaby reproductive tissues; Characterization of corpus luteum receptors.

Currently, four relaxin peptide family receptors are known: LGR7 is the relaxin receptor, although it also interacts specifically with relaxin-3; LGR8 is the insulin-like factor 3 (INSL3) receptor; and GPCR135 or the somatostatin- and angiotensin-like peptide receptor (SALPR) and GPCR142 are both specific relaxin-3 receptors. Because these receptors coevolved together with their relaxin ligands, phylogenetic analysis of these sequences can provide insight into peptide-receptor interactions and even predict interacting partners for INSL4, INSL5, and INSL6, the receptors for which are unknown. [ABSTRACT FROM AUTHOR]

The relaxin-like peptide family consists of relaxin-1, relaxin-2, and relaxin-3 and the insulin-like peptides (INSL)-3, INSL4, INSL5, and INSL6 (human relaxin-2 is equivalent to relaxin-1 in other species). Evolution of this family has been contentious. We therefore sought to clarify the issue by performing phylogenetic analysis of all relaxin-like peptides from the genomic databases available. Surprisingly, the phylogeny, combined with previous biologic characterizations, suggest that although relaxin's original function was likely in the brain, its reproductive role was acquired just prior to the divergence of amphibians. This phylogeny also illuminates inconsistencies in relaxin evolution in invertebrates, chickens, and cows. [ABSTRACT FROM AUTHOR]

Recent molecular and pharmacologic studies have identified LGR8, a member of the leucine-rich repeat-containing G-protein-coupled receptor family, as a cognate receptor for insulin-like peptide-3 (INSL3). LGR8 mRNA has been detected in various tissues, but the precise roles of these INSL3-LGR8 systems are unknown. In this study we first investigated the presence and cellular localization of LGR8 mRNA in both adult and developing rat kidney and subsequently examined the possible role of INSL3-LGR8 signaling in cultured mesangial cells. LGR8 mRNA was detected in the kidney by polymerase chain reaction and localized by in situ hybridization in mature glomerular mesangial cells within the renal cortex, with highest levels detected at embryonic day 18 and lowest levels in adult kidney. Synthetic INSL3 inhibited the proliferation of mesangial cells in primary culture, indicating the presence of functional LGR8 on these cells. These findings suggest that INSL3/LGR8 signaling may be involved in the genesis and/or developmental maturation of renal glomeruli and in the regulation of mesangial cell density in the adult kidney. [ABSTRACT FROM AUTHOR]

LGR8 was recently identified as a cognate receptor for insulin-like peptide-3 (INSL3), and INSL3-LGR8 signaling is best known for its role in testis descent during development. LGR8 mRNA has been detected in various human tissues including brain, but the regional and cellular distribution of LGR8 expression in the mammalian central nervous system is unknown. Therefore, in this study we investigated the presence and localization of LGR8 mRNA in rat brain using reverse transcription-polymerase chain reaction and in situ hybridization histochemistry. Results revealed a distinct distribution of LGR8 in forebrain, with transcripts principally restricted to the posterior thalamus and highest densities detected in the parafascicular nucleus of both adult and developing rats. Unexpectedly, INSL3 mRNA was not detected in brain by similar methods, but preliminary electrophysiologic studies of parafascicular neurons revealed that INSL3 altered their activity. These findings suggest that LGR8 signaling may be involved in sensorimotor control in the rat and perhaps other species, particularly via actions on parafascicular neurons that project to basal ganglia and are depleted in Parkinson's disease. [ABSTRACT FROM AUTHOR]

Several orphan G-protein-coupled receptors (GPCRs), LGR7 and LGR8, GPCR135 and GPCR142, were recently identified as putative, native receptors for different relaxin-family peptides, and their cell signaling mechanisms were elucidated in stably transfected cell lines. Anatomic studies have demonstrated that discrete populations of neurons in rat brain express relaxin and relaxin-3 mRNA/peptide, relaxin and relaxin-3 binding sites, and LGR7 and GPCR135 mRNAs. Thus, we began to assess the ability of relaxin-family peptides to alter cAMP production in brain and the involvement of the different native receptors. In mouse cortical membranes, a fixed concentration of relaxin peptides (100 nM) inhibited forskolin-induced cAMP production, but further studies in normal and receptor knockout mouse strains are required to assess the specificity of these effects. In addition, whole-cell signaling mechanisms are being investigated in a mouse hypothalamic cell line, GT1-7. Such studies will help to establish the actions of relaxin-family peptides via their different GPCRs in different brain pathways. [ABSTRACT FROM AUTHOR]

Discrete neuronal populations in brain express relaxin and relaxin- 3, and molecular studies have identified former-orphan, G-protein-coupled receptors LGR7 and GPCR135 as their native receptors. To better understand the role of central relaxin systems, we began to assess the anatomic distribution of these receptors and ligands in brain. This study documents the widespread distribution of LGR7 mRNA and LGR7-like immunoreactivity (LI) throughout adult rat forebrain areas shown to contain specific [33P]-relaxin binding sites. High densities of LGR7 mRNA hybridization were detected in deep layers of neocortex, hypothalamic paraventricular and supraoptic nuclei and within hippocampal subiculum and CA3, the basolateral amygdala and subfornical organ. Low to moderate hybridization was detected in septum, midline thalamic nuclei, arcuate and supramammillary nuclei, and regions of the midbrain pons. Complementary expression of LGR7-LI was observed in cortical pyramidal neurons, hypothalamic magnocellular neurons, and hippocampal pyramidal and interneurons. These findings provide further evidence for actions of relaxin as a modulator in somatosensory, autonomic, and neuroendocrine pathways. [ABSTRACT FROM AUTHOR]

Relaxin is well known for its actions on collagen remodeling. To improve our understanding of the physiologic role(s) of relaxin, the relaxin gene-knockout (RLX-KO) mouse was established by our group and subsequently phenotyped. Pregnant RLX-KO mice underwent inadequate development of the pubic symphysis as well as the mammary glands and nipples compared to wild-type mice, thus preventing lactation. Later studies showed that these deficiencies were associated with increased collagen, primarily in the nipple and vagina. Analysis of male RLX-KO mice also demonstrated inadequate reproductive tract development. The testis, epididymis, and prostate of RLX-KO mice showed delayed tissue maturation and growth associated with increased collagen deposition. In nonreproductive tissues, an age-related increase in interstitial collagen (fibrosis) was also detected in the lung, heart, and kidneys of RLX-KO mice and was associated with organ dysfunction. From 6-9 months of age and onwards, all organs of RLX-KO mice, particularly male mice, underwent progressive increases in tissue weight and collagen content (all P <.05) compared with wild-type animals. The increased fibrosis contributed to bronchiole epithelium thickening and alveolar congestion (lung), atrial hypertrophy and increased ventricular chamber stiffness (heart) in addition to glomerulosclerosis (kidney). Treatment of RLX-KO mice with recombinant human relaxin in early and developed stages of fibrosis caused the reversal of collagen deposition in the lung, heart, and kidneys. Together, these findings suggest that relaxin is a naturally occurring inhibitor of collagen deposition during normal development, aging, and pregnancy and can be used to prevent the progression of fibrosis. [ABSTRACT FROM AUTHOR]

The ectodomains of both the relaxin (LGR7) and the INSL3 (LGR8) receptors can be expressed on the cell surface using only a single transmembrane domain. These membrane-anchored proteins retain the ability to bind relaxin and can be cleaved from the cell surface. The subsequent LGR7 protein, 7BP, binds relaxin and can act as a functional relaxin antagonist. By contrast, the equivalent LGR8 protein 8BP does not bind relaxin or antagonize LGR8 activity. The 7BP protein has been successfully immobilized onto chemically derivatized surfaces for the capture of relaxin peptides and subsequent identification via SELDI-MS analysis. [ABSTRACT FROM AUTHOR]

Relaxin/insulin-like family peptide receptor 3 (RXFP3) belongs to class A G protein-coupled receptor family. RXFP3 and its endogenous ligand relaxin-3 are mainly expressed in the brain with important roles in the regulation of appetite, energy metabolism, endocrine homeostasis and emotional processing. It is therefore implicated as a potential target for treatment of various central nervous system diseases. Since selective agonists of RXFP3 are restricted to relaxin-3 and its analogs, we conducted a high-throughput screening campaign against 32,021 synthetic and natural product-derived compounds using a cyclic adenosine monophosphate (cAMP) measurement-based method. Only one compound, WNN0109-C011, was identified following primary screening, secondary screening and dose-response studies. Although displayed agonistic effect in cells overexpressing the human RXFP3, it also showed cross-reactivity with the human RXFP4. This hit compound may provide not only a chemical probe to investigate the function of RXFP3/4, but also a novel scaffold for the development of RXFP3/4 agonists. [ABSTRACT FROM AUTHOR]

Chemogenetics enables manipulation of neuronal activity in experimental animals. While providing information about the transduced neuron expressing a ligand-activated molecule, chemogenetics does not provide understanding about the antecedent circuit that drives that neuron's activity. For current approaches, this is not feasible, because the activating molecules are not genetically encoded. The insect allatostatin/allatostatin receptor system, a highly specific, powerful inhibitory chemogenetic approach, has this advantage, because the ligand, being a peptide, is genetically encoded. We developed viral vector-based systems to express biologically active allatostatin in neurons in vivo and allatostatin receptors in subpopulations of postsynaptic neurons. We demonstrate that activity-dependent release of allatostatin induces inhibition of allatostatin receptor-expressing neurons. We validate the approach in the vagal viscerosensory system where inhibitory, rather than the usual excitatory, viscerosensory input leads to sustained decreases in baroreceptor reflex sensitivity and bodyweight. • Oxytocin is replaced in its precursor with the insect peptide allatostatin (Ast) • Viral expression induces activity-dependent, synaptic release of bioactive Ast • Ast is expressed in vagal afferents and Ast receptor in Phox2 NTS neurons • This induces a long-term reduction in baroreceptor sensitivity and bodyweight gain Chemogenetics provides understanding of the function of specific neurons transduced to express an actuating receptor that is affected by global ligand administration. Ngo et al. generate a viral vector that induces neuronal expression and synaptic release of the inhibitory chemogenetic ligand, allatostatin, enabling circuit-based inhibition of allatostatin receptor-expressing neurons. [ABSTRACT FROM AUTHOR]