The neuroimmune CGRP-RAMP1 axis tunes cutaneous adaptive immunity to the microbiota.

The somatosensory nervous system surveils external stimuli at barrier tissues, regulating innate immune cells under infection and inflammation. The roles of sensory neurons in controlling the adaptive immune system, and more specifically immunity to the microbiota, however, remain elusive. Here, we identified a mechanism for direct neuroimmune communication between commensal-specific T lymphocytes and somatosensory neurons mediated by the neuropeptide calcitonin gene-related peptide (CGRP) in the skin. Intravital imaging revealed that commensal-specific T cells are in close proximity to cutaneous nerve fibers in vivo. Correspondingly, we observed upregulation of the receptor for the neuropeptide CGRP, RAMP1, in CD8+ T lymphocytes induced by skin commensal colonization. The neuroimmune CGRP-RAMP1 signaling axis functions in commensal-specific T cells to constrain Type 17 responses and moderate the activation status of microbiota-reactive lymphocytes at homeostasis. As such, modulation of neuroimmune CGRP-RAMP1 signaling in commensal-specific T cells shapes the overall activation status of the skin epithelium, thereby impacting the outcome of responses to insults such as wounding. The ability of somatosensory neurons to control adaptive immunity to the microbiota via the CGRP-RAMP1 axis underscores the various layers of regulation and multisystem coordination required for optimal microbiota-reactive T cell functions under steady state and pathology.

Signalling of the neuropeptide calcitonin gene-related peptide (CGRP) through RAMP1 promotes liver fibrosis via TGFß1/Smad2 and YAP pathways.

The liver is innervated by primary sensory nerve fibres releasing the neuropeptide calcitonin gene-related peptide (CGRP). Elevated plasma levels of CGRP have been found in patients with liver fibrosis or cirrhosis. We hypothesised that signalling of CGRP and its receptors might regulate liver fibrosis and propose a novel potential target for the treatment. In this study, hepatic expression of CGRP and its receptor component, the receptor activity-modifying protein 1 (RAMP1), was dramatically increased in diseased livers of patients. In a murine liver fibrosis model, deficiency of RAMP1 resulted in attenuated fibrogenesis characterized by less collagen deposition and decreased activity of hepatic stellate cells (HSC). Mechanistically, activity of the TGFß1 signalling core component Smad2 was severely impaired in the absence of RAMP1, and Yes-associated protein (YAP) activity was found to be diminished in RAMP1-deficient liver parenchyma. In vitro, stimulation of the HSC line LX-2 cells with CGRP induces TGFß1 production and downstream signalling as well as HSC activation documented by increased α-SMA expression and collagen synthesis. We further demonstrate in LX-2 cells that CGRP promotes YAP activation and its nuclear translocation subsequent to TGFß1/Smad2 signals. These data support a promotive effect of CGRP signalling in liver fibrosis via stimulation of TGFß1/Smad2 and YAP activity.

Sensory nerves drive migration of dental pulp stem cells via the CGRP-Ramp1 axis in pulp repair.

Dental pulp stem cells (DPSCs) are responsible for maintaining pulp structure and function after pulp injury. DPSCs migrate directionally to the injury site before differentiating into odontoblast-like cells, which is a prerequisite and a determinant in pulp repair. Increasing evidence suggests that sensory neuron-stem cell crosstalk is critical for maintaining normal physiological functions, and sensory nerves influence stem cells mainly by neuropeptides. However, the role of sensory nerves on DPSC behaviors after pulp injury is largely unexplored. Here, we find that sensory nerves released significant amounts of calcitonin gene-related peptide (CGRP) near the injury site, acting directly on DPSCs via receptor activity modifying protein 1 (RAMP1) to promote collective migration of DPSCs to the injury site, and ultimately promoting pulp repair. Specifically, sensory denervation leads to poor pulp repair and ectopic mineralization, in parallel with that DPSCs failed to be recruited to the injury site. Furthermore, in vitro evidence shows that sensory nerve-deficient microenvironment suppressed DPSC migration prominently among all related behaviors. Mechanistically, the CGRP-Ramp1 axis between sensory neurons and DPSCs was screened by single-cell RNA-seq analysis and immunohistochemical studies confirmed that the expression of CGRP rather than Ramp1 increases substantially near the damaged site. We further demonstrated that CGRP released by sensory nerves binds the receptor Ramp1 on DPSCs to facilitate cell collective migration by an indirect co-culture system using conditioned medium from trigeminal neurons, CGRP recombinant protein and antagonists BIBN4096. The treatment with exogenous CGRP promoted the recruitment of DPSCs, and ultimately enhanced the quality of pulp repair. Targeting the sensory nerve could therefore provide a new strategy for stem cell-based pulp repair and regeneration.

Regulation of Cutaneous Immunity In Vivo by Calcitonin Gene-Related Peptide Signaling through Endothelial Cells.

Calcitonin gene-related peptide (CGRP) can bias the outcome of Ag presentation to responsive T cells in vitro away from Th1-type immunity and toward the Th2 and Th17 poles through actions on endothelial cells (ECs). To test the in vivo significance of this observation, we engineered a mouse lacking functional CGRP receptors on ECs (EC receptor activity modifying protein 1 [RAMP1] knockout mice). On percutaneous immunization to 1-fluoro-2,4-dinitrobenzene, stimulated CD4+ T cells from draining lymph nodes showed significantly reduced IL-17A expression with significantly increased IFN-γ, IL-4, and IL-22 expression at the protein and mRNA levels compared with control mice. Retinoic acid receptor-related orphan receptor γ t mRNA was significantly reduced, while mRNAs for T-box expressed in T cells and GATA binding protein 3 were significantly increased. In addition, EC RAMP1 knockout mice had significantly reduced contact hypersensitivity responses, and systemic administration of a CGRP receptor antagonist similarly inhibited contact hypersensitivity in wild-type mice. These observations provide compelling evidence that CGRP is a key regulator of cutaneous immunity through effects on ECs and suggest a novel pathway for potential therapeutic manipulation.

Structural Basis for Receptor Activity-Modifying Protein-Dependent Selective Peptide Recognition by a G Protein-Coupled Receptor.

Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a ß-turn structure near their C termini rather than the α-helical structure common to peptides that bind related GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. The structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes.

Receptor activity-modifying protein 1 regulates mouse skin fibroblast proliferation via the Gαi3-PKA-CREB-YAP axis.

BACKGROUND: Skin innervation is crucial for normal wound healing. However, the relationship between nerve receptors and wound healing and the intrinsic mechanism remains to be further identified. In this study, we investigated the role of a calcitonin gene-related peptide (CGRP) receptor component, receptor activity-modifying protein 1 (RAMP1), in mouse skin fibroblast (MSF) proliferation. METHODS: In vivo, Western blotting and immunohistochemical (IHC) staining of mouse skin wounds tissue was used to detect changes in RAMP1 expression. In vitro, RAMP1 was overexpressed in MSF cell lines by infection with Tet-On-Flag-RAMP1 lentivirus and doxycycline (DOX) induction. An IncuCyte S3 Live-Cell Analysis System was used to assess and compare the proliferation rate differences between different treatment groups. Total protein and subcellular extraction Western blot analysis, quantitative real-time-polymerase chain reaction (qPCR) analysis, and immunofluorescence (IF) staining analysis were conducted to detect signalling molecule expression and/or distribution. The CUT & RUN assay and dual-luciferase reporter assay were applied to measure protein-DNA interactions. RESULTS: RAMP1 expression levels were altered during skin wound healing in mice. RAMP1 overexpression promoted MSF proliferation. Mechanistically, total Yes-associated protein (YAP) and nuclear YAP protein expression was increased in RAMP1-overexpressing MSFs. RAMP1 overexpression increased inhibitory guanine nucleotide-binding protein (G protein) α subunit 3 (Gαi3) expression and activated downstream protein kinase A (PKA), and both elevated the expression of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) and activated it, promoting the transcription of YAP, elevating the total YAP level and promoting MSF proliferation. CONCLUSIONS: Based on these data, we report, for the first time, that changes in the total RAMP1 levels during wound healing and RAMP1 overexpression alone can promote MSF proliferation via the Gαi3-PKA-CREB-YAP axis, a finding critical for understanding RAMP1 function, suggesting that this pathway is an attractive and accurate nerve target for skin wound treatment. Video Abstract.

RAMP1 signaling in immune cells regulates inflammation-associated lymphangiogenesis.

Calcitonin gene-related peptide (CGRP) regulates inflammation via signaling through receptor activity-modifying protein (RAMP) 1. Here, we investigated the role of RAMP1 signaling in growth of lymphatic vessels during inflammation. Lymphangiogenesis in the diaphragm of RAMP1-deficient (-/-) mice or their wild-type (WT) counterparts was induced by repeated intraperitoneal injection of lipopolysaccharide (LPS). Compared with WT mice, LPS-induced lymphangiogenesis in RAMP1-/- mice was suppressed. This was accompanied by the reduced expression of vascular endothelial growth factor (VEGF)-C and VEGF-D. The number of CD4+ cells in diaphragm tissue from WT mice was greater than RAMP1-/- mice. Removing CD4+ cells attenuated lymphangiogenesis and expression of VEGF-C and VEGF-D. CD4+ cells isolated from RAMP1-/- mice exhibited reduced expression of VEGF-C and VEGF-D. The number of CD11b+ cells from RAMP1-/- mice was higher than WT mice and was associated with the upregulated expression of genes related to pro-inflammatory macrophage phenotype and downregulation of reparative macrophage phenotype-related expression. When fluorescein isothiocyanate (FITC)-dextran was injected into the peritoneal cavity, the amount of residual FITC-dextran in WT mice was lower than that in RAMP1-/- mice. The present results suggest that RAMP1 signaling in immune cells plays a critical role in inflammation-related lymphangiogenesis; therefore, it represents a novel target for controlling lymphangiogenesis.

Activation of Calcitonin Gene-Related Peptide and Adrenomedullin Receptors by PEGylated Adrenomedullin.

Adrenomedullin (AM) improves colitis in animal models and patients with inflammatory bowel disease. We have developed a PEGylated AM derivative (PEG-AM) for clinical application because AM has a short half-life in the blood. However, modification by addition of polyethylene glycol (PEG) may compromise the function of the original peptide. In this paper, we examined the time course of cAMP accumulation induced by 5 and 60 kDa PEG-AM and compared the activation of calcitonin gene-related peptide (CGRP), AM1 and AM2 receptors by AM, 5 and 60 kDa PEG-AM. We also evaluated the effects of antagonists on the action of 5 and 60 kDa PEG-AM. PEG-AM stimulated cAMP production induced by these receptors; the increase in cAMP levels resulting from application of PEG-AM peaked at 15 min. Moreover, PEG-AM activity was antagonized by CGRP (8-37) or AM (22-52) (antagonists of CGRP and AM receptors, respectively) and the maximal response was not suppressed. These findings indicate that the effects of PEG-AM are similar to those of native AM.

The influence of receptor activity-modifying protein-1 overexpression on angiogenesis in mouse brain capillary endothelial cells.

Receptor activity-modifying protein-1 (RAMP1) is highly expressed in the heart and vasculature, indicating that it might be related to the vascular system. However, the effects of RAMP1 on angiogenesis and the intrinsic mechanisms underlying this process remain unclear. Here, we verified that RAMP1 is a critical regulator of angiogenesis in a mouse brain capillary endothelial cell line (bEnd.3). We first constructed a RAMP1 overexpression lentiviral vector system and stably transfected bEnd.3 cells. We further showed that RAMP1 overexpression could lead to bEnd.3 migration and capillary tube formation in Matrigel without exogenous calcitonin gene-related peptide (CGRP) treatment. At the same time, RAMP1 overexpression had little effect on proliferation. More importantly, vascular endothelial growth factor (VEGF) and CGRP expression levels were not significantly higher in RAMP1-overexpressing cells than in control cells (P > 0.05), indicating that RAMP1 did not function through upregulating VEGF or CGRP expression in bEnd.3 cells. Strikingly, RAMP1 transfection increased adrenomedullin 2 (AM2) expression levels ( P < 0.05). Taken together, these data contribute to a better understanding of the molecular mechanisms of RAMP1 in angiogenesis.

Expression and activity of the calcitonin receptor family in a sample of primary human high-grade gliomas.

BACKGROUND: Glioblastoma (GBM) is the most common and aggressive type of primary brain cancer. With median survival of less than 15 months, identification and validation of new GBM therapeutic targets is of critical importance. RESULTS: In this study we tested expression and performed pharmacological characterization of the calcitonin receptor (CTR) as well as other members of the calcitonin family of receptors in high-grade glioma (HGG) cell lines derived from individual patient tumours, cultured in defined conditions. Previous immunohistochemical data demonstrated CTR expression in GBM biopsies and we were able to confirm CALCR (gene encoding CTR) expression. However, as assessed by cAMP accumulation assay, only one of the studied cell lines expressed functional CTR, while the other cell lines have functional CGRP (CLR/RAMP1) receptors. The only CTR-expressing cell line (SB2b) showed modest coupling to the cAMP pathway and no activation of other known CTR signaling pathways, including ERK1/2 and p38 MAP kinases, and Ca2+ mobilization, supportive of low cell surface receptor expression. Exome sequencing data failed to account for the discrepancy between functional data and expression on the cell lines that do not respond to calcitonin(s) with no deleterious non-synonymous polymorphisms detected, suggesting that other factors may be at play, such as alternative splicing or rapid constitutive receptor internalisation. CONCLUSIONS: This study shows that GPCR signaling can display significant variation depending on cellular system used, and effects seen in model recombinant cell lines or tumour cell lines are not always reproduced in a more physiologically relevant system and vice versa.

The neuropeptide receptor subunit RAMP1 constrains the innate immune response during acute pancreatitis in mice.

OBJECTIVES: The importance of the Calcitonin-gene-related-peptide-pathway (CGRP) as neuronal modulator of innate immune responses in mice has been previously demonstrated. The CGRP-receptor is composed of two subunits: the receptor-activity-modifying-protein-1 (RAMP1) and the calcitonin-receptor-like-receptor (CLR). CGRP can influence immune cells and their capacity of producing inflammatory cytokines. Using a RAMP1 knockout-mouse (RAMP1-/-) we examined the role of the CGRP-receptor in the acute-phase of cerulein-induced pancreatitis. METHODS: Hourly cerulein-injections for a period of 8 h in RAMP1-/- and wild-type mice were performed. To compare severity and extent of inflammation in RAMP1-/- and wild-type mice, histological analyses were done and cytokine levels were assessed using qRT-PCR 8 h, 24 h, 2 days, and 7 days post-cerulein-treatment. Furthermore, serum activities of LDH and lipase were determined. RESULTS: After 8 h RAMP1-/- mice showed a higher pancreas-to-body-weight-ratio, increased tissue edema and immune cell infiltration with higher amount of F4/80-positive cells as compared to wild-type mice. Overall infiltration of immune cells at 24 h was increased in RAMP1-/- mice and composed predominantly of MPO-positive neutrophils. In addition, after 24 h RAMP1-/- mice presented a higher pancreas-to-body-weight-ratio, higher expression of Ccl3, Il6, and Il1b and increased number of cleaved caspase 3 positive cells. Serum lipase correlated with the extent of tissue damage in RAMP1-/- compared to wild-type mice 24 h post-cerulein treatment. CONCLUSION: Mice lacking RAMP1 showed increased inflammation, tissue edema, and pancreas injury particularly in the early phase of acute pancreatitis. This study highlights the essential role of CGRP for dampening the innate immune response in acute pancreatitis.

Molecular studies of CGRP and the CGRP family of peptides in the central nervous system.

BACKGROUND: Calcitonin gene-related peptide is an important target for migraine and other painful neurovascular conditions. Understanding the normal biological functions of calcitonin gene-related peptide is critical to understand the mechanisms of calcitonin gene-related peptide-blocking therapies as well as engineering improvements to these medications. Calcitonin gene-related peptide is closely related to other peptides in the calcitonin gene-related peptide family of peptides, including amylin. Relatedness in peptide sequence and in receptor biology makes it difficult to tease apart the contributions that each peptide and receptor makes to physiological processes and to disorders. SUMMARY: The focus of this review is the expression of calcitonin gene-related peptide, related peptides and their receptors in the central nervous system. Calcitonin gene-related peptide is expressed throughout the nervous system, whereas amylin and adrenomedullin have only limited expression at discrete sites in the brain. The components of two receptors that respond to calcitonin gene-related peptide, the calcitonin gene-related peptide receptor (calcitonin receptor-like receptor with receptor activity-modifying protein 1) and the AMY1 receptor (calcitonin receptor with receptor activity-modifying protein 1), are expressed throughout the nervous system. Understanding expression of the peptides and their receptors lays the foundation for more deeply understanding their physiology, pathophysiology and therapeutic use.

Induction of Migraine-Like Photophobic Behavior in Mice by Both Peripheral and Central CGRP Mechanisms.

The neuropeptide calcitonin gene-related peptide (CGRP) is a key player in migraine. Although migraine can be treated using CGRP antagonists that act peripherally, the relevant sites of CGRP action remain unknown. To address the role of CGRP both within and outside the CNS, we used CGRP-induced light-aversive behavior in mice as a measure of migraine-associated photophobia. Peripheral (intraperitoneal) injection of CGRP resulted in light-aversive behavior in wild-type CD1 mice similar to aversion seen previously after central (intracerebroventricular) injection. The phenotype was also observed in C57BL/6J mice, although to a lesser degree and with more variability. After intraperitoneal CGRP, motility was decreased in the dark only, similar to motility changes after intracerebroventricular CGRP. In addition, as with intracerebroventricular CGRP, there was no general increase in anxiety as measured in an open-field assay after intraperitoneal CGRP. Importantly, two clinically effective migraine drugs, the 5-HT1B/D agonist sumatriptan and a CGRP-blocking monoclonal antibody, attenuated the peripheral CGRP-induced light aversion and motility behaviors. To begin to address the mechanism of peripheral CGRP action, we used transgenic CGRP-sensitized mice that have elevated levels of the CGRP receptor hRAMP1 subunit in nervous tissue (nestin/hRAMP1). Surprisingly, sensitivity to low light was not seen after intraperitoneal CGRP injection, but was seen after intracerebroventricular CGRP injection. These results suggest that CGRP can act in both the periphery and the brain by distinct mechanisms and that CGRP actions may be transmitted to the CNS via indirect sensitization of peripheral nerves. SIGNIFICANCE STATEMENT: The neuropeptide calcitonin gene-related peptide (CGRP) is a central player in migraine pathogenesis, yet its site(s) of action remains unknown. Some preclinical studies have pointed to central sites in the brain and brainstem. However, a peripheral site of action is indicated by the ability of intravenous CGRP to trigger migraine in humans and the efficacy of CGRP receptor antagonists that evidently do no penetrate the CNS in effective amounts. Resolving this issue is particularly important given recent clinical trials showing that anti-CGRP monoclonal antibodies can reduce and even prevent migraine attacks. In this study, we report that CGRP can act in both the brain and the periphery of the mouse to cause migraine-like photophobia by apparently distinct mechanisms.

Photoaffinity Cross-Linking and Unnatural Amino Acid Mutagenesis Reveal Insights into Calcitonin Gene-Related Peptide Binding to the Calcitonin Receptor-like Receptor/Receptor Activity-Modifying Protein 1 (CLR/RAMP1) Complex.

Calcitonin gene-related peptide (CGRP) binds to the complex of the calcitonin receptor-like receptor (CLR) with receptor activity-modifying protein 1 (RAMP1). How CGRP interacts with the transmembrane domain (including the extracellular loops) of this family B receptor remains unclear. In this study, a photoaffinity cross-linker, p-azido l-phenylalanine (azF), was incorporated into CLR, chiefly in the second extracellular loop (ECL2) using genetic code expansion and unnatural amino acid mutagenesis. The method was optimized to ensure efficient photolysis of azF residues near the transmembrane bundle of the receptor. A CGRP analogue modified with fluorescein at position 15 was used for detection of ultraviolet-induced cross-linking. The methodology was verified by confirming the known contacts of CGRP to the extracellular domain of CLR. Within ECL2, the chief contacts were I284 on the loop itself and L291, at the top of the fifth transmembrane helix (TM5). Minor contacts were noted along the lip of ECL2 between S286 and L290 and also with M223 in TM3 and F349 in TM6. Full length molecular models of the bound receptor complex suggest that CGRP sits at the top of the TM bundle, with Thr6 of the peptide making contacts with L291 and H295. I284 is likely to contact Leu12 and Ala13 of CGRP, and Leu16 of CGRP is at the ECL/extracellular domain boundary of CLR. The reduced potency, Emax, and affinity of [Leu16Ala]-human α CGRP are consistent with this model. Contacts between Thr6 of CGRP and H295 may be particularly important for receptor activation.

Probing the Mechanism of Receptor Activity-Modifying Protein Modulation of GPCR Ligand Selectivity through Rational Design of Potent Adrenomedullin and Calcitonin Gene-Related Peptide Antagonists.

Binding of the vasodilator peptides adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) to the class B G protein-coupled receptor calcitonin receptor-like receptor (CLR) is modulated by receptor activity-modifying proteins (RAMPs). RAMP1 favors CGRP, whereas RAMP2 and RAMP3 favor AM. Crystal structures of peptide-bound RAMP1/2-CLR extracellular domain (ECD) heterodimers suggested RAMPs alter ligand preference through direct peptide contacts and allosteric modulation of CLR. Here, we probed this dual mechanism through rational structure-guided design of AM and CGRP antagonist variants. Variants were characterized for binding to purified RAMP1/2-CLR ECD and for antagonism of the full-length CGRP (RAMP1:CLR), AM1 (RAMP2:CLR), and AM2 (RAMP3:CLR) receptors. Short nanomolar affinity AM(37-52) and CGRP(27-37) variants were obtained through substitutions including AM S45W/Q50W and CGRP K35W/A36S designed to stabilize their ß-turn. K46L and Y52F substitutions designed to exploit RAMP allosteric effects and direct peptide contacts, respectively, yielded AM variants with selectivity for the CGRP receptor over the AM1 receptor. AM(37-52) S45W/K46L/Q50W/Y52F exhibited nanomolar potency at the CGRP receptor and micromolar potency at AM1 A 2.8-Å resolution crystal structure of this variant bound to the RAMP1-CLR ECD confirmed that it bound as designed. CGRP(27-37) N31D/S34P/K35W/A36S exhibited potency and selectivity comparable to the traditional antagonist CGRP(8-37). Giving this variant the ability to contact RAMP2 through the F37Y substitution increased affinity for AM1, but it still preferred the CGRP receptor. These potent peptide antagonists with altered selectivity inform the development of AM/CGRP-based pharmacological tools and support the hypothesis that RAMPs alter CLR ligand selectivity through allosteric effects and direct peptide contacts.

Neuropeptide regulation of adaptive immunity in the tibia fracture model of complex regional pain syndrome.

BACKGROUND: Both dysfunctional neuropeptide signaling and immune system activation are characteristic of complex regional pain syndrome (CRPS). Unknown is whether substance P (SP) or calcitonin gene-related peptide (CGRP) support autoantibody production and, consequently, nociceptive sensitization. METHODS: These experiments involved the use of a well-characterized tibia fracture model of CRPS. Mice deficient in SP expression (Tac1-/-) and CGRP signaling (RAMP1-/-) were used to probe the neuropeptide dependence of post-fracture sensitization and antibody production. The deposition of IgM in the spinal cord, sciatic nerves, and skin was followed using Western blotting, as was expression of the CRPS-related autoantigen cytokeratin 16 (Krt16). Passive serum transfer to B-cell-deficient muMT mice was used to assess the production of functional autoantibodies in CRPS model mice. The use of immunohistochemistry allowed us to assess neuropeptide-containing fiber distribution and Langerhans cell abundance in mouse and human CRPS patient skin, while Langerhans cell-deficient mice were used to assess the functional contributions of these cells. RESULTS: Functional SP and CGRP signaling were required both for the full development of nociceptive sensitization after fracture and the deposition of IgM in skin and neural tissues. Furthermore, the passive transfer of serum from wildtype but not neuropeptide-deficient mice to fractured muMT mice caused enhanced allodynia and postural unweighting. Langerhans cells were increased in number in the skin of fracture mice and CRPS patients, and those increases in mice were reduced in neuropeptide signaling-deficient animals. Unexpectedly, Langerhans cell-deficient mice showed normal nociceptive sensitization after fracture. However, the increased expression of Krt16 after tibia fracture was not seen in neuropeptide-deficient mice. CONCLUSIONS: Collectively, these data support the hypothesis that neuropeptide signaling in the fracture limb of mice is required for autoantigenic IgM production and nociceptive sensitization. The mechanism may be related to neuropeptide-supported autoantigen expression.

Global functions of extracellular, transmembrane and cytoplasmic domains of organic solute transporter ß-subunit.

Transport of bile acids across the basolateral membrane of the intestinal enterocyte is carried out by the organic solute transporter (Ost) composed of a seven-transmembrane domain (TMD) subunit (Ostα) and an ancillary single TMD subunit (Ostß). Although previous investigations have demonstrated the importance of the TMD of Ostß for its activity, further studies were conducted to assess the contributions of other regions of the Ostß subunit. Transport activity was retained when Ostß was truncated to contain only the TMD with 15 additional residues on each side and co-expressed with Ostα, whereas shorter fragments were inactive. To probe the broader functions of Ostß segments, chimeric proteins were constructed in which N-terminal, TMD or C-terminal regions of Ostß were fused to corresponding regions of receptor activity-modifying protein (RAMP1), a single TMD protein required by several seven-TMD G-protein-coupled receptors including the calcitonin receptor-like receptor (CLR). Ostß/RAMP1 chimeras were expressed with Ostα and CLR. As expected, replacing the Ostß TMD abolished transport activity; however, replacing either the entire N-terminal or entire C-terminal domain of Ostß with RAMP1 sequences did not prevent plasma membrane localization or the ability to support [3H]taurocholate uptake. Co-immunoprecipitation experiments revealed that the C-terminus of Ostß is a previously unrecognized site of interaction with Ostα. All chimeras containing N-terminal RAMP1 segments allowed co-expressed CLR to respond to agonists with strong increases in cyclic AMP. These results provide new insights into the structure and function of the heteromeric Ost transporter complex.

Relative Antagonism of Mutants of the CGRP Receptor Extracellular Loop 2 Domain (ECL2) Using a Truncated Competitive Antagonist (CGRP8-37): Evidence for the Dual Involvement of ECL2 in the Two-Domain Binding Model.

The second extracellular loop (ECL2) of the G protein-coupled receptor (GPCR) family is important for ligand interaction and drug discovery. ECL2 of the family B cardioprotective calcitonin gene-related peptide (CGRP) receptor is required for cell signaling. Family B GPCR ligands have two regions; the N-terminus mediates receptor activation, and the remainder confers high-affinity binding. Comparing antagonism of CGRP8-37 at a number of point mutations of ECL2 of the CGRP receptor, we show that the ECL2 potentially facilitates interaction with up to the 18 N-terminal residues of CGRP. This has implications for understanding family B GPCR activation and for drug design at the CGRP receptor.

Calcitonin and Amylin Receptor Peptide Interaction Mechanisms: INSIGHTS INTO PEPTIDE-BINDING MODES AND ALLOSTERIC MODULATION OF THE CALCITONIN RECEPTOR BY RECEPTOR ACTIVITY-MODIFYING PROTEINS.

Receptor activity-modifying proteins (RAMP1-3) determine the selectivity of the class B G protein-coupled calcitonin receptor (CTR) and the CTR-like receptor (CLR) for calcitonin (CT), amylin (Amy), calcitonin gene-related peptide (CGRP), and adrenomedullin (AM) peptides. RAMP1/2 alter CLR selectivity for CGRP/AM in part by RAMP1 Trp-84 or RAMP2 Glu-101 contacting the distinct CGRP/AM C-terminal residues. It is unclear whether RAMPs use a similar mechanism to modulate CTR affinity for CT and Amy, analogs of which are therapeutics for bone disorders and diabetes, respectively. Here, we reproduced the peptide selectivity of intact CTR, AMY1 (CTR·RAMP1), and AMY2 (CTR·RAMP2) receptors using purified CTR extracellular domain (ECD) and tethered RAMP1- and RAMP2-CTR ECD fusion proteins and antagonist peptides. All three proteins bound salmon calcitonin (sCT). Tethering RAMPs to CTR enhanced binding of rAmy, CGRP, and the AMY antagonist AC413. Peptide alanine-scanning mutagenesis and modeling of receptor-bound sCT and AC413 supported a shared non-helical CGRP-like conformation for their TN(T/V)G motif prior to the C terminus. After this motif, the peptides diverged; the sCT C-terminal Pro was crucial for receptor binding, whereas the AC413/rAmy C-terminal Tyr had little or no influence on binding. Accordingly, mutant RAMP1 W84A- and RAMP2 E101A-CTR ECD retained AC413/rAmy binding. ECD binding and cell-based signaling assays with antagonist sCT/AC413/rAmy variants with C-terminal residue swaps indicated that the C-terminal sCT/rAmy residue identity affects affinity more than selectivity. rAmy(8-37) Y37P exhibited enhanced antagonism of AMY1 while retaining selectivity. These results reveal unexpected differences in how RAMPs determine CTR and CLR peptide selectivity and support the hypothesis that RAMPs allosterically modulate CTR peptide affinity.

The central anorexigenic mechanism of amylin in Japanese quail (Coturnix japonica) involves pro-opiomelanocortin, calcitonin receptor, and the arcuate nucleus of the hypothalamus.

Amylin is a 37-amino acid peptide hormone that exerts anorexigenic effects in humans and animals. We demonstrated that central injection of amylin into chicks affected feeding and related behaviors via the hypothalamus and brainstem, although the molecular mechanisms remained elusive. Thus, the objective of this study was to investigate the molecular mechanisms underlying anorexigenic effects of amylin in 7 day-old Japanese quail. Food but not water intake was reduced after intracerebroventricular amylin injection, and the behavior analysis indicated that this was associated with decreased food pecks and preening. Whole hypothalamus and hypothalamic nuclei including the arcuate nucleus (ARC), paraventricular nucleus (PVN), ventromedial hypothalamus (VMH), dorsomedial nucleus (DMN) and lateral hypothalamic area (LH) were extracted from quail at 1h post-injection for total RNA isolation. Real time PCR was performed to quantify mRNA abundance of amylin receptors, appetite-associated neuropeptides and monoamine-synthesis-related enzymes. Central amylin injection increased the mRNA abundance of calcitonin receptor (CALCR), receptor activity modifying protein 1 (RAMP1), pro-opiomelanocortin (POMC), and aromatic l-amino acid decarboxylase (AADC) in the hypothalamus and individual hypothalamic nuclei. Relative quantities of CALCR and POMC mRNA were greater in the ARC of the amylin- than vehicle-treated group. Thus, amylin-mediated effects on food intake may involve POMC, monoamine synthesis, and amylin receptor 1 (a complex of CALCR and RAMP1) in the ARC. Together, these data provide novel insights on the hypothalamic-specific molecular mechanisms of amylin-induced food intake.