Negative allosteric modulation of the glucagon receptor by RAMP2.

Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B GPCRs. We show that RAMP2 directly interacts with the glucagon receptor (GCGR), a Family B GPCR responsible for blood sugar homeostasis, and broadly inhibits receptor-induced downstream signaling. HDX-MS experiments demonstrate that RAMP2 enhances local flexibility in select locations in and near the receptor extracellular domain (ECD) and in the 6th transmembrane helix, whereas smFRET experiments show that this ECD disorder results in the inhibition of active and intermediate states of the intracellular surface. We determined the cryo-EM structure of the GCGR-Gs complex at 2.9 Å resolution in the presence of RAMP2. RAMP2 apparently does not interact with GCGR in an ordered manner; however, the receptor ECD is indeed largely disordered along with rearrangements of several intracellular hallmarks of activation. Our studies suggest that RAMP2 acts as a negative allosteric modulator of GCGR by enhancing conformational sampling of the ECD.

Expression of the Calcitonin Receptor-like Receptor (CALCRL) in Normal and Neoplastic Tissues.

Little information is available concerning protein expression of the calcitonin receptor-like receptor (CALCRL) at the protein level. Here, we developed a rabbit monoclonal antibody, 8H9L8, which is directed against human CALCRL but cross-reacts with the rat and mouse forms of the receptor. We confirmed antibody specificity via Western blot analyses and immunocytochemistry using the CALCRL-expressing neuroendocrine tumour cell line BON-1 and a CALCRL-specific small interfering RNA (siRNA). We then used the antibody for immunohistochemical analyses of various formalin-fixed, paraffin-embedded specimens of normal and neoplastic tissues. In nearly all tissue specimens examined, CALCRL expression was detected in the capillary endothelium, smooth muscles of the arterioles and arteries, and immune cells. Analyses of normal human, rat, and mouse tissues revealed that CALCRL was primarily present in distinct cell populations in the cerebral cortex; pituitary; dorsal root ganglia; epithelia, muscles, and glands of the larger bronchi; intestinal mucosa (particularly in enteroendocrine cells); intestinal ganglia; exocrine and endocrine pancreas; arteries, capillaries, and glomerular capillary loops in the kidneys; the adrenals; Leydig cells in the testicles; and syncytiotrophoblasts in the placenta. In the neoplastic tissues, CALCRL was predominantly expressed in thyroid carcinomas, parathyroid adenomas, small-cell lung cancers, large-cell neuroendocrine carcinomas of the lung, pancreatic neuroendocrine neoplasms, renal clear-cell carcinomas, pheochromocytomas, lymphomas, and melanomas. In these tumours with strong expression of CALCRL, the receptor may represent a useful target structure for future therapies.

A narrative review of the calcitonin peptide family and associated receptors as migraine targets: Calcitonin gene-related peptide and beyond.

OBJECTIVE: To summarize the pharmacology of the calcitonin peptide family of receptors and explore their relationship to migraine and current migraine therapies. BACKGROUND: Therapeutics that dampen calcitonin gene-related peptide (CGRP) signaling are now in clinical use to prevent or treat migraine. However, CGRP belongs to a broader peptide family, including the peptides amylin and adrenomedullin. Receptors for this family are complex, displaying overlapping pharmacologic profiles. Despite the focus on CGRP and the CGRP receptor in migraine research, recent evidence implicates related peptides and receptors in migraine. METHODS: This narrative review summarizes literature encompassing the current pharmacologic understanding of the calcitonin peptide family, and the evidence that links specific members of this family to migraine and migraine-like behaviors. RESULTS: Recent work links amylin and adrenomedullin to migraine-like behavior in rodent models and migraine-like attacks in individuals with migraine. We collate novel information that suggests females may be more sensitive to amylin and CGRP in the context of migraine-like behaviors. We report that drugs designed to antagonize the canonical CGRP receptor also antagonize a second CGRP-responsive receptor and speculate as to whether this influences therapeutic efficacy. We also discuss the specificity of current drugs with regards to CGRP isoforms and how this may influence therapeutic profiles. Lastly, we emphasize that receptors related to, but distinct from, the canonical CGRP receptor may represent underappreciated and novel drug targets. CONCLUSION: Multiple peptides within the calcitonin family have been linked to migraine. The current focus on CGRP and its canonical receptor may be obscuring pathways to further therapeutics. Drug discovery schemes that take a wider view of the receptor family may lead to the development of new anti-migraine drugs with favorable clinical profiles. We also propose that understanding these related peptides and receptors may improve our interpretation regarding the mechanism of action of current drugs.

Evolution of calcitonin/calcitonin gene-related peptide family in chordates: Identification of CT/CGRP family peptides in cartilaginous fish genome.

The calcitonin (CT)/CT gene-related peptide (CGRP) family is a peptide gene family that is widely found in bilaterians. CT, CGRP, adrenomedullin (AM), amylin (AMY), and CT receptor-stimulating peptide (CRSP) are members of the CT/CGRP family. In mammals, CT is involved in calcium homeostasis, while CGRP and AM primarily function in vasodilation. AMY and CRSP are associated with anorectic effects. Diversification of the molecular features and physiological functions of the CT/CGRP family in vertebrate lineages have been extensively reported. However, the origin and diversification mechanisms of the vertebrate CT/CGRP family of peptides remain unclear. In this review, the molecular characteristics of CT/CGRP family peptides and their receptors, along with their major physiological functions in mammals and teleosts, are introduced. Furthermore, novel candidates of the CT/CGRP family in cartilaginous fish are presented based on genomic information. The CT/CGRP family peptides and receptors in urochordates and cephalochordates, which are closely related to vertebrates, are also described. Finally, a putative evolutionary scenario of the CT/CGRP family peptides and receptors in chordates is discussed.

Possible involvement of neuropeptide and neurotransmitter receptors in Adenomyosis.

BACKGROUND: Accumulating data indicate that sensory nerve derived neuropeptides such as substance P and calcitonin gene related-protein (CGRP) can accelerate the progression of endometriosis via their respective receptors, so can agonists to their respective receptors receptor 1 (NK1R), receptor activity modifying protein 1 (RAMP-1) and calcitonin receptor-like receptor (CRLR). Adrenergic ß2 receptor (ADRB2) agonists also can facilitate lesional progression. In contrast, women with endometriosis appear to have depressed vagal activity, concordant with reduced expression of α7 nicotinic acetylcholine receptor (α7nAChR). The roles of these receptors in adenomyosis are completely unknown. METHODS: Adenomyotic tissue samples from 30 women with adenomyosis and control endometrial tissue samples from 24 women without adenomyosis were collected and subjected to immunohistochemistry analysis of RAMP1, CRLR, NK1R, ADRB2 and α7nAChR, along with their demographic and clinical information. The extent of tissue fibrosis was evaluated by Masson trichrome staining. RESULTS: We found that the staining levels of NK1R, CRLR, RAMP1 and ADRB2 were all significantly elevated in adenomyotic lesions as compared with control endometrium. In contrast, α7nAChR staining levels were significantly reduced. The severity of dysmenorrhea correlated positively with lesional ADRB2 staining levels. CONCLUSIONS: Our results suggest that SP, CGRP and noradrenaline may promote, while acetylcholine may stall, the progression of adenomyosis through their respective receptors on adenomyotic lesions. Additionally, through the activation of the hypothalamic-pituitary-adrenal (HPA)-sympatho-adrenal-medullary (SAM) axes and the lesional overexpression of ADRB2, adenomyosis-associated dysmenorrhea and adenomyotic lesions may be mutually promotional, forming a viscous feed-forward cycle.

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.

GPCRs globally coevolved with receptor activity-modifying proteins, RAMPs.

Receptor activity-modifying proteins (RAMPs) are widely expressed in human tissues and, in some cases, have been shown to affect surface expression or ligand specificity of G-protein-coupled receptors (GPCRs). However, whether RAMP-GPCR interactions are widespread, and the nature of their functional consequences, remains largely unknown. In humans, there are three RAMPs and over 800 expressed GPCRs, making direct experimental approaches challenging. We analyzed relevant genomic data from all currently available sequenced organisms. We discovered that RAMPs and GPCRs tend to have orthologs in the same species and have correlated phylogenetic trees to the same extent, or higher than other interacting protein pairs that play key roles in cellular signaling. In addition, the resulting RAMP-GPCR interaction map suggests that RAMP1 and RAMP3 interact with the same set of GPCRs, which implies functional redundancy. We next analyzed human transcriptomes and found expression correlation for GPCRs and RAMPs. Our results suggest global coevolution of GPCRs and RAMPS and support the hypothesis that GPCRs interact globally with RAMPs in cellular signaling pathways.

Structure-function analyses reveal a triple ß-turn receptor-bound conformation of adrenomedullin 2/intermedin and enable peptide antagonist design.

The cardioprotective vasodilator peptide adrenomedullin 2/intermedin (AM2/IMD) and the related adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) signal through three heterodimeric receptors comprising the calcitonin receptor-like class B G protein-coupled receptor (CLR) and a variable receptor activity-modifying protein (RAMP1, -2, or -3) that determines ligand selectivity. The CGRP receptor (RAMP1:CLR) favors CGRP binding, whereas the AM1 (RAMP2:CLR) and AM2 (RAMP3:CLR) receptors favor AM binding. How AM2/IMD binds the receptors and how RAMPs modulate its binding is unknown. Here, we show that AM2/IMD binds the three purified RAMP-CLR extracellular domain (ECD) complexes with a selectivity profile that is distinct from those of CGRP and AM. AM2/IMD bound all three ECD complexes but preferred the CGRP and AM2 receptor complexes. A 2.05 Å resolution crystal structure of an AM2/IMD antagonist fragment-bound RAMP1-CLR ECD complex revealed that AM2/IMD binds the complex through a unique triple ß-turn conformation that was confirmed by peptide and receptor mutagenesis. Comparisons of the receptor-bound conformations of AM2/IMD, AM, and a high-affinity CGRP analog revealed differences that may have implications for biased signaling. Guided by the structure, enhanced-affinity AM2/IMD antagonist variants were developed, including one that discriminates the AM1 and AM2 receptors with ∼40-fold difference in affinities and one stabilized by an intramolecular disulfide bond. These results reveal differences in how the three peptides engage the receptors, inform development of AM2/IMD-based pharmacological tools and therapeutics, and provide insights into RAMP modulation of receptor pharmacology.

Receptor activity-modifying protein 1 regulates the phenotypic expression of BMSCs via the Hippo/Yap pathway.

Receptor activity-modifying protein 1 (RAMP1) might be a critical regulator during bone wound healing. However, the roles and mechanisms of RAMP1 in osteogenesis remain unclear. Here, we aimed to elucidate the role of RAMP1 and explore the effects of Yes-associated protein 1 (Yap1), an effector of the Hippo/Yap pathway, in this process. We used a RAMP1 overexpression lentiviral system in bone marrow mesenchymal stem cells (BMSCs), which enhanced RAMP1 expression in an effective, appropriate, and sustained manner. Alkaline phosphatase (ALP) activity assays and alizarin red staining showed that RAMP1 promoted osteogenic differentiation of BMSCs after calcitonin gene-related peptide (CGRP) treatment (10 -8 mol/L). Moreover, real-time polymerase chain reaction and Western blot analysis indicated that RAMP1 upregulated the expression of osteogenic phenotypic markers (ALP, runt-related transcription factor 2, osteopontin; p < 0.05). To further uncover the mechanism of RAMP1 in osteogenic differentiation, we used verteporfin (10 -7 mol/L) to block Yap1. Notably, verteporfin impaired RAMP1-induced osteogenesis. Taken together, our findings confirmed that RAMP1 is a key mediator of bone regeneration and indicate that RAMP1 promotes CGRP-induced osteogenic differentiation of BMSCs via regulation of the Hippo/Yap pathway.

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.

Mutant RAMP2 causes primary open-angle glaucoma via the CRLR-cAMP axis.

PURPOSE: Primary open-angle glaucoma (POAG) is the leading cause of irreversible blindness worldwide and mutations in known genes can only explain 5-6% of POAG. This study was conducted to identify novel POAG-causing genes and explore the pathogenesis of this disease. METHODS: Exome sequencing was performed in a Han Chinese cohort comprising 398 sporadic cases with POAG and 2010 controls, followed by replication studies by Sanger sequencing. A heterozygous Ramp2 knockout mouse model was generated for in vivo functional study. RESULTS: Using exome sequencing analysis and replication studies, we identified pathogenic variants in receptor activity-modifying protein 2 (RAMP2) within three genetically diverse populations (Han Chinese, German, and Indian). Six heterozygous RAMP2 pathogenic variants (Glu39Asp, Glu54Lys, Phe103Ser, Asn113Lysfs*10, Glu143Lys, and Ser171Arg) were identified among 16 of 4763 POAG patients, whereas no variants were detected in any exon of RAMP2 in 10,953 control individuals. Mutant RAMP2s aggregated in transfected cells and resulted in damage to the AM-RAMP2/CRLR-cAMP signaling pathway. Ablation of one Ramp2 allele led to cAMP reduction and retinal ganglion cell death in mice. CONCLUSION: This study demonstrated that disruption of RAMP2/CRLR-cAMP axis could cause POAG and identified a potential therapeutic intervention for POAG.

CGRP Receptor Signalling Pathways.

Calcitonin gene-related peptide (CGRP) is a promiscuous peptide, similar to many other members of the calcitonin family of peptides. The potential of CGRP to act on many different receptors with differing affinities and efficacies makes deciphering the signalling from the CGRP receptor a challenging task for researchers.Although it is not a typical G protein-coupled receptor (GPCR), in that it is composed not just of a GPCR, the CGRP receptor activates many of the same signalling pathways common for other GPCRs. This includes the family of G proteins and a variety of protein kinases and transcription factors. It is now also clear that in addition to the initiation of cell-surface signalling, GPCRs, including the CGRP receptor, also activate distinct signalling pathways as the receptor is trafficking along the endocytic conduit.Given CGRP's characteristic of activating multiple GPCRs, we will first consider the complex of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) as the CGRP receptor. We will discuss the discovery of the CGRP receptor components, the molecular mechanisms controlling its internalization and post-endocytic trafficking (recycling and degradation) and the diverse signalling cascades that are elicited by this receptor in model cell lines. We will then discuss CGRP-mediated signalling pathways in primary cells pertinent to migraine including neurons, glial cells and vascular smooth muscle cells.Investigation of all the CGRP- and CGRP receptor-mediated signalling cascades is vital if we are to fully understand CGRP's role in migraine and will no doubt unearth new targets for the treatment of migraine and other CGRP-driven diseases.

Disruption of calcitonin gene-related peptide signaling accelerates muscle denervation and dampens cytotoxic neuroinflammation in SOD1 mutant mice.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease. Neuronal vacuolization and glial activation are pathologic hallmarks in the superoxide dismutase 1 (SOD1) mouse model of ALS. Previously, we found the neuropeptide calcitonin gene-related peptide (CGRP) associated with vacuolization and astrogliosis in the spinal cord of these mice. We now show that CGRP abundance positively correlated with the severity of astrogliosis, but not vacuolization, in several motor and non-motor areas throughout the brain. SOD1 mice harboring a genetic depletion of the ßCGRP isoform showed reduced CGRP immunoreactivity associated with vacuolization, while motor functions, body weight, survival, and astrogliosis were not altered. When CGRP signaling was completely disrupted through genetic depletion of the CGRP receptor component, receptor activity-modifying protein 1 (RAMP1), hind limb muscle denervation, and loss of muscle performance were accelerated, while body weight and survival were not affected. Dampened neuroinflammation, i.e., reduced levels of astrogliosis in the brain stem already in the pre-symptomatic disease stage, and reduced microgliosis and lymphocyte infiltrations during the late disease phase were additional neuropathology features in these mice. On the molecular level, mRNA expression levels of brain-derived neurotrophic factor (BDNF) and those of the anti-inflammatory cytokine interleukin 6 (IL-6) were elevated, while those of several pro-inflammatory cytokines found reduced in the brain stem of RAMP1-deficient SOD1 mice at disease end stage. Our results thus identify an important, possibly dual role of CGRP in ALS pathogenesis.

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.

Evidence for Conservation of the Calcitonin Superfamily and Activity-regulating Mechanisms in the Basal Chordate Branchiostoma floridae: INSIGHTS INTO THE MOLECULAR AND FUNCTIONAL EVOLUTION IN CHORDATES.

The calcitonin (CT)/CT gene-related peptide (CGRP) family is conserved in vertebrates. The activities of this peptide family are regulated by a combination of two receptors, namely the calcitonin receptor (CTR) and the CTR-like receptor (CLR), and three receptor activity-modifying proteins (RAMPs). Furthermore, RAMPs act as escort proteins by translocating CLR to the cell membrane. Recently, CT/CGRP family peptides have been identified or inferred in several invertebrates. However, the molecular characteristics and relevant functions of the CTR/CLR and RAMPs in invertebrates remain unclear. In this study, we identified three CT/CGRP family peptides (Bf-CTFPs), one CTR/CLR-like receptor (Bf-CTFP-R), and three RAMP-like proteins (Bf-RAMP-LPs) in the basal chordate amphioxus (Branchiostoma floridae). The Bf-CTFPs were shown to possess an N-terminal circular region typical of the CT/CGRP family and a C-terminal Pro-NH2. The Bf-CTFP genes were expressed in the central nervous system and in endocrine cells of the midgut, indicating that Bf-CTFPs serve as brain and/or gut peptides. Cell surface expression of the Bf-CTFP-R was enhanced by co-expression with each Bf-RAMP-LP. Furthermore, Bf-CTFPs activated Bf-CTFP-R·Bf-RAMP-LP complexes, resulting in cAMP accumulation. These results confirmed that Bf-RAMP-LPs, like vertebrate RAMPs, are prerequisites for the function and translocation of the Bf-CTFP-R. The relative potencies of the three peptides at each receptor were similar. Bf-CTFP2 was a potent ligand at all receptors in cAMP assays. Bf-RAMP-LP effects on ligand potency order were distinct to vertebrate CGRP/adrenomedullin/amylin receptors. To the best of our knowledge, this is the first molecular and functional characterization of an authentic invertebrate CT/CGRP family receptor and RAMPs.

Receptor Activity-modifying Proteins 2 and 3 Generate Adrenomedullin Receptor Subtypes with Distinct Molecular Properties.

Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM1 and AM2 receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins 2 and 3 (RAMP2 and RAMP3), respectively. These different CLR-RAMP interactions yield discrete receptor pharmacology and physiological effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the molecular details of the effects of RAMPs on CLR. To understand the role of RAMP2 and -3 on the activation and conformation of the CLR subunit of AM receptors, we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors, and determined the effects on cAMP signaling. Sixteen CLR mutations had differential effects between the AM1 and AM2 receptors. Accompanying this, independent molecular modeling of the full-length AM-bound AM1 and AM2 receptors predicted differences in the binding pocket and differences in the electrostatic potential of the two AM receptors. Druggability analysis indicated unique features that could be used to develop selective small molecule ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function.

Hyperoxia exposure disrupts adrenomedullin signaling in newborn mice: Implications for lung development in premature infants.

Hyperoxia contributes to the development of bronchopulmonary dysplasia (BPD), a chronic lung disease of human infants that is characterized by disrupted lung angiogenesis. Adrenomedullin (AM) is a multifunctional peptide with angiogenic and vasoprotective properties. AM signals via its cognate receptors, calcitonin receptor-like receptor (Calcrl) and receptor activity-modifying protein 2 (RAMP2). Whether hyperoxia affects the pulmonary AM signaling pathway in neonatal mice and whether AM promotes lung angiogenesis in human infants are unknown. Therefore, we tested the following hypotheses: (1) hyperoxia exposure will disrupt AM signaling during the lung development period in neonatal mice; and (2) AM will promote angiogenesis in fetal human pulmonary artery endothelial cells (HPAECs) via extracellular signal-regulated kinases (ERK) 1/2 activation. We initially determined AM, Calcrl, and RAMP2 mRNA levels in mouse lungs on postnatal days (PND) 3, 7, 14, and 28. Next we determined the mRNA expression of these genes in neonatal mice exposed to hyperoxia (70% O2) for up to 14 d. Finally, using HPAECs, we evaluated if AM activates ERK1/2 and promotes tubule formation and cell migration. Lung AM, Calcrl, and RAMP2 mRNA expression increased from PND 3 and peaked at PND 14, a time period during which lung development occurs in mice. Interestingly, hyperoxia exposure blunted this peak expression in neonatal mice. In HPAECs, AM activated ERK1/2 and promoted tubule formation and cell migration. These findings support our hypotheses, emphasizing that AM signaling axis is a potential therapeutic target for human infants with BPD.

ß-arrestins negatively control human adrenomedullin type 1-receptor internalization.

Adrenomedullin (AM) is a potent hypotensive peptide that exerts a powerful variety of protective effects against multiorgan damage through the AM type 1 receptor (AM1 receptor), which consists of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 2 (RAMP2). Two ß-arrestin (ß-arr) isoforms, ß-arr-1 and ß-arr-2, play a central role in the agonist-induced internalization of many receptors for receptor resensitization. Notably, ß-arr-biased agonists are now being tested in phase II clinical trials, targeting acute pain and acute heart failure. Here, we examined the effects of ß-arr-1 and ß-arr-2 on human AM1 receptor internalization. We constructed a V5-tagged chimera in which the cytoplasmic C-terminal tail (C-tail) of CLR was replaced with that of the ß2-adrenergic receptor (ß2-AR), and it was transiently transfected into HEK-293 cells that stably expressed RAMP2. The cell-surface expression and internalization of the wild-type or chimeric receptor were quantified by flow cytometric analysis. The [125I]AM binding and the AM-induced cAMP production of these receptors were also determined. Surprisingly, the coexpression of ß-arr-1 or -2 resulted in significant decreases in AM1 receptor internalization without affecting AM binding and signaling prior to receptor internalization. Dominant-negative (DN) ß-arr-1 or -2 also significantly decreased AM-induced AM1 receptor internalization. In contrast, the AM-induced internalization of the chimeric AM1 receptor was markedly augmented by the cotransfection of ß-arr-1 or -2 and significantly reduced by the coexpression of DN-ß-arr-1 or -2. These results were consistent with those seen for ß2-AR. Thus, both ß-arrs negatively control AM1 receptor internalization, which depends on the C-tail of CLR.

CGRP and its receptors.

The calcitonin gene-related peptide (CGRP) neuropeptide system is an important but still evolving target for migraine. A fundamental consideration for all of the current drugs in clinical trials and for ongoing development in this area is the identity, expression pattern, and function of CGRP receptors because this knowledge informs safety and efficacy considerations. In recent years, only the calcitonin receptor-like receptor/receptor activity-modifying protein 1 (RAMP1) complex, known as the CGRP receptor, has generally been considered relevant. However, CGRP is capable of activating multiple receptors and could have more than one endogenous receptor. The recent identification of the CGRP-responsive calcitonin receptor/RAMP1 complex (AMY1 receptor - amylin subtype 1 receptor) in the trigeminovascular system warrants a deeper consideration of the molecular identity of CGRP receptor(s) involved in the pathophysiology, and thus potential treatment of migraine. This perspective considers some of the issues and implications.

Amylin.

Amylin is a 37 amino acid peptide hormone that is closely related to calcitonin gene-related peptide (CGRP). Amylin and CGRP share a receptor and are reported to have several similar biological actions. Given the important role of CGRP in migraine and intense efforts to develop drugs against this target, it is important to consider potential areas of overlap between the amylin and CGRP systems. This short review provides a brief introduction to amylin biology, the use of an amylin analog to treat diabetes, and consideration of whether amylin could have any role in headache disorders. Finally, this review informs readers about the AMY1 (amylin subtype 1) receptor, which is a dual receptor for amylin and CGRP and potentially plays a role in the bioactivity of both of these peptides.