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.

Clinical Potential of Adrenomedullin Signaling in the Cardiovascular System.

Numerous clinical studies have revealed the utility of circulating AM (adrenomedullin) or MR-proAM (mid-regional proAM 45-92) as an effective prognostic and diagnostic biomarker for a variety of cardiovascular-related pathophysiologies. Thus, there is strong supporting evidence encouraging the exploration of the AM-CLR (calcitonin receptor-like receptor) signaling pathway as a therapeutic target. This is further bolstered because several drugs targeting the shared CGRP (calcitonin gene-related peptide)-CLR pathway are already Food and Drug Administration-approved and on the market for the treatment of migraine. In this review, we summarize the AM-CLR signaling pathway and its modulatory mechanisms and provide an overview of the current understanding of the physiological and pathological roles of AM-CLR signaling and the yet untapped potentials of AM as a biomarker or therapeutic target in cardiac and vascular diseases and provide an outlook on the recently emerged strategies that may provide further boost to the possible clinical applications of AM signaling.

Functional modulation of PTH1R activation and signaling by RAMP2.

Receptor-activity-modifying proteins (RAMPs) are ubiquitously expressed membrane proteins that associate with different G protein-coupled receptors (GPCRs), including the parathyroid hormone 1 receptor (PTH1R), a class B GPCR and an important modulator of mineral ion homeostasis and bone metabolism. However, it is unknown whether and how RAMP proteins may affect PTH1R function. Using different optical biosensors to measure the activation of PTH1R and its downstream signaling, we describe here that RAMP2 acts as a specific allosteric modulator of PTH1R, shifting PTH1R to a unique preactivated state that permits faster activation in a ligand-specific manner. Moreover, RAMP2 modulates PTH1R downstream signaling in an agonist-dependent manner, most notably increasing the PTH-mediated Gi3 signaling sensitivity. Additionally, RAMP2 increases both PTH- and PTHrP-triggered ß-arrestin2 recruitment to PTH1R. Employing homology modeling, we describe the putative structural molecular basis underlying our functional findings. These data uncover a critical role of RAMPs in the activation and signaling of a GPCR that may provide a new venue for highly specific modulation of GPCR function and advanced drug design.

Itch receptor MRGPRX4 interacts with the receptor activity-modifying proteins.

Cholestatic itch is a severe and debilitating symptom in liver diseases with limited treatment options. The class A G protein-coupled receptor (GPCR) Mas-related GPCR subtype X4 (MRGPRX4) has been identified as a receptor for bile acids, which are potential cholestatic pruritogens. An increasing number of GPCRs have been shown to interact with receptor activity-modifying proteins (RAMPs), which can modulate different aspects of GPCR biology. Using a combination of multiplexed immunoassay and proximity ligation assay, we show that MRGPRX4 interacts with RAMPs. The interaction of MRGPRX4 with RAMP2, but not RAMP1 or 3, causes attenuation of basal and agonist-dependent signaling, which correlates with a decrease of MRGPRX4 cell surface expression as measured using a quantitative NanoBRET pulse-chase assay. Finally, we use AlphaFold Multimer to predict the structure of the MRGPRX4-RAMP2 complex. The discovery that RAMP2 regulates MRGPRX4 may have direct implications for future drug development for cholestatic itch.

Differential expression of components of the CGRP-receptor family in human coronary and human middle meningeal arteries: functional implications.

BACKGROUND: Different responses in human coronary arteries (HCA) and human middle meningeal arteries (HMMA) were observed for some of the novel CGRP receptor antagonists, the gepants, for inhibiting CGRP-induced relaxation. These differences could be explained by the presence of different receptor populations in the two vascular beds. Here, we aim to elucidate which receptors are involved in the relaxation to calcitonin gene-related peptide (CGRP), adrenomedullin (AM) and adrenomedullin 2 (AM2) in HCA and HMMA. METHODS: RNA was isolated from homogenized human arteries (23 HCAs; 12 F, 11 M, age 50 ± 3 years and 26 HMMAs; 14 F, 12 M, age 51 ± 3 years) and qPCR was performed for different receptor subunits. Additionally, relaxation responses to CGRP, AM or AM2 of the human arteries were quantified using a Mulvany myograph system, in the presence or absence of the adrenomedullin 1 receptor antagonist AM22-52 and/or olcegepant. RESULTS: Calcitonin-like receptor (CLR) mRNA was expressed equally in both vascular beds, while calcitonin receptor (CTR) and receptor activity-modifying protein 3 (RAMP3) expression was low and could not be detected in all samples. RAMP1 expression was similar in HCA and HMMA, while RAMP2 expression was higher in HMMA. Moreover, receptor component protein (RCP) expression was higher in HMMA than in HCA. Functional experiments showed that olcegepant inhibits relaxation to all three agonists in both vascular beds. In HCA, antagonist AM22-52 did not inhibit relaxation to any of the agonists, while a trend for blocking relaxation to AM and AM2 could be observed in HMMA. CONCLUSION: Based on the combined results from receptor subunit mRNA expression and the functional responses in both vascular tissues, relaxation of HCA is mainly mediated via the canonical CGRP receptor (CLR-RAMP1), while relaxation of HMMA can be mediated via both the canonical CGRP receptor and the adrenomedullin 1 receptor (CLR-RAMP2). Future research should investigate whether RAMP2 predominance over RAMP1 in the meningeal vasculature results in altered migraine susceptibility or in a different response to anti-migraine medication in these patients. Moreover, the exact role of RCP in CGRP receptor signalling should be elucidated in future research.

RAMP2-AS1 stabilized RAPM2 mRNA through TIA1 to inhibit the progression of non-small cell lung cancer.

As the most common subtype of lung cancer, non-small cell lung cancer (NSCLC)is responsible for a large proportion of global cancer-caused deaths. The implication of long non-coding RNAs (lncRNAs) as tumor-suppressor or carcinogenic genes in NSCLC has been widely documented. Our study sought to investigate the performance of lncRNA RAMP2 antisense RNA1 (RAMP2-AS1) in NSCLC. GEPIA bioinformatics tool and RT-qPCR were applied for assessing the expression of RAMP2-AS1 and its neighboring gene receptor activity-modifying protein 2 (RAMP2) in NSCLC. Functional assays including CCK-8 assay, colony formation assay as well as caspase-3 activity analysis and Transwell invasion assays were applied for detecting the biological phenotypes of NSCLC cells. Interaction among RAMP2-AS1, RAMP2 and T-cell intracellular antigen 1cytotoxic granule associated RNA binding protein (TIA1) was evaluated by RNA immunoprecipitation and pulldown assays. We found that RAMP2-AS1 and RAMP2 were downregulated in NSCLC. Overexpression of RAMP2-AS1 hampered proliferation and invasion, whereas induced apoptosis of NSCLC cells. Mechanistically, RAMP2-AS1 interacted with TIA1 to stabilize the mRNA of RAMP2. In conclusion, we first uncovered that RAMP2-AS1 stabilized RAPM2 mRNA through TIA1 to inhibit the progression of NSCLC, providing new insight to improve the treatment efficacy of NSCLC.

RAMP2-AS1 inhibits CXCL11 expression to suppress malignant phenotype of breast cancer by recruiting DNMT1 and DNMT3B.

BACKGROUND: Breast cancer is the most common malignancy in women populations. METHODS: RAMP2-AS1 and CXCL11 expression in breast cancer tissues and cells were determined using RT-qPCR or Western blot. RIP analysis confirmed the interaction between DNMT1, DNMT3B and RAMP2-AS1. ChIP assay verified that RAMP2-AS1 recruited DNMT1 and DNMT3B to the promoter region of CXCL11. FISH detected the sub-localization of RAMP2-AS1 in breast cancer cells. Bisulfite sequencing PCR (BSP) tested the methylation level of CXCL11. The cell viability, proliferation, migration and apoptosis were assessed by CCK-8, colony formation, transwell and flow cytometry assays, respectively. IHC was performed to evaluate the expression of Ki67, CXCL11, MMP2 in tumor tissues. RESULTS: The level of RAMP2-AS1 was decreased in breast cancer tissues and cells, whereas CXCL11 was highly expressed. Patients with decreased RAMP2-AS1 had a poor prognosis. RAMP2-AS1 inhibited breast cancer cell malignant phenotype. Besides, RAMP2-AS1 regulated the methylation of CXCL11 by recruiting DNMT1 and DNMT3B to the promoter region of CXCL11. RAMP2-AS1 overexpression suppressed the malignant phenotype through CXCL11 and inhibited tumor growth in vivo. CONCLUSION: RAMP2-AS1 suppresses breast cancer malignant phenotype via DNMT1 and DNMT3B mediated inhibition of CXCL11.

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.

Adrenomedullin-Receptor Activity-Modifying Protein 2 System Ameliorates Subretinal Fibrosis by Suppressing Epithelial-Mesenchymal Transition in Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a leading cause of visual impairment. Anti-vascular endothelial growth factor drugs used to treat AMD carry the risk of inducing subretinal fibrosis. We investigated the use of adrenomedullin (AM), a vasoactive peptide, and its receptor activity-modifying protein 2, RAMP2, which regulate vascular homeostasis and suppress fibrosis. The therapeutic potential of the AM-RAMP2 system was evaluated after laser-induced choroidal neovascularization (LI-CNV), a mouse model of AMD. Neovascular formation, subretinal fibrosis, and macrophage invasion were all enhanced in both AM and RAMP2 knockout mice compared with those in wild-type mice. These pathologic changes were suppressed by intravitreal injection of AM. Comprehensive gene expression analysis of the choroid after LI-CNV with or without AM administration revealed that fibrosis-related molecules, including Tgfb, Cxcr4, Ccn2, and Thbs1, were all down-regulated by AM. In retinal pigment epithelial cells, co-administration of transforming growth factor-ß and tumor necrosis factor-α induced epithelial-mesenchymal transition, which was also prevented by AM. Finally, transforming growth factor-ß and C-X-C chemokine receptor type 4 (CXCR4) inhibitors eliminated the difference in subretinal fibrosis between RAMP2 knockout and wild-type mice. These findings suggest the AM-RAMP2 system suppresses subretinal fibrosis in LI-CNV by suppressing epithelial-mesenchymal transition.

Adrenomedullin Is Necessary to Resolve Hyperoxia-Induced Experimental Bronchopulmonary Dysplasia and Pulmonary Hypertension in Mice.

Bronchopulmonary dysplasia (BPD)-associated pulmonary hypertension (PH) is an infantile lung disease characterized by aberrant angiogenesis and impaired resolution of lung injury. Adrenomedullin (AM) signals through calcitonin receptor-like receptor and receptor activity-modifying protein 2 and modulates lung injury initiation. However, its role in lung injury resolution and the mechanisms by which it regulates angiogenesis remain unclear. Consequently, we hypothesized that AM resolves hyperoxia-induced BPD and PH via endothelial nitric oxide synthase (NOS3). AM-sufficient (ADM+/+) or -deficient (ADM+/-) mice were exposed to normoxia or hyperoxia through postnatal days (PNDs) 1 to 14, and the hyperoxia-exposed mice were allowed to recover in normoxia for an additional 56 days. Lung injury and development and PH were quantified at different time points. Human pulmonary microvascular endothelial cells were also used to examine the effects of AM signaling on the NOS3 pathway and angiogenesis. Lung blood vessels and NOS3 expression decreased and the extent of hyperoxia-induced BPD and PH increased in ADM+/- mice compared with ADM+/+ mice. Hyperoxia-induced apoptosis and PH resolved by PND14 and PND70, respectively, in ADM+/+ mice but not in ADM+/- mice. Knockdown of ADM, calcitonin receptor-like receptor, and receptor activity-modifying protein 2 in vitro decreased NOS3 expression, nitric oxide generation, and angiogenesis. Furthermore, NOS3 knockdown abrogated the angiogenic effects of AM. Collectively, these results indicate that AM resolves hyperoxic lung injury via NOS3.

Development of a Novel Model of Central Retinal Vascular Occlusion and the Therapeutic Potential of the Adrenomedullin-Receptor Activity-Modifying Protein 2 System.

Central retinal vein occlusion (CRVO) is an intractable disease that causes visual acuity loss with retinal ischemia, hemorrhage, and edema. In this study, we developed an experimental CRVO model in mice and evaluated the therapeutic potential of the pleiotropic peptide adrenomedullin (ADM) and its receptor activity-modifying protein 2 (RAMP2). The CRVO model, which had phenotypes resembling those seen in the clinic, was produced by combining i.p. injection of Rose bengal, a photoactivator dye enhancing thrombus formation, with laser photocoagulation. Retinal vascular area, analyzed using fluorescein angiography and fluorescein isothiocyanate-perfused retinal flat mounts, was decreased after induction of CRVO but gradually recovered from day 1 to 7. Measurements of retinal thickness using optical coherence tomography and histology revealed prominent edema early after CRVO, followed by gradual atrophy. Reperfusion after CRVO was diminished in Adm and Ramp2 knockout (KO) mice but was increased by exogenous ADM administration. CRVO also increased expression of a coagulation factor, oxidative stress markers, and a leukocyte adhesion molecule in both wild-type and Adm KO mice, and the effect was more pronounced in Adm KO mice. Using retinal capillary endothelial cells, ADM was found to directly suppress retinal endothelial injury. The retinoprotective effects of the Adm-Ramp2 system make it a novel therapeutic target for the treatment of CRVO.

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.

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.

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.

Intermedin protects HUVECs from ischemia reperfusion injury via Wnt/ß-catenin signaling pathway.

Intermedin (IMD) is a member of the calcitonin gene-related peptide (CGRP) superfamily and a pro-angiogenic factor. In the present study, we identified activation of the Wnt/ß-catenin signaling pathway by IMD. Adding CoCl2 HUVECs was used to establish an in vitro model. The migration of HUVECs was measured by wound healing assays and transwell migration assays. Capillary formation was measured using tube formation assays. Immunocytochemistry (ICC) analysis was used to evaluate VEGF and RAMP2 expression in HUVECs. The relevant signaling molecules were detected with western blot. Our study shows that IMD could promote H/R impaired HUVECs migration and tube formation in vitro. On the other hand, inhibition of Wnt/ß-catenin signaling led to the suppression of this promotion of migration and tube formation. This result suggests that Wnt/ß-catenin signaling is correlated to IMD induced angiogenesis. Analysis of results from ICC assays indicated that IMD works through increasing levels of VEGF and RAMP2. Meanwhile, the Wnt/ß-catenin signaling specific inhibitor IWR-1-endo was shown to down-regulate VEGF and RAMP2 expression. Western blot results further confirmed the signaling mechanism by which IMD promotes angiogenesis. Thus, Wnt/ß-catenin signaling plays an important role in IMD induced neovascularization. The data further suggest that the PI3K axis contributes positively downstream.

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.

N-Glycosylation of Asparagine 130 in the Extracellular Domain of the Human Calcitonin Receptor Significantly Increases Peptide Hormone Affinity.

The calcitonin receptor (CTR) is a class B G protein-coupled receptor that is activated by the peptide hormones calcitonin and amylin. Calcitonin regulates bone remodeling through CTR, whereas amylin regulates blood glucose and food intake by activating CTR in complex with receptor activity-modifying proteins (RAMPs). These receptors are targeted clinically for the treatment of osteoporosis and diabetes. Here, we define the role of CTR N-glycosylation in hormone binding using purified calcitonin and amylin receptor extracellular domain (ECD) glycoforms and fluorescence polarization/anisotropy and isothermal titration calorimetry peptide-binding assays. N-Glycan-free CTR ECD produced in Escherichia coli exhibited ∼10-fold lower peptide affinity than CTR ECD produced in HEK293T cells, which yield complex N-glycans, or in HEK293S GnTI- cells, which yield core N-glycans (Man5GlcNAc2). PNGase F-catalyzed removal of N-glycans at N73, N125, and N130 in the CTR ECD decreased peptide affinity ∼10-fold, whereas Endo H-catalyzed trimming of the N-glycans to single GlcNAc residues had no effect on peptide binding. Similar results were observed for an amylin receptor RAMP2-CTR ECD complex. Characterization of peptide-binding affinities of purified N → Q CTR ECD glycan site mutants combined with PNGase F and Endo H treatment strategies and mass spectrometry to define the glycan species indicated that a single GlcNAc residue at CTR N130 was responsible for the peptide affinity enhancement. Molecular modeling suggested that this GlcNAc functions through an allosteric mechanism rather than by directly contacting the peptide. These results reveal an important role for N-linked glycosylation in the peptide hormone binding of a clinically relevant class B GPCR.

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.

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.

Effect of Valsartan on Cerebellar Adrenomedullin System Dysregulation During Hypertension.

Adrenomedullin (AM) and its receptors components, calcitonin-receptor-like receptor (CRLR), and receptor activity-modifying protein (RAMP1, RAMP2, and RAMP3) are expressed in cerebellum. Cerebellar AM, AM binding sites and receptor components are altered during hypertension, suggesting a role for cerebellar AM in blood pressure regulation. Thus, we assessed the effect of valsartan, on AM and its receptor components expression in the cerebellar vermis of Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Additionally, we evaluated AM action on superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activity, and thiobarbituric acid reactive substances (TBARS) production in cerebellar vermis. Animals were treated with valsartan or vehicle for 11 days. Rats were sacrificed by decapitation; cerebellar vermis was dissected; and AM, CRLR, RAMP1, RAMP2, and RAMP3 expression was quantified by Western blot analysis. CAT, SOD, and GPx activity was determined spectrophotometrically and blood pressure by non-invasive plethysmography. We demonstrate that AM and RAMP2 expression was lower in cerebellum of SHR rats, while CRLR, RAMP1, and RAMP3 expression was higher than those of WKY rats. AM reduced cerebellar CAT, SOD, GPx activities, and TBARS production in WKY rats, but not in SHR rats. Valsartan reduced blood pressure and reversed the altered expression of AM and its receptors components, as well the loss of AM capacity to reduce antioxidant enzyme activity and TBARS production in SHR rats. These findings demonstrate that valsartan is able to reverse the dysregulation of cerebellar adrenomedullinergic system; and they suggest that altered AM system in the cerebellum could represent the primary abnormality leading to hypertension.