Deconvoluting the Molecular Control of Binding and Signaling at the Amylin 3 Receptor: RAMP3 Alters Signal Propagation through Extracellular Loops of the Calcitonin Receptor

Amylin is coexpressed with insulin in pancreatic islet β-cells and has potent effects on gastric emptying and food intake. The effect of amylin on satiation has been postulated to involve AMY3 receptors (AMY3R) that are heteromers of the calcitonin receptor (CTR) and receptor activity-modifying protein 3 (RAMP3). Understanding the molecular control of signaling through the AMY3R is thus important for peptide drug targeting of this receptor. We have previously used alanine scanning mutagenesis to study the contribution of the extracellular surface of the CTR to binding and signaling initiated by calcitonin (CT) and related peptides (Dal Maso, E., et al. (2019) The molecular control of calcitonin receptor signaling. ACS Pharmacol. Transl. Sci.2, 31–51). That work revealed ligand- and pathway-specific effects of mutation, with extracellular loops (ECLs) 2 and 3 particularly important in the distinct propagation of signaling mediated by individual peptides. In the current study, we have used equivalent alanine scanning of ECL2 and ECL3 of the CTR in the context of coexpression with RAMP3 to form AMY3Rs, to examine functional affinity and efficacy of peptides in cAMP accumulation and extracellular signal-regulated kinase (ERK) phosphorylation (pERK). The effect of mutation was determined on representatives of the three major distinct classes of CT peptide, salmon CT (sCT), human CT (hCT), and porcine CT (pCT), as well as rat amylin (rAmy) or human α-CGRP (calcitonin gene-related peptide, hCGRP) whose potency is enhanced by RAMP interaction. We demonstrate that the dynamic nature of CTR ECL2 and ECL3 in propagation of signaling is fundamentally altered when complexed with RAMP3 to form the AMY3R, despite only having predicted direct interactions with ECL2. Moreover, the work shows that the role of these loops in receptor signaling is highly peptide dependent, illustrating that even subtle changes to peptide sequence may change signaling output downstream of the receptor

Extracellular loops 2 and 3 of the calcitonin receptor selectively modify agonist binding and efficacy.

Class B peptide hormone GPCRs are targets for the treatment of major chronic disease. Peptide ligands of these receptors display biased agonism and this may provide future therapeutic advantage. Recent active structures of the calcitonin (CT) and glucagon-like peptide-1 (GLP-1) receptors reveal distinct engagement of peptides with extracellular loops (ECLs) 2 and 3, and mutagenesis of the GLP-1R has implicated these loops in dynamics of receptor activation. In the current study, we have mutated ECLs 2 and 3 of the human CT receptor (CTR), to interrogate receptor expression, peptide affinity and efficacy. Integration of these data with insights from the CTR and GLP-1R active structures, revealed marked diversity in mechanisms of peptide engagement and receptor activation between the CTR and GLP-1R. While the CTR ECL2 played a key role in conformational propagation linked to Gs/cAMP signalling this was mechanistically distinct from that of GLP-1R ECL2. Moreover, ECL3 was a hotspot for distinct ligand- and pathway- specific effects, and this has implications for the future design of biased agonists of class B GPCRs

The Molecular Control of Calcitonin Receptor Signaling

The calcitonin receptor (CTR) is a class B G protein-coupled receptor (GPCR) that responds to the peptide hormone calcitonin (CT). CTs are clinically approved for the treatment of bone diseases. We previously reported a 4.1 Å structure of the activated CTR bound to salmon CT (sCT) and heterotrimeric Gs protein by cryo-electron microscopy (Liang, Y.-L., et al. Phase-plate cryo- EM structure of a class B GPCR-G protein complex. Nature 2017, 546, 118–123). In the current study, we have reprocessed the electron micrographs to yield a 3.3 Å map of the complex. This has allowed us to model extracellular loops (ECLs) 2 and 3, and the peptide N-terminus that previously could not be resolved. We have also performed alanine scanning mutagenesis of ECL1 and the upper segment of transmembrane helix 1 (TM1) and its extension into the receptor extracellular domain (TM1 stalk), with effects on peptide binding and function assessed by cAMP accumulation and ERK1/2 phosphorylation. These data were combined with previously published alanine scanning mutagenesis of ECL2 and ECL3 and the new structural information to provide a comprehensive 3D map of the molecular surface of the CTR that controls binding and signaling of distinct CT and related peptides. The work highlights distinctions in how different, related, class B receptors may be activated. The new mutational data on the TM1 stalk and ECL1 have also provided critical insights into the divergent control of cAMP versus pERK signaling and, collectively with previous mutagenesis data, offer evidence that the conformations linked to these different signaling pathways are, in many ways, mutually exclusive. This study furthers our understanding of the complex nature of signaling elicited by GPCRs and, in particular, that of the therapeutically important class B subfamily

The costs and impacts of the winter 2013/14 floods in England and Wales

Between December 2013 and March 2014, the UK witnessed heavy and prolonged rainfall, including the wettest January on record in parts of the country and around twice the average monthly rainfall in other locations. The East Coast was also affected by the largest coastal surge since 1953. These events resulted in significant coastal damage and prolonged fluvial and groundwater flooding affecting a large expanse of England and Wales. During this period many properties, crucial transport infrastructure and farmland were flooded. This study for the UK Environment Agency, led by Risk & Policy Analysts (RPA) in association with HaskoningDHV UK, John Chatterton Associates and Morris Resource Economics, reviewed more than 500 documents, articles and reports and requested information from more than 640 individuals and organisations. Through careful consideration of the reliability of each and every piece of data, we were able to calculate a best estimate for the economic costs of the winter 2013/14 floods (in 2014 values) of €1.7 billion, with uncertainty resulting in a range of between €1.3 billion to €1.9 billion. Residential properties suffered the greatest proportion of damages, with 25% of total damages (best estimate of €410 million incurred by 10,465 properties)

The costs and impacts of the winter 2013/14 floods in England and Wales

Between December 2013 and March 2014, the UK witnessed heavy and prolonged rainfall, including the wettest January on record in parts of the country and around twice the average monthly rainfall in other locations. The East Coast was also affected by the largest coastal surge since 1953. These events resulted in significant coastal damage and prolonged fluvial and groundwater flooding affecting a large expanse of England and Wales. During this period many properties, crucial transport infrastructure and farmland were flooded. This study for the UK Environment Agency, led by Risk & Policy Analysts (RPA) in association with HaskoningDHV UK, John Chatterton Associates and Morris Resource Economics, reviewed more than 500 documents, articles and reports and requested information from more than 640 individuals and organisations. Through careful consideration of the reliability of each and every piece of data, we were able to calculate a best estimate for the economic costs of the winter 2013/14 floods (in 2014 values) of €1.7 billion, with uncertainty resulting in a range of between €1.3 billion to €1.9 billion. Residential properties suffered the greatest proportion of damages, with 25% of total damages (best estimate of €410 million incurred by 10,465 properties)

The costs and impacts of the winter 2013/14 floods in England and Wales

Between December 2013 and March 2014, the UK witnessed heavy and prolonged rainfall, including the wettest January on record in parts of the country and around twice the average monthly rainfall in other locations. The East Coast was also affected by the largest coastal surge since 1953. These events resulted in significant coastal damage and prolonged fluvial and groundwater flooding affecting a large expanse of England and Wales. During this period many properties, crucial transport infrastructure and farmland were flooded. This study for the UK Environment Agency, led by Risk & Policy Analysts (RPA) in association with HaskoningDHV UK, John Chatterton Associates and Morris Resource Economics, reviewed more than 500 documents, articles and reports and requested information from more than 640 individuals and organisations. Through careful consideration of the reliability of each and every piece of data, we were able to calculate a best estimate for the economic costs of the winter 2013/14 floods (in 2014 values) of €1.7 billion, with uncertainty resulting in a range of between €1.3 billion to €1.9 billion. Residential properties suffered the greatest proportion of damages, with 25% of total damages (best estimate of €410 million incurred by 10,465 properties)

Extracellular loops 2 and 3 of the calcitonin G protein-coupled receptor selectively modify agonist binding and efficacy

Supporting information for: Del Maso et al., Extracellular loops 2 and 3 of the calcitonin G protein-coupled receptor selectively modify agonist binding and efficac

超高強度短パルスレーザーの時間波形がイオン加速へ与える影響

超高強度レーザーを用いたイオン加速の研究に関して発表する2021年春の物理学会年

A Mouse Model Suggests Two Mechanisms for Thyroid Alterations in Infantile Cystinosis: Decreased Thyroglobulin Synthesis Due to Endoplasmic Reticulum Stress/Unfolded Protein Response and Impaired Lysosomal Processing

Thyroid hormones are released from thyroglobulin (Tg) in lysosomes, which is impaired in infantile/nephropathic cystinosis. Cystinosis is a lysosomal cystine storage disease due to defective cystine exporter, cystinosin (CTNS). Cystinotic children develop subclinical then overt hypothyroidism. Why hypothyroidism is the most frequent and earliest endocrine complication of cystinosis is unknown. We here defined early alterations in Ctns(-/-) mice thyroid and identified subcellular and molecular mechanisms. At 9 months, T4 and T3 plasma levels were normal and TSH was moderately increased (∼4-fold). By histology, hyperplasia and hypertrophy of most follicles preceded colloid exhaustion. Increased immunolabelling for thyrocyte proliferation and apoptotic shedding indicated accelerated cell turnover. Electron microscopy revealed endoplasmic reticulum (ER) dilation, apical lamellipodia indicating macropinocytic colloid uptake, and lysosomal cystine crystals. Tg accumulation in dilated ER contrasted with mRNA down-regulation. Increased expression of ER chaperones, GRP78 and PDI, associated with alternative XBP-1 splicing, revealed unfolded protein response (UPR) activation by ER stress. Decreased Tg mRNA and ER stress suggested reduced Tg synthesis. Coordinated increase of UPR markers, ATF-4 and CHOP, linked ER stress to apoptosis. Hormonogenic cathepsins were not altered, but LAMP-1 immunolabelling disclosed enlarged vesicles containing iodo-Tg and impaired lysosomal fusion. Isopycnic fractionation showed iodo-Tg accumulation in denser lysosomes, suggesting defective lysosomal processing and hormone release. In conclusion, Ctns(-/-) mice show (i) compensated primary hypothyroidism and accelerated thyrocyte turnover; (ii) impaired Tg production linked to ER stress/UPR response; and (iii) altered endolysosomal trafficking and iodo-Tg processing. The Ctns(-/-) thyroid is useful to study disease progression and evaluate novel therapies