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

Conformational changes in proteins caused by ligand binding

L'obiettivo della ricerca è determinare quando è possibile giudicare e predire le modifiche conformazionali del sito attivo indotte dal ligando. I risultati ottenuti dimostrano che tali modifiche non sono correlabili nè al numero di ponti a H, nè al B-factor, nè alla superficie libera di residui a ligando all'interno del sito attivo

Internalisation of sCT(1-32)-ROX and 9E10-AF647 in COS-7 cells stably expressing the hCTRaLeu.

Confocal field images of COS-7 stably expressing hCTR_aLeu were incubated with 1 μM sCT-ROX and 1 µg/ml anti-cMyc antibody (9E10-AF647) on ice. Cells were extensively washed. Imaged were acquired every 1 min for 60 min at 37 ̊C. Images are presented as maximum intensity projections for 9E10-AF647 emission (green), sCT-ROX (red) emission. Representative images of 2 independent experiments.<br

Internalisation of 9E10-AF647 in COS-7 cells stably expressing the hCTRaLeu

Confocal field images of COS-7 stably expressing hCTR_aLeu were incubated with 1 µg/ml anti-cMyc antibody (9E10-AF647) on ice. Cells were extensively washed. Imaged were acquired every 1 min for 60 min at 37 ̊C. Images are presented as maximum intensity projections for 9E10-AF647 emission Representative images of 2 independent experiments.<br

Internalisation of sCT(8-32)-AF568 and 9E10-AF647 in COS-7 cells stably expressing the hCTRaLeu

Confocal field images of COS-7 stably expressing hCTR_aLeu were incubated with 1 μM sCT(8-32)-AF568 and 1 µg/ml anti-cMyc antibody (9E10-AF647) on ice. Cells were extensively washed. Imaged were acquired every 1 min for 60 min at 37 ̊C. Images are presented as maximum intensity projections for 9E10-AF647 emission (green), sCT-(8-32)-AF568 (red) emission. Representative images of 2 independent experiments

Effects of hospital facilities on patient outcomes after cancer surgery: an international, prospective, observational study

© 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 licenseBackground: Early death after cancer surgery is higher in low-income and middle-income countries (LMICs) compared with in high-income countries, yet the impact of facility characteristics on early postoperative outcomes is unknown. The aim of this study was to examine the association between hospital infrastructure, resource availability, and processes on early outcomes after cancer surgery worldwide. Methods: A multimethods analysis was performed as part of the GlobalSurg 3 study—a multicentre, international, prospective cohort study of patients who had surgery for breast, colorectal, or gastric cancer. The primary outcomes were 30-day mortality and 30-day major complication rates. Potentially beneficial hospital facilities were identified by variable selection to select those associated with 30-day mortality. Adjusted outcomes were determined using generalised estimating equations to account for patient characteristics and country-income group, with population stratification by hospital. Findings: Between April 1, 2018, and April 23, 2019, facility-level data were collected for 9685 patients across 238 hospitals in 66 countries (91 hospitals in 20 high-income countries; 57 hospitals in 19 upper-middle-income countries; and 90 hospitals in 27 low-income to lower-middle-income countries). The availability of five hospital facilities was inversely associated with mortality: ultrasound, CT scanner, critical care unit, opioid analgesia, and oncologist. After adjustment for case-mix and country income group, hospitals with three or fewer of these facilities (62 hospitals, 1294 patients) had higher mortality compared with those with four or five (adjusted odds ratio [OR] 3·85 [95% CI 2·58–5·75]; p<0·0001), with excess mortality predominantly explained by a limited capacity to rescue following the development of major complications (63·0% vs 82·7%; OR 0·35 [0·23–0·53]; p<0·0001). Across LMICs, improvements in hospital facilities would prevent one to three deaths for every 100 patients undergoing surgery for cancer. Interpretation: Hospitals with higher levels of infrastructure and resources have better outcomes after cancer surgery, independent of country income. Without urgent strengthening of hospital infrastructure and resources, the reductions in cancer-associated mortality associated with improved access will not be realised. Funding: National Institute for Health and Care Research