Actin cytoskeleton-dependent regulation of corticotropin-releasing factor receptor heteromers

Stress responses are highly nuanced and variable, but how this diversity is achieved by modulating receptor function is largely unknown. Corticotropin-releasing factor receptors (CRFRs), class B G protein–coupled receptors, are pivotal in mediating stress responses. Here we show that the two known CRFRs interact to form heteromeric complexes in HEK293 cells coexpressing both CRFRs and in vivo in mouse pancreas. Coimmunoprecipitation and mass spectrometry confirmed the presence of both CRF1R and CRF2βR, along with actin in these heteromeric complexes. Inhibition of actin filament polymerization prevented the transport of CRF2βR to the cell surface but had no effect on CRF1R. Transport of CRF1R when coexpressed with CRF2βR became actin dependent. Simultaneous stimulation of cells coexpressing CRF1R+CRF2βR with their respective high-affinity agonists, CRF+urocortin2, resulted in approximately twofold increases in peak Ca2+responses, whereas stimulation with urocortin1 that binds both receptors with 10-fold higher affinity did not. The ability of CRFRs to form heteromeric complexes in association with regulatory proteins is one mechanism to achieve diverse and nuanced function

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

Numerical simulation of CO2 dispersion from punctures and ruptures of buried high-pressure dense phase CO2 pipelines with experimental validation

Carbon capture and storage (CCS) presents an option for significantly reducing the amount of carbon dioxide (CO2) released into the atmosphere and mitigating the effects of climate change. Pipelines are considered to be the most likely method for transporting captured CO2 and their safe operation is of paramount importance as their contents are likely to be in the region of several thousand tonnes and CO2 poses a number of concerns upon release due to its unusual physical properties. To this end, National Grid initiated the COOLTRANS (CO2 Liquid Pipeline Transportation) research programme to consider the pipeline transportation of high-pressure dense phase CO2. Part of this work involved the development of a mathematical model for predicting the dispersion of pure CO2 following the venting, puncture, or rupture, of such a transportation pipeline during normal operational conditions. In this paper, we describe the use of a computational fluid dynamic (CFD) tool that can be used to numerically simulate the near-field sonic dispersion from such releases, above and below ground. The model is shown to qualitatively and quantitatively reproduce observed experimental results. Validated flows at the top of the crater formed by below ground releases presented here for a range of scenarios provide the basis for developing robust source conditions for use in CFD studies of far-field dispersion, and for use with pragmatic quantified risk assessment (QRA) models

The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach

The calcitonin gene-related peptide (CGRP) receptor is a complex of a calcitonin receptor-like receptor (CLR), which is a family B G-protein-coupled receptor (GPCR) and receptor activity modifying protein 1. The role of the second extracellular loop (ECL2) of CLR in binding CGRP and coupling to Gs was investigated using a combination of mutagenesis and modelling. An alanine scan of residues 271–294 of CLR showed that the ability of CGRP to produce cAMP was impaired by point mutations at 13 residues; most of these also impaired the response to adrenomedullin (AM). These data were used to select probable ECL2-modelled conformations that are involved in agonist binding, allowing the identification of the likely contacts between the peptide and receptor. The implications of the most likely structures for receptor activation are discussed.</jats:p

Understanding the molecular functions of the second extracellular loop (ECL2) of the calcitonin gene-related peptide (CGRP) receptor using a comprehensive mutagenesis approach

The extracellular loop 2 (ECL2) region is the most conserved of the three ECL domains in family B G protein-coupled receptors (GPCRs) and has a fundamental role in ligand binding and activation across the receptor super-family. ECL2 is fundamental for ligand-induced activation of the calcitonin gene related peptide (CGRP) receptor, a family B GPCR implicated in migraine and heart disease. In this study we apply a comprehensive targeted non-alanine substitution analysis method and molecular modelling to the functionally important residues of ECL2 to reveal key molecular interactions. We identified an interaction network between R274/Y278/D280/W283. These amino acids had the biggest reduction in signalling following alanine substitution analysis and comprise a group of basic, acidic and aromatic residues conserved in the wider calcitonin family of class B GPCRs. This study identifies key and varied constraints at each locus, including diverse biochemical requirements for neighbouring tyrosine residues and a W283H substitution that recovered wild-type (WT) signalling, despite the strictly conserved nature of the central ECL2 tryptophan and the catastrophic effects on signalling of W283A substitution. In contrast, while the distal end of ECL2 requires strict conservation of hydrophobicity or polarity in each position, mutation of these residues never has a large effect. This approach has revealed linked networks of amino acids, consistent with structural models of ECL2 and likely to represent a shared structural framework at an important ligand-receptor interface that is present across the family B GPCRs

High pressure CO2 CCS pipelines: Comparing dispersion models with multiple experimental datasets

Carbon capture and storage (CCS) presents the short-term option for significantly reducing the amount of carbon dioxide (CO2) released into the atmosphere from the combustion of fossil fuels, thereby mitigating the effects of climate change. Enabling CCS requires the development of capture, storage and transport methodologies. The safe transport of CO2 in CCS scenarios can be achieved through pipelines or by shipping. Either way, transport and temporary storage of pressurised liquid CO2 will be required and subject to quantitative risk assessment, which includes the consideration of the low-risk, low-probability puncture or rupture scenario of such a pipeline, ship or storage facility. In this work, we combine multiple experimental datasets all concerned with the atmospheric free release of pure and impure liquid CO2 from CCS-transport-chain-relevant high pressure reservoirs and perform the first multiple dataset comparison to numerical models for both pure and impure jets in dry ambient air with no water vapour. The results validate the numerical approach adopted and for the prediction of such releases, highlight the significance of the mixture fraction at the release point, over the mixture composition itself. A new method for impure CO2 dispersion modelling is introduced and limited preliminary comparisons of impure CO2 data and predictions are performed. No clear difference between pure and impure releases is found for the cases considered