CGRP-receptor family reveals endogenous GPCR agonist bias and its significance in primary human cardiovascular cells

AbstractAgonist bias at G protein-coupled receptors has attracted considerable interest, although its relevance for physiologically-produced agonists is not always clear. Here, using primary human cells and gene editing techniques, we demonstrate for the first time, endogenous agonist bias with physiological consequences for the calcitonin-like receptor (CLR). We reveal that by switching the accessory protein: receptor activity-modifying protein (RAMP) associated with CLR we can re-route the physiological pathways activated by the stimulating peptide agonists. These results have revealed a unique role in calcium-mediated nitric oxide signalling for the little-understood peptide adrenomedullin 2 and distinct pro-proliferative effects of calcitonin-gene related peptide (CGRP) and adrenomedullin in cardiovascular cells. This work reveals that CLR-based agonist bias occurs naturally in human cells and has a fundamental purpose for its existence. We anticipate this will be a starting point for more studies into RAMP function in native environments and its importance in endogenous GPCR signalling.

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CGRP, adrenomedullin and adrenomedullin 2 display endogenous GPCR agonist bias in primary human cardiovascular cells

Communications Biology ◽

10.1038/s42003-021-02293-w ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Ashley J. Clark ◽

Niamh Mullooly ◽

Dewi Safitri ◽

Matthew Harris ◽

Tessa de Vries ◽

...

Keyword(s):

Gene Editing ◽

Human Cells ◽

Calcitonin Receptor ◽

Physiological Relevance ◽

Starting Point ◽

Calcitonin Gene ◽

Ramp Function ◽

Physiological Pathways ◽

Cardiovascular Cells ◽

Primary Human Cells

AbstractAgonist bias occurs when different ligands produce distinct signalling outputs when acting at the same receptor. However, its physiological relevance is not always clear. Using primary human cells and gene editing techniques, we demonstrate endogenous agonist bias with physiological consequences for the calcitonin receptor-like receptor, CLR. By switching the receptor-activity modifying protein (RAMP) associated with CLR we can “re-route” the physiological pathways activated by endogenous agonists calcitonin gene-related peptide (CGRP), adrenomedullin (AM) and adrenomedullin 2 (AM2). AM2 promotes calcium-mediated nitric oxide signalling whereas CGRP and AM show pro-proliferative effects in cardiovascular cells, thus providing a rationale for the expression of the three peptides. CLR-based agonist bias occurs naturally in human cells and has a fundamental purpose for its existence. We anticipate this will be a starting point for more studies into RAMP function in native environments and their importance in endogenous GPCR signalling.

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Exploring Ligand Binding to Calcitonin Gene-Related Peptide Receptors

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.720561 ◽

2021 ◽

Vol 8 ◽

Author(s):

Giuseppe Deganutti ◽

Silvia Atanasio ◽

Roxana-Maria Rujan ◽

Patrick M. Sexton ◽

Denise Wootten ◽

...

Keyword(s):

Molecular Dynamics ◽

Ligand Binding ◽

Calcitonin Gene Related Peptide ◽

Related Peptide ◽

Calcitonin Gene ◽

Approved Drugs ◽

G Protein Coupled ◽

Dynamic Docking ◽

Receptor Family

Class B1 G protein-coupled receptors (GPCRs) are important targets for many diseases, including cancer, diabetes, and heart disease. All the approved drugs for this receptor family are peptides that mimic the endogenous activating hormones. An understanding of how agonists bind and activate class B1 GPCRs is fundamental for the development of therapeutic small molecules. We combined supervised molecular dynamics (SuMD) and classic molecular dynamics (cMD) simulations to study the binding of the calcitonin gene-related peptide (CGRP) to the CGRP receptor (CGRPR). We also evaluated the association and dissociation of the antagonist telcagepant from the extracellular domain (ECD) of CGRPR and the water network perturbation upon binding. This study, which represents the first example of dynamic docking of a class B1 GPCR peptide, delivers insights on several aspects of ligand binding to CGRPR, expanding understanding of the role of the ECD and the receptor-activity modifying protein 1 (RAMP1) on agonist selectivity.

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Mapping the Heart

Microscopy Today ◽

10.1017/s155192951000043x ◽

2010 ◽

Vol 18 (4) ◽

pp. 6-8

Author(s):

Stephen W. Carmichael

Keyword(s):

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

G Protein Coupled Receptors ◽

Receptor Subtypes ◽

Cardiac Muscle Cells ◽

Guanine Nucleotide Binding Protein ◽

The Common ◽

Guanine Nucleotide Binding ◽

First Time ◽

G Protein Coupled

Some of the receptors on the surface of cardiac muscle cells (cardiomyocytes) mediate the response of these cells to catecholamines by causing the production of the common second messenger cyclic adenosine monophosphate (cAMP). An example of such receptors are the β1- and β2-adrenergic receptors (βARs) that are heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors. Selective stimulation of these two receptor subtypes leads to distinct physiological and pathophysiological responses, but their precise location on the surface of cardiomyocytes has not been correlated with these responses. In an ingenious combination of techniques, Viacheslav Nikolaev, Alexey Moshkov, Alexander Lyon, Michele Miragoli, Pavel Novak, Helen Paur, Martin Lohse, Yuri Korchev, Sian Harding, and Julia Gorelik have mapped the function of these receptors for the first time.

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Adhesion GPCRs in Glioblastoma

Neuro-Oncology Advances ◽

10.1093/noajnl/vdab046 ◽

2021 ◽

Author(s):

Gabriele Stephan ◽

Niklas Ravn-Boess ◽

Dimitris G Placantonakis

Keyword(s):

G Protein ◽

G Protein Coupled Receptors ◽

The Past ◽

Novel Treatment ◽

The Family ◽

Health And Disease ◽

Basic Biology ◽

G Protein Coupled ◽

Potential Use ◽

Receptor Family

Abstract Members of the adhesion family of G protein-coupled receptors (GPCRs) have received attention for their roles in health and disease, including cancer. Over the past decade, several members of the family have been implicated in the pathogenesis of glioblastoma. Here, we discuss the basic biology of adhesion GPCRs and review in detail specific members of the receptor family with known functions in glioblastoma. Finally, we discuss the potential use of adhesion GPCRs as novel treatment targets in neuro-oncology.

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Advances on the Implementation of Circular Economy Techniques in Rural Areas in Colombia under a Sustainable Development Framework

Sustainability ◽

10.3390/su13073816 ◽

2021 ◽

Vol 13 (7) ◽

pp. 3816

Author(s):

Javier Rodrigo-Ilarri ◽

Camilo-A. Vargas-Terranova ◽

María-Elena Rodrigo-Clavero ◽

Paula-A. Bustos-Castro

Keyword(s):

Sustainable Development ◽

Waste Management ◽

Circular Economy ◽

Rural Areas ◽

Development Framework ◽

Starting Point ◽

Management Plans ◽

Good Starting Point ◽

Depressed Regions ◽

First Time

For the first time in the scientific literature, this research shows an analysis of the implementation of circular economy techniques under sustainable development framework in six municipalities with a depressed economy in Colombia. The analysis is based on solid waste data production at a local scale, the valuation of the waste for subsequent recycling, and the identification and quantification of the variables associated with the treatment and final disposal of waste, in accordance with the Colombian regulatory framework. Waste generation data are obtained considering three different scenarios, in which a comparison between the simulated values and those established in the management plans are compared. Important differences have been identified between the waste management programs of each municipality, specifically regarding the components of waste collection, transportation and disposal, participation of environmental reclaimers, and potential use of materials. These differences are fundamentally associated with the different administrative processes considered for each individual municipality. This research is a good starting point for the development of waste management models based on circular economy techniques, through the subsequent implementation of an office tool in depressed regions such as those studied.

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Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development

Signal Transduction and Targeted Therapy ◽

10.1038/s41392-021-00558-8 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Miao-Miao Zhao ◽

Wei-Li Yang ◽

Fang-Yuan Yang ◽

Li Zhang ◽

Wei-Jin Huang ◽

...

Keyword(s):

Drug Development ◽

Cathepsin L ◽

Human Cells ◽

New Drugs ◽

Disease Course ◽

Promising Target ◽

First Time

AbstractTo discover new drugs to combat COVID-19, an understanding of the molecular basis of SARS-CoV-2 infection is urgently needed. Here, for the first time, we report the crucial role of cathepsin L (CTSL) in patients with COVID-19. The circulating level of CTSL was elevated after SARS-CoV-2 infection and was positively correlated with disease course and severity. Correspondingly, SARS-CoV-2 pseudovirus infection increased CTSL expression in human cells in vitro and human ACE2 transgenic mice in vivo, while CTSL overexpression, in turn, enhanced pseudovirus infection in human cells. CTSL functionally cleaved the SARS-CoV-2 spike protein and enhanced virus entry, as evidenced by CTSL overexpression and knockdown in vitro and application of CTSL inhibitor drugs in vivo. Furthermore, amantadine, a licensed anti-influenza drug, significantly inhibited CTSL activity after SARS-CoV-2 pseudovirus infection and prevented infection both in vitro and in vivo. Therefore, CTSL is a promising target for new anti-COVID-19 drug development.

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Characterization of the G protein-coupled receptor family SREB across fish evolution

Scientific Reports ◽

10.1038/s41598-021-91590-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Timothy S. Breton ◽

William G. B. Sampson ◽

Benjamin Clifford ◽

Anyssa M. Phaneuf ◽

Ilze Smidt ◽

...

Keyword(s):

G Protein ◽

Genomic Structure ◽

Integrated Approach ◽

Orphan Receptor ◽

Specific Gene ◽

Testicular Development ◽

Brain Expression ◽

And Function ◽

G Protein Coupled ◽

Receptor Family

AbstractThe SREB (Super-conserved Receptors Expressed in Brain) family of G protein-coupled receptors is highly conserved across vertebrates and consists of three members: SREB1 (orphan receptor GPR27), SREB2 (GPR85), and SREB3 (GPR173). Ligands for these receptors are largely unknown or only recently identified, and functions for all three are still beginning to be understood, including roles in glucose homeostasis, neurogenesis, and hypothalamic control of reproduction. In addition to the brain, all three are expressed in gonads, but relatively few studies have focused on this, especially in non-mammalian models or in an integrated approach across the entire receptor family. The purpose of this study was to more fully characterize sreb genes in fish, using comparative genomics and gonadal expression analyses in five diverse ray-finned (Actinopterygii) species across evolution. Several unique characteristics were identified in fish, including: (1) a novel, fourth euteleost-specific gene (sreb3b or gpr173b) that likely emerged from a copy of sreb3 in a separate event after the teleost whole genome duplication, (2) sreb3a gene loss in Order Cyprinodontiformes, and (3) expression differences between a gar species and teleosts. Overall, gonadal patterns suggested an important role for all sreb genes in teleost testicular development, while gar were characterized by greater ovarian expression that may reflect similar roles to mammals. The novel sreb3b gene was also characterized by several unique features, including divergent but highly conserved amino acid positions, and elevated brain expression in puffer (Dichotomyctere nigroviridis) that more closely matched sreb2, not sreb3a. These results demonstrate that SREBs may differ among vertebrates in genomic structure and function, and more research is needed to better understand these roles in fish.

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Development of a Novel SNAP-Epitope Tag/Near-Infrared Imaging Assay to Quantify G Protein-Coupled Receptor Degradation in Human Cells

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555220979793 ◽

2021 ◽

pp. 247255522097979

Author(s):

Kyung-Soon Lee ◽

Edelmar Navaluna ◽

Nicole M. Marsh ◽

Eric M. Janezic ◽

Chris Hague

Keyword(s):

G Protein ◽

Adrenergic Receptor ◽

Near Infrared ◽

Human Cells ◽

Receptor Subtypes ◽

G Protein Coupled Receptor ◽

Functional Responses ◽

Epitope Tag ◽

Nir Imaging ◽

G Protein Coupled

We have developed a novel reporter assay that leverages SNAP-epitope tag/near-infrared (NIR) imaging technology to monitor G protein-coupled receptor (GPCR) degradation in human cell lines. N-terminal SNAP-tagged GPCRs were subcloned and expressed in human embryonic kidney (HEK) 293 cells and then subjected to 24 h of cycloheximide (CHX)-chase degradation assays to quantify receptor degradation half-lives ( t1/2) using LICOR NIR imaging–polyacrylamide gel electrophoresis (PAGE) analysis. Thus far, we have used this method to quantify t1/2 for all nine adrenergic (ADRA1A, ADRA1B, ADRA1D, ADRA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, ADRB3), five somatostatin (SSTR1, SSTR2, SSTR3, SSTR4, SSTR5), four chemokine (CXCR1, CXCR2, CXCR3, CXCR5), and three 5-HT2 (5HT2A, 5HT2B, 5HT2C) receptor subtypes. SNAP-GPCR-CHX degradation t1/2 values ranged from 0.52 h (ADRA1D) to 5.5 h (SSTR3). On the contrary, both the SNAP-tag alone and SNAP-tagged and endogenous β-actin were resistant to degradation with CHX treatment. Treatment with the proteasome inhibitor bortezomib produced significant but variable increases in SNAP-GPCR protein expression levels, indicating that SNAP-GPCR degradation primarily occurs through the proteasome. Remarkably, endogenous β2-adrenergic receptor/ADRB2 dynamic mass redistribution functional responses to norepinephrine were significantly decreased following CHX treatment, with a time course equivalent to that observed with the SNAP-ADRB2 degradation assay. We subsequently adapted this assay into a 96-well glass-bottom plate format to facilitate high-throughput GPCR degradation screening. t1/2 values quantified for the α1-adrenergic receptor subtypes (ADRA1A, ADRA1B, ADR1D) using the 96-well-plate format correlated with t1/2 values quantified using NIR-PAGE imaging analysis. In summary, this novel assay permits precise quantitative analysis of GPCR degradation in human cells and can be readily adapted to quantify degradation for any membrane protein of interest.

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Genome-wide identification of the GATA transcription factor family and their expression patterns under temperature and salt stress in Aspergillus oryzae

AMB Express ◽

10.1186/s13568-021-01212-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Chunmiao Jiang ◽

Gongbo Lv ◽

Jinxin Ge ◽

Bin He ◽

Zhe Zhang ◽

...

Keyword(s):

Salt Stress ◽

Expression Patterns ◽

Interaction Network ◽

Protein Protein Interaction ◽

Genome Wide ◽

Gata Transcription Factor ◽

Starting Point ◽

Evolutionary Features ◽

First Time

AbstractGATA transcription factors (TFs) are involved in the regulation of growth processes and various environmental stresses. Although GATA TFs involved in abiotic stress in plants and some fungi have been analyzed, information regarding GATA TFs in Aspergillusoryzae is extremely poor. In this study, we identified and functionally characterized seven GATA proteins from A.oryzae 3.042 genome, including a novel AoSnf5 GATA TF with 20-residue between the Cys-X2-Cys motifs which was found in Aspergillus GATA TFs for the first time. Phylogenetic analysis indicated that these seven A. oryzae GATA TFs could be classified into six subgroups. Analysis of conserved motifs demonstrated that Aspergillus GATA TFs with similar motif compositions clustered in one subgroup, suggesting that they might possess similar genetic functions, further confirming the accuracy of the phylogenetic relationship. Furthermore, the expression patterns of seven A.oryzae GATA TFs under temperature and salt stresses indicated that A. oryzae GATA TFs were mainly responsive to high temperature and high salt stress. The protein–protein interaction network of A.oryzae GATA TFs revealed certain potentially interacting proteins. The comprehensive analysis of A. oryzae GATA TFs will be beneficial for understanding their biological function and evolutionary features and provide an important starting point to further understand the role of GATA TFs in the regulation of distinct environmental conditions in A.oryzae.

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SNARE phosphorylation: a control mechanism for insulin-stimulated glucose transport and other regulated exocytic events

Biochemical Society Transactions ◽

10.1042/bst20170202 ◽

2017 ◽

Vol 45 (6) ◽

pp. 1271-1277 ◽

Cited By ~ 6

Author(s):

Kamilla M.E. Laidlaw ◽

Rachel Livingstone ◽

Mohammed Al-Tobi ◽

Nia J. Bryant ◽

Gwyn W. Gould

Keyword(s):

Membrane Fusion ◽

Molecular Mechanisms ◽

Membrane Receptors ◽

Multivesicular Bodies ◽

Attachment Protein ◽

Protein Receptor ◽

Sensitive Factor ◽

Plasma Membrane Receptors ◽

Regulated Trafficking ◽

Receptor Family

Trafficking within eukaryotic cells is a complex and highly regulated process; events such as recycling of plasma membrane receptors, formation of multivesicular bodies, regulated release of hormones and delivery of proteins to membranes all require directionality and specificity. The underpinning processes, including cargo selection, membrane fusion, trafficking flow and timing, are controlled by a variety of molecular mechanisms and engage multiple families of lipids and proteins. Here, we will focus on control of trafficking processes via the action of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family of proteins, in particular their regulation by phosphorylation. We will describe how these proteins are controlled in a range of regulated trafficking events, with particular emphasis on the insulin-stimulated delivery of glucose transporters to the surface of adipose and muscle cells. Here, we focus on a few examples of SNARE phosphorylation which exemplify distinct ways in which SNARE machinery phosphorylation may regulate membrane fusion.

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