scholarly journals Regulation of ER Composition and Extent, and Putative Action in Protein Networks by ER/NE Protein TMEM147

2021 ◽  
Vol 22 (19) ◽  
pp. 10231
Author(s):  
Giannis Maimaris ◽  
Andri Christodoulou ◽  
Niovi Santama ◽  
Carsten Werner Lederer
Keyword(s):  
Transmembrane Protein ◽  
Protein Networks ◽  
Oxidoreductase Activity ◽  
Chromatin Dynamics ◽  
Ribosome Binding ◽  
Functional Studies ◽  
Pathway Analyses ◽  
G Protein Coupled ◽  
Comprehensive Compilation ◽  
Hub Proteins

Nuclear envelope (NE) and endoplasmic reticulum (ER) collaborate to control a multitude of nuclear and cytoplasmic actions. In this context, the transmembrane protein TMEM147 localizes to both NE and ER, and through direct and indirect interactions regulates processes as varied as production and transport of multipass membrane proteins, neuronal signaling, nuclear-shape, lamina and chromatin dynamics and cholesterol synthesis. Aiming to delineate the emerging multifunctionality of TMEM147 more comprehensively, we set as objectives, first, to assess potentially more fundamental effects of TMEM147 on the ER and, second, to identify significantly TMEM147-associated cell-wide protein networks and pathways. Quantifying curved and flat ER markers RTN4 and CLIMP63/CKAP4, respectively, we found that TMEM147 silencing causes area and intensity increases for both RTN4 and CLIMP63, and the ER in general, with a profound shift toward flat areas, concurrent with reduction in DNA condensation. Protein network and pathway analyses based on comprehensive compilation of TMEM147 interactors, targets and co-factors then served to manifest novel and established roles for TMEM147. Thus, algorithmically simplified significant pathways reflect TMEM147 function in ribosome binding, oxidoreductase activity, G protein-coupled receptor activity and transmembrane transport, while analysis of protein factors and networks identifies hub proteins and corresponding pathways as potential targets of TMEM147 action and of future functional studies.

scholarly journals Constitutive signal bias mediated by the human GHRHR splice variant 1

2021 ◽  
Vol 118 (40) ◽  
pp. e2106606118
Author(s):  
Zhaotong Cong ◽  
Fulai Zhou ◽  
Chao Zhang ◽  
Xinyu Zou ◽  
Huibing Zhang ◽  
...  
Keyword(s):  
Splice Variant ◽  
Human Growth ◽  
Bound State ◽  
G Protein Coupled Receptors ◽  
Cancer Cell Proliferation ◽  
Downstream Signaling ◽  
Functional Studies ◽  
Dynamics Simulations ◽  
Protein Molecular Dynamics ◽  
G Protein Coupled

Alternative splicing of G protein–coupled receptors has been observed, but their functions are largely unknown. Here, we report that a splice variant (SV1) of the human growth hormone–releasing hormone receptor (GHRHR) is capable of transducing biased signal. Differing only at the receptor N terminus, GHRHR predominantly activates Gs while SV1 selectively couples to β-arrestins. Based on the cryogenic electron microscopy structures of SV1 in the apo state or GHRH-bound state in complex with the Gs protein, molecular dynamics simulations reveal that the N termini of GHRHR and SV1 differentiate the downstream signaling pathways, Gs versus β-arrestins. As suggested by mutagenesis and functional studies, it appears that GHRH-elicited signal bias toward β-arrestin recruitment is constitutively mediated by SV1. The level of SV1 expression in prostate cancer cells is also positively correlated with ERK1/2 phosphorylation but negatively correlated with cAMP response. Our findings imply that constitutive signal bias may be a mechanism that ensures cancer cell proliferation.

scholarly journals G-protein-coupled receptor 30 interacts with receptor activity-modifying protein 3 and confers sex-dependent cardioprotection

2013 ◽  
Vol 51 (1) ◽  
pp. 191-202 ◽  
Author(s):  
Patricia M Lenhart ◽  
Stefan Broselid ◽  
Cordelia J Barrick ◽  
L M Fredrik Leeb-Lundberg ◽  
Kathleen M Caron
Keyword(s):  
G Protein ◽  
Transmembrane Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cardiac Cells ◽  
Receptor Activity ◽  
Hek293 Cells ◽  
G Protein Coupled

Receptor activity-modifying protein 3 (RAMP3) is a single-pass transmembrane protein known to interact with and affect the trafficking of several G-protein-coupled receptors (GPCRs). We sought to determine whether RAMP3 interacts with GPR30, also known as G-protein-coupled estrogen receptor 1. GPR30 is a GPCR that binds estradiol and has important roles in cardiovascular and endocrine physiology. Using bioluminescence resonance energy transfer titration studies, co-immunoprecipitation, and confocal microscopy, we show that GPR30 and RAMP3 interact. Furthermore, the presence of GPR30 leads to increased expression of RAMP3 at the plasma membrane in HEK293 cells. In vivo, there are marked sex differences in the subcellular localization of GPR30 in cardiac cells, and the hearts of Ramp3−/− mice also show signs of GPR30 mislocalization. To determine whether this interaction might play a role in cardiovascular disease, we treated Ramp3+/+ and Ramp3−/− mice on a heart disease-prone genetic background with G-1, a specific agonist for GPR30. Importantly, this in vivo activation of GPR30 resulted in a significant reduction in cardiac hypertrophy and perivascular fibrosis that is both RAMP3 and sex dependent. Our results demonstrate that GPR30–RAMP3 interaction has functional consequences on the localization of these proteins both in vitro and in vivo and that RAMP3 is required for GPR30-mediated cardioprotection.

scholarly journals Transcriptomic Profiling of Tumor-Infiltrating CD4+TIM-3+ T Cells Reveals Their Suppressive, Exhausted, and Metastatic Characteristics in Colorectal Cancer Patients

Vaccines ◽  
2020 ◽  
Vol 8 (1) ◽  
pp. 71 ◽  
Author(s):  
Varun Sasidharan Nair ◽  
Salman M Toor ◽  
Rowaida Z Taha ◽  
Ayman A Ahmed ◽  
Mohamed A Kurer ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
T Cells ◽  
T Cell ◽  
Mononuclear Cells ◽  
T Regulatory Cell ◽  
Peripheral Blood Mononuclear ◽  
Functional Studies ◽  
Normal Tissues ◽  
Pathway Analyses ◽  
Colorectal Cancer Patients

T cell immunoglobulin mucin-3 (TIM-3) is an immune checkpoint identified as one of the key players in regulating T-cell responses. Studies have shown that TIM-3 is upregulated in the tumor microenvironment (TME). However, the precise role of TIM-3 in colorectal cancer (CRC) TME is yet to be elucidated. We performed phenotypic and molecular characterization of TIM-3+ T cells in the TME and circulation of CRC patients by analyzing tumor tissues (TT, TILs), normal tissues (NT, NILs), and peripheral blood mononuclear cells (PBMC). TIM-3 was upregulated on both CD4+ and CD3+CD4− (CD8+) TILs. CD4+TIM-3+ TILs expressed higher levels of T regulatory cell (Tregs)-signature genes, including FoxP3 and Helios, compared with their TIM-3− counterparts. Transcriptomic and ingenuity pathway analyses showed that TIM-3 potentially activates inflammatory and tumor metastatic pathways. Moreover, NF-κB-mediated transcription factors were upregulated in CD4+TIM-3+ TILs, which could favor proliferation/invasion and induce inflammatory and T-cell exhaustion pathways. In addition, we found that CD4+TIM-3+ TILs potentially support tumor invasion and metastasis, compared with conventional CD4+CD25+ Tregs in the CRC TME. However, functional studies are warranted to support these findings. In conclusion, this study discloses some of the functional pathways of TIM-3+ TILs, which could improve their targeting in more specific therapeutic approaches in CRC patients.

scholarly journals GWAS-driven pathway analyses and functional studies reveals GLIS1 to predispose to mitral valve prolapse

2017 ◽  
Vol 9 (1) ◽  
pp. 96
Author(s):  
M. Yu ◽  
C. Dina ◽  
N. Tucker ◽  
F. Delling ◽  
S. Slaugenhaupt ◽  
...  
Keyword(s):  
Mitral Valve ◽  
Mitral Valve Prolapse ◽  
Functional Studies ◽  
Pathway Analyses

scholarly journals MOLECULAR EVOLUTION OF GPCRS: 26Rfa/GPR103

2014 ◽  
Vol 52 (3) ◽  
pp. T119-T131 ◽  
Author(s):  
Kazuyoshi Ukena ◽  
Tomohiro Osugi ◽  
Jérôme Leprince ◽  
Hubert Vaudry ◽  
Kazuyoshi Tsutsui
Keyword(s):  
Energy Homeostasis ◽  
Hormone Secretion ◽  
Pituitary Hormone ◽  
Nociceptive Transmission ◽  
Functional Studies ◽  
Cognate Receptor ◽  
Current State ◽  
Representative Species ◽  
Peptide Family ◽  
G Protein Coupled

Neuropeptides possessing the Arg-Phe-NH2 (RFamide) motif at their C-termini (designated as RFamide peptides) have been characterized in a variety of animals. Among these, neuropeptide 26RFa (also termed QRFP) is the latest member of the RFamide peptide family to be discovered in the hypothalamus of vertebrates. The neuropeptide 26RFa/QRFP is a 26-amino acid residue peptide that was originally identified in the frog brain. It has been shown to exert orexigenic activity in mammals and to be a ligand for the previously identified orphan G protein-coupled receptor, GPR103 (QRFPR). The cDNAs encoding 26RFa/QRFP and QRFPR have now been characterized in representative species of mammals, birds, and fish. Functional studies have shown that, in mammals, the 26RFa/QRFP–QRFPR system may regulate various functions, including food intake, energy homeostasis, bone formation, pituitary hormone secretion, steroidogenesis, nociceptive transmission, and blood pressure. Several biological actions have also been reported in birds and fish. This review summarizes the current state of identification, localization, and understanding of the functions of 26RFaQRFP and its cognate receptor, QRFPR, in vertebrates.

scholarly journals Identification and characterization of an atypical Gαs-biased β2AR agonist that fails to evoke airway smooth muscle cell tachyphylaxis

2021 ◽  
Vol 118 (49) ◽  
pp. e2026668118
Author(s):  
Donghwa Kim ◽  
Alina Tokmakova ◽  
Lauren K. Lujan ◽  
Hannah R. Strzelinski ◽  
Nicholas Kim ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Chemical Space ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Airway Smooth Muscle Cell ◽  
Functional Studies ◽  
G Protein Coupled ◽  
Identification And Characterization

G protein–coupled receptors display multifunctional signaling, offering the potential for agonist structures to promote conformational selectivity for biased outputs. For β2-adrenergic receptors (β2AR), unbiased agonists stabilize conformation(s) that evoke coupling to Gαs (cyclic adenosine monophosphate [cAMP] production/human airway smooth muscle [HASM] cell relaxation) and β-arrestin engagement, the latter acting to quench Gαs signaling, contributing to receptor desensitization/tachyphylaxis. We screened a 40-million-compound scaffold ranking library, revealing unanticipated agonists with dihydroimidazolyl-butyl-cyclic urea scaffolds. The S-stereoisomer of compound C1 shows no detectable β-arrestin engagement/signaling by four methods. However, C1-S retained Gαs signaling—a divergence of the outputs favorable for treating asthma. Functional studies with two models confirmed the biasing: β2AR-mediated cAMP signaling underwent desensitization to the unbiased agonist albuterol but not to C1-S, and desensitization of HASM cell relaxation was observed with albuterol but not with C1-S. These HASM results indicate biologically pertinent biasing of C1-S, in the context of the relevant physiologic response, in the human cell type of interest. Thus, C1-S was apparently strongly biased away from β-arrestin, in contrast to albuterol and C5-S. C1-S structural modeling and simulations revealed binding differences compared with unbiased epinephrine at transmembrane (TM) segments 3,5,6,7 and ECL2. C1-S (R2 = cyclohexane) was repositioned in the pocket such that it lost a TM6 interaction and gained a TM7 interaction compared with the analogous unbiased C5-S (R2 = benzene group), which appears to contribute to C1-S biasing away from β-arrestin. Thus, an agnostic large chemical-space library identified agonists with receptor interactions that resulted in relevant signal splitting of β2AR actions favorable for treating obstructive lung disease.

scholarly journals HIV-1 Envelope and MPER antibody structures in lipid assemblies

2019 ◽  
Author(s):  
Kimmo Rantalainen ◽  
Zachary T. Berndsen ◽  
Aleksandar Antanasijevic ◽  
Torben Schiffner ◽  
Xi Zhang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmembrane Protein ◽  
Full Length ◽  
Structural Studies ◽  
Wild Type ◽  
Functional Studies ◽  
Antibody Complex ◽  
Modular Platform ◽  
Lipid Assemblies ◽  
Hiv 1

SummaryStructural and functional studies of HIV Env as a transmembrane protein have long been complicated by challenges associated with inherent flexibility of the molecule and the membrane-embedded hydrophobic regions. Thus, most structural studies have utilized soluble forms where the regions C-terminal to the ectodomain are deleted. Here, we present approaches for incorporating full-length, wild-type HIV-1 Env, as well as C-terminally truncated and stabilized versions, into lipid assemblies, providing a modular platform for Env structural studies by single particle electron microscopy. We reconstituted a full-length Env clone into a nanodisc with MSP1D1 scaffold, complexed it with an MPER targeting antibody 10E8, and structurally defined the full quaternary epitope of 10E8 consisting of lipid, MPER and ectodomain contacts. By aligning this and other Env-MPER antibody complex reconstructions with the lipid bilayer, we observe evidence of Env tilting as part of the neutralization mechanism for MPER-targeting antibodies. We also adapted the platform toward vaccine design purposes by introducing stabilizing mutations that allow purification of unliganded Env with peptidisc scaffold.

scholarly journals Highly Selective, Amine-Derived Cannabinoid Receptor 2 Probes

2019 ◽  
Author(s):  
Matthias V. Westphal ◽  
Roman C. Sarott ◽  
Elisabeth A. Zirwes ◽  
Anja Osterwald ◽  
Wolfgang Guba ◽  
...  
Keyword(s):  
Cannabinoid Receptor ◽  
Synthetic Cannabinoids ◽  
Structural Features ◽  
Pharmacological Characterization ◽  
Cannabinoid Receptor 2 ◽  
Functional Studies ◽  
Design And Synthesis ◽  
Downstream Effects ◽  
G Protein Coupled

The endocannabinoid (eCB) system is implied in various human diseases ranging from central nervous system to autoimmune disorders. Cannabinoid receptor 2 (CB<sub>2</sub>R) is an integral component of the eCB system. Yet, the downstream effects elicited by this G protein-coupled receptor upon binding of endogenous or synthetic ligands are insufficiently understood—likely due to the limited arsenal of reliable biological and chemical tools. Herein, we report the design and synthesis of CB<sub>2</sub>R-selective cannabinoids along with their <i>in vitro</i> pharmacological characterization (binding and functional studies). They combine structural features of HU-308 and AM841 to give chimeric ligands that emerge as potent CB<sub>2</sub>R agonists with high selectivity over the closely related cannabinoid receptor 1 (CB<sub>1</sub>R). The synthesis work includes convenient preparation of substituted resorcinols often found in cannabinoids. The utility of the synthetic cannabinoids in this study is showcased by preparation of the most selective high-affinity fluorescent probe for CB<sub>2</sub>R to date.

scholarly journals Differential Effects of Modified Batrachotoxins on Voltage-gated Sodium Channel Fast and Slow Inactivation

2021 ◽  
Author(s):  
Tim M. G. MacKenzie ◽  
Fayal Abderemane-Ali ◽  
Catherine E. Garrison ◽  
Daniel L. Minor Jr. ◽  
Justin Du Bois
Keyword(s):  
De Novo ◽  
Protein Complexes ◽  
Transmembrane Protein ◽  
Ion Selectivity ◽  
Allosteric Modulators ◽  
Slow Inactivation ◽  
Voltage Gated ◽  
Functional Studies ◽  
Voltage Gated Sodium Channels ◽  
Initiation And Propagation

Voltage-gated sodium channels (Na<sub>V</sub>s), large transmembrane protein complexes responsible for the initiation and propagation of action potentials, are targets for a number of acute poisons. Many of these agents act as allosteric modulators of channel activity and serve as powerful chemical tools for understanding channel function. Batrachotoxin (BTX) is a steroidal amine derivative most commonly associated with poison dart frogs and is unique as a Na<sub>V</sub> ligand in that it alters every property of the channel, including threshold potential of activation, inactivation, ion selectivity, and ion conduction. Structure-function studies with BTX are limited, however, by the inability to access preparative quantities of this compound from natural sources. We have addressed this problem through <i>de novo</i> synthesis of BTX, which gives access to modified toxin structures. In this report, we detail electrophysiology studies of three BTX C20-ester derivatives against recombinant Na<sub>V</sub> subtypes (rat Na<sub>V</sub>1.4 and human Na<sub>V</sub>1.5). Two of these compounds, BTX-B and BTX-<sup>c</sup>Hx, are functionally equivalent to BTX, hyperpolarizing channel activation and blocking both fast and slow inactivation. BTX-yne—a C20-<i>n</i>-heptynoate ester—is a conspicuous outlier, eliminating fast but not slow inactivation. This unique property qualifies BTX-yne as the first reported Na<sub>V</sub> modulator that separates inactivation processes. These findings are supported by functional studies with bacterial Na<sub>V</sub>s (BacNa<sub>V</sub>s) that lack a fast inactivation gate. The availability of BTX-yne should advance future efforts aimed at understanding Na<sub>V</sub> gating mechanisms and designing allosteric regulators of Na<sub>V</sub> activity.

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