scholarly journals Overexpression of Orphan Receptor GPR61 Increases cAMP Levels upon Forskolin Stimulation in HEK293 Cells: in vitro and in silico Validation of 5-(Nonyloxy)Tryptamine as a Low-Affinity Inverse Agonist

Pharmacology ◽  
2019 ◽  
Vol 104 (5-6) ◽  
pp. 377-382
Author(s):  
Pawel Kozielewicz ◽  
Gillian Grafton ◽  
Joanna J. Sajkowska-Kozielewicz ◽  
Nicholas M. Barnes
Keyword(s):  
Docking Studies ◽  
G Protein Coupled Receptors ◽  
Orphan Receptor ◽  
Hek293 Cells ◽  
Intrinsic Activity ◽  
Camp Synthesis ◽  
Class A ◽  
G Protein Coupled ◽  
Camp Levels

GPR61 is an orphan receptor that belongs to Class A of G-protein-coupled receptors. It has been reported that GPR61 has a constitutive activity and couples to Gαs. In the present study, we characterized GPR61 function and ligand binding by experimental and molecular docking studies. We demonstrated that heterologous expression of GPR61 in HEK293 cells enhanced the cAMP synthesis response to forskolin, whereas the basal cAMP synthesis was unaffected. 5-(Nonyloxy)tryptamine inhibited forskolin-stimulated cAMP production in GPR61-expressing HEK293 cells. These studies highlight that the intrinsic activity of this receptor is only measurable following its synergy with Gαs.

scholarly journals cAMP levels regulate macrophage alternative activation marker expression

2020 ◽  
pp. 175342592097508
Author(s):  
Swamy Polumuri ◽  
Darren J Perkins ◽  
Stefanie N Vogel
Keyword(s):  
G Protein Coupled Receptors ◽  
Alternative Activation ◽  
Marker Genes ◽  
Marker Expression ◽  
Inflammatory Milieu ◽  
G Protein Coupled ◽  
Camp Levels ◽  
Dependent Pathway

The capacity for macrophages to polarize into distinct functional activation states (e.g., M1, M2) is critical to tune an inflammatory response to the relevant infection or injury. Alternative or M2 polarization of macrophages is most often achieved in vitro in response to IL-4/IL-13 and results in the transcriptional up-regulation of a constellation of characteristic M2 marker genes. In vivo, additional signals from the inflammatory milieu can further increase or decrease M2 marker expression. Particularly, activation of cAMP-generating G protein-coupled receptors is reported to increase M2 markers, but whether this is strictly dependent upon cAMP production is unclear. We report herein that increased cAMP alone can increase IL-4-dependent M2 marker expression through a PKA/C/EBPβ/CREB dependent pathway in murine macrophages.

scholarly journals The evolution and mechanism of GPCR proton sensing

2020 ◽  
pp. jbc.RA120.016352
Author(s):  
Jacob B. Rowe ◽  
Nicholas J. Kapolka ◽  
Geoffrey J. Taghon ◽  
William M. Morgan ◽  
Daniel G. Isom
Keyword(s):  
Therapeutic Potential ◽  
G Protein Coupled Receptors ◽  
Hek293 Cells ◽  
Ph Sensing ◽  
Physiological Importance ◽  
Class A ◽  
Acidic Residues ◽  
Ph Range ◽  
G Protein Coupled ◽  
Direct Regulation

Of the 800 G protein-coupled receptors (GPCRs) in humans, only three (GPR4, GPR65, and GPR68) regulate signaling in acidified microenvironments by sensing protons (H+). How these receptors have uniquely obtained this ability is unknown. Here we show these receptors evolved the capability to sense H+ signals by acquiring buried acidic residues. Using our informatics platform pHinder, we identified a triad of buried acidic residues shared by all three receptors, a feature distinct from all other human GPCRs. Phylogenetic analysis shows the triad emerged in GPR65, the immediate ancestor of GPR4 and GPR68. To understand the evolutionary and mechanistic importance of these triad residues, we developed Deep Variant Profiling (DVP), a yeast-based technology that utilizes high-throughput CRISPR to build and profile large libraries of GPCR variants. Using DVP and GPCR assays in HEK293 cells, we assessed the pH-sensing contributions of each triad residue in all three receptors. As predicted by our calculations, most triad mutations had profound effects consistent with direct regulation of receptor pH sensing. Additionally, we found that an allosteric modulator of many class A GPCRs, Na+, synergistically regulated pH sensing by maintaining the pKa values of triad residues within the physiologically relevant pH range. As such, we show that all three receptors function as coincidence detectors of H+ and Na+. Taken together, these findings elucidate the molecular evolution and long-sought mechanism of GPR4, GPR65, and GPR68 pH sensing, and provide pH-insensitive variants that should be valuable for assessing the therapeutic potential and (patho)physiological importance of GPCR pH sensing.

scholarly journals Carbazole and Simplified Derivatives: Novel Tools toward β-Adrenergic Receptors Targeting

2021 ◽  
Vol 11 (12) ◽  
pp. 5486
Author(s):  
Fedora Grande ◽  
Anna De Bartolo ◽  
Maria Antonietta Occhiuzzi ◽  
Anna Caruso ◽  
Carmine Rocca ◽  
...  
Keyword(s):  
Adrenergic Receptors ◽  
Cardiac Activity ◽  
Docking Studies ◽  
G Protein Coupled Receptors ◽  
Carbazole Derivative ◽  
Biological Assays ◽  
H9c2 Cardiomyocytes ◽  
Β Adrenergic Receptors ◽  
G Protein Coupled

β-Adrenergic receptors (β-ARs) are G protein-coupled receptors involved in important physiological and pathological processes related to blood pressure and cardiac activity. The inhibition of cardiac β1-ARs could be beneficial in myocardial hypertrophy, ischemia and failure. Several carbazole-based compounds have been described as promising β-blockers. Herein, we investigate the capability of a carbazole derivative and three simplified indole analogs to interact with the active binding site of β1-AR by molecular docking studies. In the light of the obtained results, our compounds were tested by biological assays in H9c2 cardiomyocytes exposed to isoproterenol (ISO) to confirm their potential as β1-blockers agents, and two of them (8 and 10) showed interesting and promising properties. In particular, these compounds were effective against ISO-dependent in vitro cardiac hypertrophy, even at concentrations lower than the known β-AR antagonist propranolol. Overall, the data suggest that the indole derivatives 8 and 10 could act as potent β1-blockers and, active at low doses, could elicit limited side effects.

scholarly journals Adhesion G Protein–Coupled Receptors: From In Vitro Pharmacology to In Vivo Mechanisms

2015 ◽  
Vol 88 (3) ◽  
pp. 617-623 ◽  
Author(s):  
Kelly R. Monk ◽  
Jörg Hamann ◽  
Tobias Langenhan ◽  
Saskia Nijmeijer ◽  
Torsten Schöneberg ◽  
...  
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
In Vitro Pharmacology ◽  
G Protein Coupled

scholarly journals Constitutive Activity among Orphan Class-A G Protein Coupled Receptors

PLoS ONE ◽  
2015 ◽  
Vol 10 (9) ◽  
pp. e0138463 ◽  
Author(s):  
Adam L. Martin ◽  
Michael A. Steurer ◽  
Robert S. Aronstam
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Constitutive Activity ◽  
Class A ◽  
G Protein Coupled

Selective recruitment of arrestin-3 to clathrin coated pits upon stimulation of G protein-coupled receptors

2000 ◽  
Vol 113 (13) ◽  
pp. 2463-2470 ◽  
Author(s):  
F. Santini ◽  
R.B. Penn ◽  
A.W. Gagnon ◽  
J.L. Benovic ◽  
J.H. Keen
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Coated Pits ◽  
Over Expression ◽  
Physiological Conditions ◽  
A Cell ◽  
G Protein Coupled ◽  
Comparable Magnitude

Non-visual arrestins (arrestin-2 and arrestin-3) play critical roles in the desensitization and internalization of many G protein-coupled receptors. In vitro experiments have shown that both non-visual arrestins bind with high and approximately comparable affinities to activated, phosphorylated forms of receptors. They also exhibit high affinity binding, again of comparable magnitude, to clathrin. Further, agonist-promoted internalization of many receptors has been found to be stimulated by exogenous over-expression of either arrestin2 or arrestin3. The existence of multiple arrestins raises the question whether stimulated receptors are selective for a specific endogenous arrestin under more physiological conditions. Here we address this question in RBL-2H3 cells, a cell line that expresses comparable levels of endogenous arrestin-2 and arrestin-3. When (beta)(2)-adrenergic receptors are stably expressed in these cells the receptors internalize efficiently following agonist stimulation. However, by immunofluorescence microscopy we determine that only arrestin-3, but not arrestin-2, is rapidly recruited to clathrin coated pits upon receptor stimulation. Similarly, in RBL-2H3 cells that stably express physiological levels of m1AChR, the addition of carbachol selectively induces the localization of arrestin-3, but not arrestin-2, to coated pits. Thus, this work demonstrates coupling of G protein-coupled receptors to a specific non-visual arrestin in an in vivo setting.

GPCR activation mechanisms across classes and macro/microscales

2021 ◽  
Author(s):  
David Gloriam ◽  
Alexander Hauser ◽  
Albert Kooistra ◽  
Christian Munk ◽  
M. Madan Babu
Keyword(s):  
Residue Level ◽  
G Protein Coupled Receptors ◽  
Conformational Selection ◽  
Class A ◽  
New Methods ◽  
Allosteric Communication ◽  
Gpcr Activation ◽  
Activation Mechanisms ◽  
Clinical Drugs ◽  
G Protein Coupled

Abstract Two-thirds of human hormones and one-third of clinical drugs activate ~350 G protein-coupled receptors belonging to four classes: A, B1, C and F. Whereas a model of activation has been described for class A, very little is known about the activation of the other classes which differ by being activated by endogenous ligands bound mainly or entirely extracellularly. Here, we show that although they use the same structural scaffold and share several helix macroswitches, the GPCR classes differ in their microswitch residue positions and contacts. We present molecular mechanistic maps of activation for each GPCR class and new methods for contact analysis applicable for any functional determinants. This is the first superfamily residue-level rationale for conformational selection and allosteric communication by ligands and G proteins laying the foundation for receptor-function studies and drugs with the desired modality.

N-ethyl-N-nitrosourea-based generation of mouse models for mutant G protein-coupled receptors

2006 ◽  
Vol 26 (3) ◽  
pp. 209-217 ◽  
Author(s):  
Johannes Grosse ◽  
Patrick Tarnow ◽  
Holger Römpler ◽  
Boris Schneider ◽  
Reinhard Sedlmeier ◽  
...  
Keyword(s):  
G Protein ◽  
Mouse Models ◽  
Germ Line ◽  
Random Mutagenesis ◽  
G Protein Coupled Receptors ◽  
In Vitro Assays ◽  
Vitro Fertilization ◽  
Type 3 ◽  
G Protein Coupled

Chemical random mutagenesis techniques with the germ line supermutagen N-ethyl- N-nitrosourea (ENU) have been established to provide comprehensive collections of mouse models, which were then mined and analyzed in phenotype-driven studies. Here, we applied ENU mutagenesis in a high-throughput fashion for a gene-driven identification of new mutations. Selected members of the large superfamily of G protein-coupled receptors (GPCR), melanocortin type 3 (Mc3r) and type 4 (Mc4r) receptors, and the orphan chemoattractant receptor GPR33, were used as model targets to prove the feasibility of this approach. Parallel archives of DNA and sperm from mice mutagenized with ENU were screened for mutations in these GPCR, and in vitro assays served as a preselection step before in vitro fertilization was performed to generate the appropriate mouse model. For example, mouse models for inherited obesity were established by selecting fully or partially inactivating mutations in Mc4r. Our technology described herein has the potential to provide mouse models for a GPCR dysfunction of choice within <4 mo and can be extended to other gene classes of interest.

scholarly journals Prospects for use of peptides and their derivatives, structurally corresponding to the G protein-coupled receptors, in medicine

2015 ◽  
Vol 61 (1) ◽  
pp. 19-29 ◽  
Author(s):  
A.O. Shpakov ◽  
E.A. Shpakova
Keyword(s):  
G Protein ◽  
Intracellular Signaling ◽  
High Efficiency ◽  
Clinical Medicine ◽  
Inflammatory Diseases ◽  
G Protein Coupled Receptors ◽  
Signaling Cascades ◽  
G Protein Coupled

The regulation of signaling pathways involved in the control of many physiological functions is carried out via the heterotrimeric G protein-coupled receptors (GPCR). The search of effective and selective regulators of GPCR and intracellular signaling cascades coupled with them is one of the important problems of modern fundamental and clinical medicine. Recently data suggest that synthetic peptides and their derivatives, structurally corresponding to the intracellular and transmembrane regions of GPCR, can interact with high efficiency and selectivity with homologous receptors and influence, thus, the functional activity of intracellular signaling cascades and fundamental cellular processes controlled by them. GPCR-peptides are active in both in vitro and in vivo. They regulate hematopoiesis, angiogenesis and cell proliferation, inhibit tumor growth and metastasis, and prevent the inflammatory diseases and septic shock. These data show greatest prospects in the development of the new generations of drugs based on GPCR-derived peptides, capable of regulating the important functions of the organism.

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