Mapping the Heart

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.

GPCR Screening via ERK 1/2: A Novel Platform for Screening G Protein–Coupled Receptors

2005 ◽  
Vol 10 (7) ◽  
pp. 730-737 ◽  
Author(s):  
Ronald I. W. Osmond ◽  
Antony Sheehan ◽  
Romana Borowicz ◽  
Emma Barnett ◽  
Georgina Harvey ◽  
...  
Keyword(s):  
G Protein ◽  
High Throughput ◽  
Measurement Techniques ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
G Protein Coupled Receptors ◽  
Drug Candidates ◽  
Gpcr Activation ◽  
Intracellular Ca ◽  
G Protein Coupled

Discovery of novel agonists and antagonists for G protein–coupled receptors (GPCRs) relies heavily on cell-based assays because determination of functional consequences of receptor engagement is often desirable. Currently, there are several key parameters measured to achieve this, including mobilization of intracellular Ca2+ and formation of cyclic adenosine monophosphate or inositol triphosphate. However, no single assay platform is suitable for all situations, and all of the assays have limitations. The authors have developed a new high-throughput homogeneous assay platform for GPCR discovery as an alternative to current assays, which employs detection of phosphorylation of the key signaling molecule p42/44 MAP kinase (ERK 1/2). The authors show that ERK 1/2 is consistently activated in cells stimulated by Gq-coupled GPCRs and provides a new high-throughput platform for screening GPCR drug candidates. The activation of ERK 1/2 in Gq-coupled GPCR systems generates comparable pharmacological data for receptor agonist and antagonist data obtained by other GPCR activation measurement techniques.

scholarly journals Extensive crosstalk of G protein-coupled receptors with the Hedgehog signalling pathway

Development ◽  
2021 ◽  
pp. dev.189258
Author(s):  
Farah Saad ◽  
David R. Hipfner
Keyword(s):  
G Protein ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
G Protein Coupled Receptors ◽  
Signalling Pathway ◽  
Protein Activity ◽  
Cellular Sensitivity ◽  
Hedgehog Signalling Pathway ◽  
G Protein Coupled ◽  
Camp Levels

Hedgehog (Hh) ligands orchestrate tissue patterning and growth by acting as morphogens, dictating different cellular responses depending on ligand concentration. Cellular sensitivity to Hh ligands is influenced by heterotrimeric G protein activity, which controls production of the second messenger 3',5'-cyclic adenosine monophosphate (cAMP). cAMP in turn activates Protein kinase A (PKA), which functions as an inhibitor and (uniquely in Drosophila) an activator of Hh signalling. A few mammalian Gαi- and Gαs-coupled G protein-coupled receptors (GPCRs) have been shown to influence Sonic Hh (Shh) responses in this way. To determine if this is a more general phenomenon, we carried out an RNAi screen targeting GPCRs in Drosophila. RNAi-mediated depletion of more than 40% of GPCRs tested either decreased or increased Hh responsiveness in the developing Drosophila wing, closely matching the effects of Gαs and Gαi depletion, respectively. Genetic analysis indicated that the orphan GPCR Mthl5 lowers cAMP levels to attenuate Hh responsiveness. Our results identify Mthl5 as a new Hh signalling pathway modulator in Drosophila and suggest that many GPCRs may crosstalk with the Hh pathway in mammals.

Photoinduced receptor confinement drives ligand-independent GPCR signaling

Science ◽  
2021 ◽  
Vol 371 (6536) ◽  
pp. eabb7657
Author(s):  
M. Florencia Sánchez ◽  
Sylvia Els-Heindl ◽  
Annette G. Beck-Sickinger ◽  
Ralph Wieneke ◽  
Robert Tampé
Keyword(s):  
Cell Communication ◽  
Cellular Responses ◽  
Receptor Activation ◽  
G Protein Coupled Receptors ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Lateral Membrane ◽  
Guanine Nucleotide Binding ◽  
G Protein Coupled

Cell-cell communication relies on the assembly of receptor-ligand complexes at the plasma membrane. The spatiotemporal receptor organization has a pivotal role in evoking cellular responses. We studied the clustering of heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) and established a photoinstructive matrix with ultrasmall lock-and-key interaction pairs to control lateral membrane organization of hormone neuropeptide Y2 receptors in living cells by light. Within seconds, receptor clustering was modulated in size, location, and density. After in situ confinement, changes in cellular morphology, motility, and calcium signaling revealed ligand-independent receptor activation. This approach may enhance the exploration of mechanisms in cell signaling and mechanotransduction.

scholarly journals Noncanonical scaffolding of Gαi and β-arrestin by G protein–coupled receptors

Science ◽  
2021 ◽  
pp. eaay1833
Author(s):  
Jeffrey S. Smith ◽  
Thomas F. Pack ◽  
Asuka Inoue ◽  
Claudia Lee ◽  
Kevin Zheng ◽  
...  
Keyword(s):  
G Protein ◽  
Drug Targets ◽  
Direct Interaction ◽  
Adaptor Proteins ◽  
G Protein Coupled Receptors ◽  
Guanine Nucleotide Binding Protein ◽  
Signaling Mechanism ◽  
Protein Activation ◽  
Guanine Nucleotide Binding ◽  
G Protein Coupled

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) are common drug targets and canonically couple to specific Gα protein subtypes and β-arrestin adaptor proteins. G protein- and β-arrestin-mediated signaling have been considered separable. We show GPCRs promote a direct interaction between Gαi protein subtype family members and β-arrestins, regardless of their canonical Gαi protein subtype coupling. Gαi:β-arrestin complexes bound extracellular signal-regulated kinase (ERK) and their disruption impaired both ERK activation and cell migration, consistent with β-arrestins requiring a functional interaction with Gαi for certain signaling events. These results introduce a GPCR signaling mechanism distinct from canonical G protein activation in which GPCRs cause the formation of Gαi:β-arrestin signaling complexes.

scholarly journals GRKs as Modulators of Neurotransmitter Receptors

Cells ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 52
Author(s):  
Eugenia V. Gurevich ◽  
Vsevolod V. Gurevich
Keyword(s):  
G Protein ◽  
General Model ◽  
G Protein Coupled Receptors ◽  
Receptor Internalization ◽  
Neurotransmitter Receptors ◽  
Receptor Subtype ◽  
Receptor Subtypes ◽  
Receptor Kinase ◽  
G Protein Coupled ◽  
Homologous Desensitization

Many receptors for neurotransmitters, such as dopamine, norepinephrine, acetylcholine, and neuropeptides, belong to the superfamily of G protein-coupled receptors (GPCRs). A general model posits that GPCRs undergo two-step homologous desensitization: the active receptor is phosphorylated by kinases of the G protein-coupled receptor kinase (GRK) family, whereupon arrestin proteins specifically bind active phosphorylated receptors, shutting down G protein-mediated signaling, facilitating receptor internalization, and initiating distinct signaling pathways via arrestin-based scaffolding. Here, we review the mechanisms of GRK-dependent regulation of neurotransmitter receptors, focusing on the diverse modes of GRK-mediated phosphorylation of receptor subtypes. The immediate signaling consequences of GRK-mediated receptor phosphorylation, such as arrestin recruitment, desensitization, and internalization/resensitization, are equally diverse, depending not only on the receptor subtype but also on phosphorylation by GRKs of select receptor residues. We discuss the signaling outcome as well as the biological and behavioral consequences of the GRK-dependent phosphorylation of neurotransmitter receptors where known.

CCK receptor phosphorylation exposes regulatory domains affecting phosphorylation and receptor trafficking

2000 ◽  
Vol 279 (6) ◽  
pp. C1986-C1992 ◽  
Author(s):  
Rammohan V. Rao ◽  
Eileen L. Holicky ◽  
Susan M. Kuntz ◽  
Laurence J. Miller
Keyword(s):  
G Protein ◽  
Chinese Hamster ◽  
G Protein Coupled Receptors ◽  
Receptor Internalization ◽  
Ovary Cell ◽  
Guanine Nucleotide Binding Protein ◽  
Receptor Phosphorylation ◽  
Regulatory Domains ◽  
Ovary Cell Line ◽  
G Protein Coupled

Agonist-stimulated phosphorylation of guanine nucleotide-binding protein (G protein)-coupled receptors has been recognized as an important mechanism for desensitization by interfering with coupling of the activated receptor with its G protein. We recently described a mutant of the CCK receptor that modified two of five key sites of phosphorylation (S260,264A) and eliminated agonist-stimulated receptor phosphorylation, despite normal ligand binding and signaling (20). As expected, this nonphosphorylated mutant had impaired rapid desensitization but was ultimately able to be desensitized by normal receptor internalization. Here we demonstrate that this mutant receptor is also defective in resensitization, with abnormal recycling to the cell surface. To explore this, another receptor mutant was prepared, replacing the same serines with aspartates to mimic the charge of serine-phosphate (S260,264D). This mutant was expressed in a Chinese hamster ovary cell line and shown to bind CCK normally. It had accelerated kinetics of signaling and desensitization and was phosphorylated in response to agonist occupation, with all other normal sites of phosphorylation modified. It was internalized like wild-type receptors and was resensitized and trafficked normally. This provides evidence for an additional important function for phosphorylation of G protein-coupled receptors. Phosphorylation may induce a conformational change in the receptor to expose other potential sites of phosphorylation and to expose domains involved in the targeting and trafficking of endosomes. The hierarchical phosphorylation of these sites may play a key role in receptor regulation.

scholarly journals Homo- and hetero-oligomeric interactions between G-protein-coupled receptors in living cells monitored by two variants of bioluminescence resonance energy transfer (BRET): hetero-oligomers between receptor subtypes form more efficiently than between less closely related sequences

2002 ◽  
Vol 365 (2) ◽  
pp. 429-440 ◽  
Author(s):  
Douglas RAMSAY ◽  
Elaine KELLETT ◽  
Mary McVEY ◽  
Stephen REES ◽  
Graeme MILLIGAN
Keyword(s):  
Opioid Receptor ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
G Protein Coupled Receptors ◽  
Receptor Subtypes ◽  
Bioluminescence Resonance Energy Transfer ◽  
Oligomer Formation ◽  
G Protein Coupled ◽  
Energy Acceptor

Homo- and hetero-oligomerization of G-protein-coupled receptors (GPCRs) were examined in HEK-293 cells using two variants of bioluminescence resonance energy transfer (BRET). BRET2 (a variant of BRET) offers greatly improved separation of the emission spectra of the donor and acceptor moieties compared with traditional BRET. Previously recorded homo-oligomerization of the human δ-opioid receptor was confirmed using BRET2. Homo-oligomerization of the κ-opioid receptor was observed using both BRET techniques. Both homo- and hetero-oligomers, containing both δ- and κ-opioid receptors, were unaffected by the presence of receptor ligands. BRET detection of opioid receptor homo- and hetero-oligomers required expression of 50000–100000 copies of the receptor energy acceptor construct per cell. The effectiveness of δ—κ-opioid receptor hetero-oligomer formation was as great as for homomeric interactions. The capacity of the two opioid receptors to form oligomeric complexes with the β2-adrenoceptor was also assessed. Although such interactions were detected, at least 250000 copies per cell of the energy acceptor were required. Requirement for high levels of receptor expression was equally pronounced in attempts to measure hetero-oligomer formation between the κ-opioid receptor and the thyrotropin-releasing hormone receptor-1. These studies indicate that constitutively formed homo- and hetero-oligomers of opioid receptor subtypes can be detected in living cells containing less than 100000 copies of the receptors. However, although hetero-oligomeric interactions between certain less closely related GPCRs can be detected, they appear to be of lower affinity than homo- or hetero-oligomers containing closely related sequences. Interactions recorded between certain GPCR family members in heterologous expression systems are likely to be artefacts of extreme levels of overexpression.

scholarly journals Insights into the molecular evolution of oxytocin receptor ligand binding

2013 ◽  
Vol 41 (1) ◽  
pp. 197-204 ◽  
Author(s):  
Johannes Koehbach ◽  
Thomas Stockner ◽  
Christian Bergmayr ◽  
Markus Muttenthaler ◽  
Christian W. Gruber
Keyword(s):  
Molecular Evolution ◽  
Peptide Ligands ◽  
Receptor Subtypes ◽  
Ligand Interaction ◽  
Interaction Sites ◽  
Selective Ligands ◽  
Vasopressin Receptors ◽  
The Common ◽  
G Protein Coupled ◽  
Selection Of

The design and development of selective ligands for the human OT (oxytocin) and AVP (arginine vasopressin) receptors is a big challenge since the different receptor subtypes and their native peptide ligands display great similarity. Detailed understanding of the mechanism of OT's interaction with its receptor is important and may assist in the ligand- or structure-based design of selective and potent ligands. In the present article, we compared 69 OT- and OT-like receptor sequences with regards to their molecular evolution and diversity, utilized an in silico approach to map the common ligand interaction sites of recently published G-protein-coupled receptor structures to a model of the human OTR (OT receptor) and compared these interacting residues within a selection of different OTR sequences. Our analysis suggests the existence of a binding site for OT peptides within the common transmembrane core region of the receptor, but it appears extremely difficult to identify receptor or ligand residues that could explain the selectivity of OT to its receptors. We remain confident that the presented evolutionary overview and modelling approach will aid interpretation of forthcoming OTR crystal structures.

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