scholarly journals Muscarinic receptors stimulate AC2 by novel phosphorylation sites, whereas Gβγ subunits exert opposing effects depending on the G-protein source

2012 ◽  
Vol 447 (3) ◽  
pp. 393-405 ◽  
Author(s):  
Jia X. Shen ◽  
Sebastian Wachten ◽  
Michelle L. Halls ◽  
Katy L. Everett ◽  
Dermot M. F. Cooper
Keyword(s):  
Muscarinic Receptor ◽  
Somatostatin Receptors ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Phosphorylation Sites ◽  
Canonical Pathways ◽  
Pkc Protein Kinase C ◽  
Opposite Manner ◽  
Opposing Effects

Direct phosphorylation of AC2 (adenylyl cyclase 2) by PKC (protein kinase C) affords an opportunity for AC2 to integrate signals from non-canonical pathways to produce the second messenger, cyclic AMP. The present study shows that stimulation of AC2 by pharmacological activation of PKC or muscarinic receptor activation is primarily the result of phosphorylation of Ser490 and Ser543, as opposed to the previously proposed Thr1057. A double phosphorylation-deficient mutant (S490/543A) of AC2 was insensitive to PMA (phorbol myristic acid) and CCh (carbachol) stimulation, whereas a double phosphomimetic mutant (S490/543D) mimicked the activity of PKC-activated AC2. Putative Gβγ-interacting sites are in the immediate environment of these PKC phosphorylation sites (Ser490 and Ser543) that are located within the C1b domain of AC2, suggesting a significant regulatory importance of this domain. Consequently, we examined the effect of both Gq-coupled muscarinic and Gi-coupled somatostatin receptors. Employing pharmacological and FRET (fluorescence resonance energy transfer)-based real-time single cell imaging approaches, we found that Gβγ released from the Gq-coupled muscarinic receptor or Gi-coupled somatostatin receptors exert inhibitory or stimulatory effects respectively. These results underline the sophisticated regulatory capacities of AC2, in not only being subject to regulation by PKC, but also and in an opposite manner to Gβγ subunits, depending on their source.

scholarly journals Identification of key phosphorylation sites in PTH1R that determine arrestin3 binding and fine-tune receptor signaling

2016 ◽  
Vol 473 (22) ◽  
pp. 4173-4192 ◽  
Author(s):  
Diana Zindel ◽  
Sandra Engel ◽  
Andrew R. Bottrill ◽  
Jean-Philippe Pin ◽  
Laurent Prézeau ◽  
...  
Keyword(s):  
Energy Transfer ◽  
G Protein ◽  
Mutational Analysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Phosphorylation Sites ◽  
Parathyroid Hormone Receptor ◽  
Extracellular Signal ◽  
G Protein Coupled

The parathyroid hormone receptor 1 (PTH1R) is a member of family B of G-protein-coupled receptors (GPCRs), predominantly expressed in bone and kidney where it modulates extracellular Ca2+ homeostasis and bone turnover. It is well established that phosphorylation of GPCRs constitutes a key event in regulating receptor function by promoting arrestin recruitment and coupling to G-protein-independent signaling pathways. Mapping phosphorylation sites on PTH1R would provide insights into how phosphorylation at specific sites regulates cell signaling responses and also open the possibility of developing therapeutic agents that could target specific receptor functions. Here, we have used mass spectrometry to identify nine sites of phosphorylation in the C-terminal tail of PTH1R. Mutational analysis revealed identified two clusters of serine and threonine residues (Ser489–Ser495 and Ser501–Thr506) specifically responsible for the majority of PTH(1–34)-induced receptor phosphorylation. Mutation of these residues to alanine did not affect negatively on the ability of the receptor to couple to G-proteins or activate extracellular-signal-regulated kinase 1/2. Using fluorescence resonance energy transfer and bioluminescence resonance energy transfer to monitor PTH(1–34)-induced interaction of PTH1R with arrestin3, we show that the first cluster Ser489–Ser495 and the second cluster Ser501–Thr506 operated in concert to mediate both the efficacy and potency of ligand-induced arrestin3 recruitment. We further demonstrate that Ser503 and Thr504 in the second cluster are responsible for 70% of arrestin3 recruitment and are key determinants for interaction of arrestin with the receptor. Our data are consistent with the hypothesis that the pattern of C-terminal tail phosphorylation on PTH1R may determine the signaling outcome following receptor activation.

scholarly journals Minireview: GPCR and G Proteins: Drug Efficacy and Activation in Live Cells

2009 ◽  
Vol 23 (5) ◽  
pp. 590-599 ◽  
Author(s):  
Jean-Pierre Vilardaga ◽  
Moritz Bünemann ◽  
Timothy N. Feinstein ◽  
Nevin Lambert ◽  
Viacheslav O. Nikolaev ◽  
...  
Keyword(s):  
Energy Transfer ◽  
G Protein ◽  
G Proteins ◽  
Intracellular Signaling ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Live Cells ◽  
Mechanical Stimuli ◽  
G Protein Coupled

Abstract Many biochemical pathways are driven by G protein-coupled receptors, cell surface proteins that convert the binding of extracellular chemical, sensory, and mechanical stimuli into cellular signals. Their interaction with various ligands triggers receptor activation that typically couples to and activates heterotrimeric G proteins, which in turn control the propagation of secondary messenger molecules (e.g. cAMP) involved in critically important physiological processes (e.g. heart beat). Successful transfer of information from ligand binding events to intracellular signaling cascades involves a dynamic interplay between ligands, receptors, and G proteins. The development of Förster resonance energy transfer and bioluminescence resonance energy transfer-based methods has now permitted the kinetic analysis of initial steps involved in G protein-coupled receptor-mediated signaling in live cells and in systems as diverse as neurotransmitter and hormone signaling. The direct measurement of ligand efficacy at the level of the receptor by Förster resonance energy transfer is also now possible and allows intrinsic efficacies of clinical drugs to be linked with the effect of receptor polymorphisms.

scholarly journals GPCR-dependent biasing of GIRK channel signaling dynamics by RGS6 in mouse sinoatrial nodal cells

2020 ◽  
Vol 117 (25) ◽  
pp. 14522-14531
Author(s):  
Allison Anderson ◽  
Ikuo Masuho ◽  
Ezequiel Marron Fernandez de Velasco ◽  
Atsushi Nakano ◽  
Lutz Birnbaumer ◽  
...  
Keyword(s):  
G Protein ◽  
Coupling Efficiency ◽  
Resonance Energy Transfer ◽  
Cardiac Pacemaker ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Protein Isoforms ◽  
Minimal Impact ◽  
Signaling Dynamics ◽  
The Impact

How G protein-coupled receptors (GPCRs) evoke specific biological outcomes while utilizing a limited array of G proteins and effectors is poorly understood, particularly in native cell systems. Here, we examined signaling evoked by muscarinic (M2R) and adenosine (A1R) receptor activation in the mouse sinoatrial node (SAN), the cardiac pacemaker. M2R and A1R activate a shared pool of cardiac G protein-gated inwardly rectifying K+(GIRK) channels in SAN cells from adult mice, but A1R-GIRK responses are smaller and slower than M2R-GIRK responses. Recordings from mice lacking Regulator of G protein Signaling 6 (RGS6) revealed that RGS6 exerts a GPCR-dependent influence on GIRK-dependent signaling in SAN cells, suppressing M2R-GIRK coupling efficiency and kinetics and A1R-GIRK signaling amplitude. Fast kinetic bioluminescence resonance energy transfer assays in transfected HEK cells showed that RGS6 prefers Gαoover Gαias a substrate for its catalytic activity and that M2R signals preferentially via Gαo, while A1R does not discriminate between inhibitory G protein isoforms. The impact of atrial/SAN-selective ablation of Gαoor Gαi2was consistent with these findings. Gαi2ablation had minimal impact on M2R-GIRK and A1R-GIRK signaling in SAN cells. In contrast, Gαoablation decreased the amplitude and slowed the kinetics of M2R-GIRK responses, while enhancing the sensitivity and prolonging the deactivation rate of A1R-GIRK signaling. Collectively, our data show that differences in GPCR-G protein coupling preferences, and the Gαosubstrate preference of RGS6, shape A1R- and M2R-GIRK signaling dynamics in mouse SAN cells.

Mapping the Protease Activated Receptor 4 (PAR4) Homodimer Interface.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 773-773
Author(s):  
Marvin T Nieman
Keyword(s):  
Conflicts Of Interest ◽  
Specific Interaction ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Dependent Manner ◽  
Intracellular Loop ◽  
Concentration Dependent Manner ◽  
Platelet Surface

Abstract Abstract 773 Thrombin activates platelets by binding and cleaving protease activated receptors 1 and 4 (PAR1 and PAR4). PAR1 and PAR4 communicate with each other to lower the concentration of thrombin required for PAR4 activation (Nieman Biochemistry, 2008). In addition, PAR1 and PAR4 form homo and heterodimers. However, where these receptors interact has not been defined and it is not known if dimerization influences receptor activation, downstream signaling, or both. Since PAR4 activation is important on human and mouse platelets, we sought to characterize the interaction site between PAR4 homodimers. Using bioluminescence resonance energy transfer (BRET), we mapped the PAR4 homodimer interface. The PAR4 homodimers show a specific interaction as indicated by a hyperbolic BRET signal in response to increasing PAR4-GFP expression with a fixed concentration of PAR4-Rluc. The threshold maximum BRET signal was disrupted in a concentration-dependent manner by unlabeled PAR4. In contrast, the unrelated G-protein coupled receptor, rhodopsin, was unable to disrupt the BRET signal indicating that the disruption of the PAR4 homodimer is a specific interaction. We have mapped the region required for PAR4 homodimer formation using chimeras between rhodopsin and PAR4. PAR4 does not interact with rhodopsin in BRET assays. Using a library of rho-PAR4 chimeras that have the junction at the beginning of transmembrane (TM) 2, 3, 4, 5, 6 or 7, we determined where dimer formation is restored. When the junction is placed at the beginning of TM4 or TM5, the chimera does not interact with PAR4-WT. In contrast, when the junction is moved to the end of TM2, the BRET signal is restored. These results indicate that the region on PAR4 required for homodimer formation encompasses a 63 amino acid region that includes the first extracellular loop, TM3 and the second intracellular loop. These studies establish techniques that may be used to define the interactions between other GPCRs found on the platelet surface. These receptor-receptor interactions may be another level of regulation of agonist activity and platelet function in vivo and may provide novel targets for anti-platelet therapies. Disclosures: No relevant conflicts of interest to declare.

Thrombin Modulates PAR1-PAR4 Heterodimers

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2203-2203 ◽  
Author(s):  
Maria de la Fuente ◽  
Amal Arachiche ◽  
Marvin T. Nieman
Keyword(s):  
Conflicts Of Interest ◽  
Transmembrane Helix ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Bimolecular Fluorescence Complementation ◽  
Physical Interaction ◽  
Signaling Specificity ◽  
C Terminus ◽  
The Stability

Abstract Abstract 2203 Thrombin is a potent platelet agonist. Thrombin activates platelets and other cells of the cardiovascular system by cleaving its receptors, protease activated receptor 1 (PAR1), PAR4 or both. PARs are G-protein coupled receptors that activate cellular signaling through Gq and G12/13. There is structural evidence that GPCRs, as a class, function as dimers and that dimerization can alter signaling specificity. Our previous studies have determined that PAR4 forms homodimers and have mapped the homodimer interface to transmembrane helix 4 (TM4). We have also shown that coexpression of PAR1 with PAR4 lowers the threshold for PAR4 activation by thrombin ∼10-fold. The purpose of the current study is to examine the physical interaction between PAR1 and PAR4 and how these interactions influence PAR1's ability to enhance PAR4 activation. The PAR1-PAR4 heterodimers were examined by bioluminescence resonance energy transfer (BRET) and bimolecular fluorescence complementation (BiFC). Similar to our previous studies with PAR4 homodimers, PAR1 homodimers were constitutive and did not require receptor activation. In contrast, PAR1-PAR4 heterodimers were not detected under basal conditions. However, when the cells were stimulated with 10 nM thrombin, we were able to detect a strong interaction between PAR1 and PAR4. We next examined if PAR1-PAR4 heterodimers would be induced by stimulating PAR1 or PAR4 individually with their agonist peptides TFLLRN (100 μM) or AYPGKF (500 μM), respectively. The agonist peptides were unable to induce heterodimers when added to the cells individually or simultaneously. These data demonstrate that PAR1 and PAR4 require allosteric changes induced by receptor cleavage by thrombin to mediate heterodimer formation. To examine this further, we removed 37 amino acids from the C-terminus of PAR1, which disrupts the eighth helix. The truncated PAR1 was able to form constitutive heterodimers with PAR4 and these heterodimers were unaffected by thrombin. These data suggest that PAR1 is the allosteric modulator of the PAR1-PAR4 heterodimers. Finally, the stability of the constitutive PAR1 and PAR4 homodimers was unchanged in response to thrombin or the agonist peptides. Taken together, these data suggest that PAR1 and PAR4 have a dynamic interaction depending on the context of their expression. Since PAR1 is an attractive antiplatelet target, the molecular interactions of this receptor on the cells surface must be taken into account when developing and characterizing these antagonists. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Frizzled BRET sensors based on bioorthogonal labeling of unnatural amino acids reveal WNT-induced dynamics of the cysteine-rich domain.

2021 ◽  
Author(s):  
Maria Kowalski-Jahn ◽  
Hannes Schihada ◽  
Ainoleena Turku ◽  
Thomas Huber ◽  
Thomas P. Sakmar ◽  
...  
Keyword(s):  
Amino Acids ◽  
Unnatural Amino Acids ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Membrane Receptors ◽  
Rich Domain ◽  
Linker Domain ◽  
Cysteine Rich Domain

Frizzleds (FZD1-10) comprise a class of G protein-coupled receptors containing an extracellular cysteine-rich domain (CRD) that binds lipoglycoproteins of the Wingless/Int-1 family (WNTs). Despite the prominent role of the WNT/FZD system in health and disease, our understanding of how WNT binding to the FZD CRD is translated into receptor activation and transmembrane signaling remains limited. Current hypotheses dispute the roles for conformational dynamics and the involvement of the linker domain connecting the CRD with the seven-helical transmembrane core of FZD. To clarify the mechanism of WNT binding to FZD and to elucidate how WNT/FZD complexes achieve signaling pathway specificity, we devised conformational FZD-CRD biosensors based on bioluminescence-resonance-energy-transfer (BRET). Using FZD engineered with N-terminal nanoluciferase and fluorescently-labeled unnatural amino acids in the linker domain and extracellular loop 3, we show that WNT-3A and WNT-5A induce similar CRD conformational rearrangements despite promoting distinct downstream signaling pathways, and that CRD dynamics are not required for WNT/β-catenin signaling. Thus, the novel FZD-CRD biosensors we report provide insights into the stepwise binding, activation and signaling processes in FZDs. The sensor design is broadly applicable to explore fundamental events in signal transduction mediated by other membrane receptors.

scholarly journals Agonist-induced formation of unproductive receptor-G12complexes

2020 ◽  
Vol 117 (35) ◽  
pp. 21723-21730
Author(s):  
Najeah Okashah ◽  
Shane C. Wright ◽  
Kouki Kawakami ◽  
Signe Mathiasen ◽  
Joris Zhou ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Receptor Internalization ◽  
Protein Association ◽  
Protein Activation ◽  
Protein Receptor ◽  
G Protein Activation

G proteins are activated when they associate with G protein-coupled receptors (GPCRs), often in response to agonist-mediated receptor activation. It is generally thought that agonist-induced receptor-G protein association necessarily promotes G protein activation and, conversely, that activated GPCRs do not interact with G proteins that they do not activate. Here we show that GPCRs can form agonist-dependent complexes with G proteins that they do not activate. Using cell-based bioluminescence resonance energy transfer (BRET) and luminescence assays we find that vasopressin V2receptors (V2R) associate with both Gsand G12heterotrimers when stimulated with the agonist arginine vasopressin (AVP). However, unlike V2R-Gscomplexes, V2R-G12complexes are not destabilized by guanine nucleotides and do not promote G12activation. Activating V2R does not lead to signaling responses downstream of G12activation, but instead inhibits basal G12-mediated signaling, presumably by sequestering G12heterotrimers. Overexpressing G12inhibits G protein receptor kinase (GRK) and arrestin recruitment to V2R and receptor internalization. Formyl peptide (FPR1 and FPR2) and Smoothened (Smo) receptors also form complexes with G12that are insensitive to nucleotides, suggesting that unproductive GPCR-G12complexes are not unique to V2R. These results indicate that agonist-dependent receptor-G protein association does not always lead to G protein activation and may in fact inhibit G protein activation.

scholarly journals Fluorescence changes reveal kinetic steps of muscarinic receptor–mediated modulation of phosphoinositides and Kv7.2/7.3 K+ channels

2009 ◽  
Vol 133 (4) ◽  
pp. 347-359 ◽  
Author(s):  
Jill B. Jensen ◽  
John S. Lyssand ◽  
Chris Hague ◽  
Bertil Hille
Keyword(s):  
Muscarinic Receptor ◽  
Neuronal Excitability ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Signaling Cascades ◽  
M Current ◽  
M1 Muscarinic ◽  
Rate Limiting ◽  
G Protein Coupled ◽  
Kinetics Of

G protein–coupled receptors initiate signaling cascades. M1 muscarinic receptor (M1R) activation couples through Gαq to stimulate phospholipase C (PLC), which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2). Depletion of PIP2 closes PIP2-requiring Kv7.2/7.3 potassium channels (M current), thereby increasing neuronal excitability. This modulation of M current is relatively slow (6.4 s to reach within 1/e of the steady-state value). To identify the rate-limiting steps, we investigated the kinetics of each step using pairwise optical interactions likely to represent fluorescence resonance energy transfer for M1R activation, M1R/Gβ interaction, Gαq/Gβ separation, Gαq/PLC interaction, and PIP2 hydrolysis. Electrophysiology was used to monitor channel closure. Time constants for M1R activation (<100 ms) and M1R/Gβ interaction (200 ms) are both fast, suggesting that neither of them is rate limiting during muscarinic suppression of M current. Gαq/Gβ separation and Gαq/PLC interaction have intermediate 1/e times (2.9 and 1.7 s, respectively), and PIP2 hydrolysis (6.7 s) occurs on the timescale of M current suppression. Overexpression of PLC accelerates the rate of M current suppression threefold (to 2.0 s) to become nearly contemporaneous with Gαq/PLC interaction. Evidently, channel release of PIP2 and closure are rapid, and the availability of active PLC limits the rate of M current suppression.

scholarly journals Real-Time Monitoring of Somatostatin Receptor-cAMP Signaling in Live Pituitary

Endocrinology ◽  
2010 ◽  
Vol 151 (9) ◽  
pp. 4560-4565 ◽  
Author(s):  
Stefan Jacobs ◽  
Davide Calebiro ◽  
Viacheslav O. Nikolaev ◽  
Martin J. Lohse ◽  
Stefan Schulz
Keyword(s):  
Real Time ◽  
Somatostatin Receptor ◽  
Somatostatin Receptors ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Pituitary Cells ◽  
Gi Protein ◽  
Camp Levels ◽  
Pituitary Receptors

Fluorescence resonance energy transfer using genetically encoded biosensors has proven to be a powerful technique to monitor the spatiotemporal dynamics of cAMP signals stimulated by Gs-coupled receptors in living cells. In contrast, real-time imaging of Gi-mediated cAMP signals under native conditions remains challenging. Here, we describe the use of transgenic mice for cAMP imaging in living pituitary slices and primary pituitary cells. This technique can be widely used to assess the contribution of various pituitary receptors, including individual Gi protein-coupled somatostatin receptors, to the regulation of cAMP levels under physiologically relevant settings.

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