scholarly journals PAC1 Receptor Internalization and Endosomal MEK/ERK Activation Is Essential for PACAP-Mediated Neuronal Excitability

2021 ◽  
Author(s):  
Victor May ◽  
Gregory C. Johnson ◽  
Sayamwong E. Hammack ◽  
Karen M. Braas ◽  
Rodney L. Parsons
Keyword(s):  
Neuronal Excitability ◽  
Receptor Internalization ◽  
Pac1 Receptor ◽  
Erk Activation

scholarly journals Src family kinase inhibitors blunt PACAP-induced PAC1 receptor endocytosis, phosphorylation of ERK, and the increase in cardiac neuron excitability

2018 ◽  
Vol 314 (2) ◽  
pp. C233-C241 ◽  
Author(s):  
John D. Tompkins ◽  
Todd A. Clason ◽  
Thomas R. Buttolph ◽  
Beatrice M. Girard ◽  
Anne K. Linden ◽  
...  
Keyword(s):  
G Protein ◽  
Neuronal Excitability ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Src Family Kinases ◽  
Receptor Internalization ◽  
Src Family Kinase ◽  
Pac1 Receptor ◽  
Neuron Excitability ◽  
Endosomal Signaling

Pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1) activation of PAC1 receptors ( Adcyap1r1) significantly increases excitability of guinea pig cardiac neurons. This modulation of excitability is mediated in part by plasma membrane G protein-dependent activation of adenylyl cyclase and downstream signaling cascades. However, additional mechanisms responsible for the enhanced excitability are activated following internalization of the PAC1 receptor and endosomal signaling. Src family kinases play critical roles mediating endocytosis of many trophic factor and G protein-coupled receptors. The present study investigated whether Src family kinases also support the PACAP-induced PAC1 receptor internalization, phosphorylation of ERK, and enhanced neuronal excitability. Using human embryonic kidney cells stably expressing a green fluorescent protein-tagged PAC1 receptor, treatment with the Src family kinase inhibitor PP2 (10 µM) markedly reduced the PACAP-induced PAC1 receptor internalization, and in parallel, both PP2 and Src inhibitor 1 (Src-1, 2 µM) reduced ERK activation determined by Western blot analysis. In contrast, Src family kinase inhibitors did not eliminate a PACAP-induced rise in global calcium generated by inositol (1,4,5)-trisphosphate-induced release of calcium from endoplasmic reticulum stores. From confocal analysis of phosphorylated ERK immunostaining, PP2 treatment significantly attenuated PACAP activation of ERK in neurons within cardiac ganglia whole mount preparations. Intracellular recordings demonstrated that PP2 also significantly blunted a PACAP-induced increase in cardiac neuron excitability. These studies demonstrate Src-related kinase activity in PAC1 receptor internalization, activation of MEK/ERK signaling, and regulation of neuronal excitability. The present results provide further support for the importance of PAC1 receptor endosomal signaling as a key mechanism regulating cellular function.

scholarly journals PACAP-induced ERK activation in HEK cells expressing PAC1 receptors involves both receptor internalization and PKC signaling

2014 ◽  
Vol 306 (11) ◽  
pp. C1068-C1079 ◽  
Author(s):  
Victor May ◽  
Thomas R. Buttolph ◽  
Beatrice M. Girard ◽  
Todd A. Clason ◽  
Rodney L. Parsons
Keyword(s):  
Adenylyl Cyclase ◽  
Receptor Internalization ◽  
Dependent Manner ◽  
Pac1 Receptor ◽  
Hek 293 ◽  
Erk Activation ◽  
Receptor Endocytosis ◽  
Erk Phosphorylation ◽  
Pkc Signaling ◽  
Pkc Activation

The pituitary adenylate cyclase-activating polypeptide (PACAP)-selective PAC1 receptor ( Adcyap1r1) is a G protein-coupled receptor (GPCR) that activates adenylyl cyclase and PLC. Similar to many other GPCRs, our previous studies showed that the PAC1 receptor is internalized after ligand binding to form signaling endosomes, which recruit additional second messenger pathways. Using a human embryonic kidney (HEK 293) PAC1Hop1-EGFP receptor cell line, we have examined how different PAC1 receptor signaling mechanisms contribute to MEK/ERK activation. Unlike PAC1 receptor-stimulated adenylyl cyclase/cAMP production in the plasma membrane, PACAP-mediated ERK phosphorylation was partly dependent on receptor internalization, as determined by treatment with pharmacological inhibitors of endocytosis or temperature reduction, which also suppressed receptor internalization. Stimulation of cAMP generation by forskolin or exposure to the cell-permeable cAMP analogs 8-bromo-cAMP and dibutyryl cAMP had minimal effects on ERK phosphorylation in this system. The ability of reduced temperature (24°C) to consistently suppress ERK activation to a greater extent than the endocytosis inhibitors Pitstop 2 and dynasore indicated that other mechanisms, in addition to PAC1 internalization/endosome activation, were involved. Inhibition of PAC1 receptor-stimulated PLC/diacylglycerol/PKC signaling by bisindoylmaleimide I also attenuated ERK phosphorylation, and direct PKC activation with phorbol ester increased ERK phosphorylation in a temperature-dependent manner. Inhibition of PAC1 receptor endocytosis and PKC activation completely blocked PACAP-stimulated ERK activation. PACAP augmented phosphorylated ERK staining uniformly over the cytoplasm and nucleus, and PKC signaling facilitated nuclear phosphorylated ERK translocation. In sum, our results show that PACAP/PAC1 receptor endocytosis and PLC/diacylglycerol/PKC activation represent two complementary mechanisms contributing to PACAP-induced ERK activation.

scholarly journals Recruitment of endosomal signaling mediates the forskolin modulation of guinea pig cardiac neuron excitability

2017 ◽  
Vol 313 (2) ◽  
pp. C219-C227 ◽  
Author(s):  
Jean C. Hardwick ◽  
Todd A. Clason ◽  
John D. Tompkins ◽  
Beatrice M. Girard ◽  
Caitlin N. Baran ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Neuronal Excitability ◽  
Receptor Activation ◽  
Erk Signaling ◽  
Pac1 Receptor ◽  
Signaling Mechanism ◽  
Erk Activation ◽  
Neuron Excitability ◽  
Endosomal Signaling ◽  
Gated Channel

Forskolin, a selective activator of adenylyl cyclase (AC), commonly is used to establish actions of G protein-coupled receptors (GPCRs) that are initiated primarily through activation of AC/cAMP signaling pathways. In the present study, forskolin was used to evaluate the potential role of AC/cAMP, which is a major signaling mechanism for the pituitary adenylate cyclase-activating polypeptide (PACAP)-selective PAC1 receptor, in the regulation of guinea pig cardiac neuronal excitability. Forskolin (5–10 µM) increases excitability in ~60% of the cardiac neurons. The forskolin-mediated increase in excitability was considered related to cAMP regulation of a cyclic nucleotide gated channel or via protein kinase A (PKA)/ERK signaling, mechanisms that have been linked to PAC1 receptor activation. However, unlike PACAP mechanisms, forskolin enhancement of excitability was not significantly reduced by treatment with cesium to block currents through hyperpolarization-activated nonselective cation channels ( Ih) or by treatment with PD98059 to block MEK/ERK signaling. In contrast, treatment with the clathrin inhibitor Pitstop2 or the dynamin inhibitor dynasore eliminated the forskolin-induced increase in excitability; treatments with the inactive Pitstop analog or PP2 treatment to inhibit Src-mediated endocytosis mechanisms were ineffective. The PKA inhibitor KT5702 significantly suppressed the forskolin-induced change in excitability; further, KT5702 and Pitstop2 reduced the forskolin-stimulated MEK/ERK activation in cardiac neurons. Collectively, the present results suggest that forskolin activation of AC/cAMP/PKA signaling leads to the recruitment of clathrin/dynamin-dependent endosomal transduction cascades, including MEK/ERK signaling, and that endosomal signaling is the critical mechanism underlying the forskolin-induced increase in cardiac neuron excitability.

scholarly journals β-Arrestin-Dependent Parathyroid Hormone-Stimulated Extracellular Signal-Regulated Kinase Activation and Parathyroid Hormone Type 1 Receptor Internalization

Endocrinology ◽  
2007 ◽  
Vol 148 (8) ◽  
pp. 4073-4079 ◽  
Author(s):  
W. Bruce Sneddon ◽  
Peter A. Friedman
Keyword(s):  
Parathyroid Hormone ◽  
Late Phase ◽  
Receptor Internalization ◽  
Rapid Phase ◽  
Kinase Activation ◽  
Erk Activation ◽  
Extracellular Signal ◽  
Wild Type Mefs ◽  
Β Adrenergic Receptors

PTH regulates renal calcium homeostasis by actions on the distal nephron. PTH-induced calcium transport in mouse distal convoluted tubule (DCT) cells requires activation of ERK1/2. ERK activation by β-adrenergic receptors occurs in a biphasic manner and involves receptor internalization. An early rapid phase is β-arrestin (βAr) independent, whereas prolonged activation is βAr dependent. We characterized PTH-stimulated ERK activation and the involvement of receptor internalization and βAr dependence. In DCT cells, PTH transiently activated ERK maximally at 5 min and then returned to baseline. βAr dependence of PTH receptor (PTH1R)-mediated ERK stimulation was assessed using mouse embryonic fibroblasts (MEFs) from βAr1- and -2-null mice. In wild-type MEFs, PTH(1–34)-stimulated ERK activation peaked after 5 min, was 50% maximal after 15 min, and then recovered to 80% of maximal stimulation by 30 min. In MEFs null for βAr1 and -2, PTH-stimulated ERK activation peaked by 5 min and returned to baseline. The effect was identical in βAr2-null MEFs. In βAr1-null MEFs, ERK exhibited delayed activation and remained elevated. PTH-stimulated ERK activation and receptor endocytosis were not inhibited by the clathrin-binding domain of βAr1 [Ar(319–418)]. Coexpression of the sodium proton exchanger regulatory factor 1 (NHERF1) with Ar(319–418) blocked PTH1R internalization. We conclude that PTH-stimulated ERK activation in DCT cells proceeds with a rapid but transient phase that may involve βAr1. Furthermore, the βAr-dependent late phase of ERK activation by PTH requires the participation of βAr2 and PTH1R internalization.

scholarly journals Kinetics of receptor tyrosine kinase activation define ERK signaling dynamics

2020 ◽  
Vol 13 (645) ◽  
pp. eaaz5267 ◽  
Author(s):  
Anatoly Kiyatkin ◽  
Iris K. van Alderwerelt van Rosenburgh ◽  
Daryl E. Klein ◽  
Mark A. Lemmon
Keyword(s):  
Growth Factor ◽  
Cell Fate ◽  
Egf Receptor ◽  
Receptor Internalization ◽  
Erk Signaling ◽  
Activation Kinetics ◽  
Kinase Activation ◽  
Erk Activation ◽  
Cell Fate Decisions ◽  
Signaling Dynamics

In responses to activation of receptor tyrosine kinases (RTKs), crucial cell fate decisions depend on the duration and dynamics of ERK signaling. In PC12 cells, epidermal growth factor (EGF) induces transient ERK activation that leads to cell proliferation, whereas nerve growth factor (NGF) promotes sustained ERK activation and cell differentiation. These differences have typically been assumed to reflect distinct feedback mechanisms in the Raf-MEK-ERK signaling network, with the receptors themselves acting as simple upstream inputs. We failed to confirm the expected differences in feedback type when investigating transient versus sustained signaling downstream of the EGF receptor (EGFR) and NGF receptor (TrkA). Instead, we found that ERK signaling faithfully followed RTK dynamics when receptor signaling was modulated in different ways. EGFR activation kinetics, and consequently ERK signaling dynamics, were switched from transient to sustained when receptor internalization was inhibited with drugs or mutations, or when cells expressed a chimeric receptor likely to have impaired dimerization. In addition, EGFR and ERK signaling both became more sustained when substoichiometric levels of erlotinib were added to reduce duration of EGFR kinase activation. Our results argue that RTK activation kinetics play a crucial role in determining MAP kinase cascade signaling dynamics and cell fate decisions, and that signaling outcome can be modified by activating a given RTK in different ways.

scholarly journals Activation of MEK/ERK signaling contributes to the PACAP-induced increase in guinea pig cardiac neuron excitability

2016 ◽  
Vol 311 (4) ◽  
pp. C643-C651 ◽  
Author(s):  
John D. Tompkins ◽  
Todd A. Clason ◽  
Jean C. Hardwick ◽  
Beatrice M. Girard ◽  
Laura A. Merriam ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Neuronal Excitability ◽  
Low Voltage ◽  
Ionic Currents ◽  
Erk Signaling ◽  
Repetitive Firing ◽  
Neuronal System ◽  
Cellular Mechanisms ◽  
Pac1 Receptor ◽  
Neuron Excitability

Pituitary adenylate cyclase (PAC)-activating polypeptide (PACAP) peptides ( Adcyap1) signaling at the selective PAC1 receptor ( Adcyap1r1) participate in multiple homeostatic and stress-related responses, yet the cellular mechanisms underlying PACAP actions remain to be completely elucidated. PACAP/PAC1 receptor signaling increases excitability of neurons within the guinea pig cardiac ganglia, and as these neurons are readily accessible, this neuronal system is particularly amenable to study of PACAP modulation of ionic conductances. The present study investigated how PACAP activation of MEK/ERK signaling contributed to the peptide-induced increase in cardiac neuron excitability. Treatment with the MEK inhibitor PD 98059 blocked PACAP-stimulated phosphorylated ERK and, in parallel, suppressed the increase in cardiac neuron excitability. However, PD 98059 did not blunt the ability of PACAP to enhance two inward ionic currents, one flowing through hyperpolarization-activated nonselective cationic channels ( Ih) and another flowing through low-voltage-activated calcium channels ( IT), which support the peptide-induced increase in excitability. Thus a PACAP - and MEK/ERK-sensitive, voltage-dependent conductance(s), in addition to Ih and IT, modulates neuronal excitability. Despite prior work implicating PACAP downregulation of the KV4.2 potassium channel in modulation of excitability in other cells, treatment with the KV4.2 current blocker 4-aminopyridine did not replicate the PACAP-induced increase in excitability in cardiac neurons. However, cardiac neurons express the ERK target, the NaV1.7 sodium channel, and treatment with the selective NaV1.7 channel inhibitor PF-04856264 decreased the PACAP modulation of excitability. From these results, PACAP/PAC1 activation of MEK/ERK signaling may phosphorylate the NaV1.7 channel, enhancing sodium currents near the threshold, an action contributing to repetitive firing of the cardiac neurons exposed to PACAP.

scholarly journals Calcium Influx, But Not Intracellular Calcium Release, Supports PACAP-Mediated ERK Activation in HEK PAC1 Receptor Cells

2014 ◽  
Vol 54 (3) ◽  
pp. 342-350 ◽  
Author(s):  
Victor May ◽  
Todd A. Clason ◽  
Thomas R. Buttolph ◽  
Beatrice M. Girard ◽  
Rodney L. Parsons
Keyword(s):  
Intracellular Calcium ◽  
Calcium Release ◽  
Calcium Influx ◽  
Pac1 Receptor ◽  
Erk Activation ◽  
Receptor Cells ◽  
Intracellular Calcium Release

scholarly journals Exploring Biased Agonism at FPR1 as a Means to Encode Danger Sensing

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 1054 ◽  
Author(s):  
Jieny Gröper ◽  
Gabriele König ◽  
Evi Kostenis ◽  
Volker Gerke ◽  
Carsten Raabe ◽  
...  
Keyword(s):  
Receptor Internalization ◽  
Formyl Peptide Receptor ◽  
Tissue Destruction ◽  
Erk Activation ◽  
Biased Agonism ◽  
Additional Layer ◽  
Pathway Activation ◽  
Biased Signaling ◽  
Formyl Peptide ◽  
Camp Formation

Ligand-based selectivity in signal transduction (biased signaling) is an emerging field of G protein-coupled receptor (GPCR) research and might allow the development of drugs with targeted activation profiles. Human formyl peptide receptor 1 (FPR1) is a GPCR that detects potentially hazardous states characterized by the appearance of N-formylated peptides that originate from either bacteria or mitochondria during tissue destruction; however, the receptor also responds to several non-formylated agonists from various sources. We hypothesized that an additional layer of FPR signaling is encoded by biased agonism, thus allowing the discrimination of the source of threat. We resorted to the comparative analysis of FPR1 agonist-evoked responses across three prototypical GPCR signaling pathways, i.e., the inhibition of cAMP formation, receptor internalization, and ERK activation, and analyzed cellular responses elicited by several bacteria- and mitochondria-derived ligands. We also included the anti-inflammatory annexinA1 peptide Ac2-26 and two synthetic ligands, the W-peptide and the small molecule FPRA14. Compared to the endogenous agonists, the bacterial agonists displayed significantly higher potencies and efficacies. Selective pathway activation was not observed, as both groups were similarly biased towards the inhibition of cAMP formation. The general agonist bias in FPR1 signaling suggests a source-independent pathway selectivity for transmission of pro-inflammatory danger signaling.

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