scholarly journals PACAP-Induced PAC1 Receptor Internalization and Recruitment of Endosomal Signaling Regulate Cardiac Neuron Excitability

2018 ◽  
Vol 68 (3) ◽  
pp. 340-347 ◽  
Author(s):  
Rodney L. Parsons ◽  
Victor May
Keyword(s):  
Receptor Internalization ◽  
Pac1 Receptor ◽  
Neuron Excitability ◽  
Endosomal Signaling

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 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 Pharmacological Programming of Endosomal Signaling Activated by Small Molecule Ligands of the Follicle Stimulating Hormone Receptor

2020 ◽  
Vol 11 ◽  
Author(s):  
Silvia Sposini ◽  
Francesco De Pascali ◽  
Rachel Richardson ◽  
Niamh S. Sayers ◽  
David Perrais ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Hormone Receptor ◽  
Follicle Stimulating Hormone ◽  
Adaptor Protein ◽  
Receptor Internalization ◽  
Camp Signaling ◽  
Pleckstrin Homology Domain ◽  
Early Endosomes ◽  
Endosomal Signaling ◽  
Stimulating Hormone

Follicle-stimulating hormone receptor (FSHR) is a G protein-coupled receptor (GPCR) with pivotal roles in reproduction. One key mechanism dictating the signal activity of GPCRs is membrane trafficking. After binding its hormone FSH, FSHR undergoes internalization to very early endosomes (VEEs) for its acute signaling and sorting to a rapid recycling pathway. The VEE is a heterogeneous compartment containing the Adaptor Protein Phosphotyrosine Interacting with Pleckstrin homology Domain and Leucine Zipper 1 (APPL1) with distinct functions in regulating endosomal Gαs/cAMP signaling and rapid recycling. Low molecular weight (LMW) allosteric FSHR ligands were developed for use in assisted reproductive technology yet could also provide novel pharmacological tools to study FSHR. Given the critical nature of receptor internalization and endosomal signaling for FSHR activity, we assessed whether these compounds exhibit differential abilities to alter receptor endosomal trafficking and signaling within the VEE. Two chemically distinct LMW agonists (benzamide, termed B3 and thiazolidinone, termed T1) were employed. T1 was able to induce a greater level of cAMP than FSH and B3. As cAMP signaling drives gonadotrophin hormone receptor recycling, rapid exocytic events were evaluated at single event resolution. Strikingly, T1 was able to induce a 3-fold increase in recycling events compared to FSH and two-fold more compared to B3. As T1-induced internalization was only marginally greater, the dramatic increase in recycling and cAMP signaling may be due to additional mechanisms. All compounds exhibited a similar requirement for receptor internalization to increase cAMP and proportion of FSHR endosomes with active Gαs, suggesting regulation of cAMP signaling induced by T1 may be altered. APPL1 plays a central role for GPCRs targeted to the VEE, and indeed, loss of APPL1 inhibited FSH-induced recycling and increased endosomal cAMP signaling. While T1-induced FSHR recycling was APPL1-dependent, its elevated cAMP signaling was only partially increased following APPL1 knockdown. Unexpectedly, B3 altered the dependence of FSHR to APPL1 in an opposing manner, whereby its endosomal signaling was negatively regulated by APPL1, while B3-induced FSHR recycling was APPL1-independent. Overall, FSHR allosteric compounds have the potential to re-program FSHR activity via altering engagement with VEE machinery and also suggests that these two distinct functions of APPL1 can potentially be selected pharmacologically.

scholarly journals Location bias contributes to functionally selective responses of biased CXCR3 agonists

2022 ◽  
Author(s):  
Dylan Scott Eiger ◽  
Noelia Boldizsar ◽  
Christopher Cole Honeycutt ◽  
Julia Gardner ◽  
Stephen Kirchner ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cd8 T Cells ◽  
Chemokine Receptors ◽  
Receptor Internalization ◽  
Contact Hypersensitivity ◽  
Transcriptional Responses ◽  
Endosomal Signaling ◽  
Specific Manner ◽  
Biased Signaling ◽  
G Protein Coupled

Some G protein-coupled receptor (GPCR) ligands act as biased agonists which preferentially activate specific signaling transducers over others. Although GPCRs are primarily found at the plasma membrane, GPCRs can traffic to and signal from many subcellular compartments. Here, we determine that differential subcellular signaling contributes to the biased signaling generated by three endogenous ligands of the chemokine GPCR CXCR3. The signaling profile of CXCR3 changed as it trafficked from the plasma membrane to endosomes in a ligand-specific manner. Endosomal signaling was critical for biased activation of G proteins, β-arrestins, and ERK1/2. In CD8+ T cells, the chemokines promoted unique transcriptional responses predicted to regulate inflammatory pathways. In a mouse model of contact hypersensitivity, β-arrestin-biased CXCR3-mediated inflammation was dependent on receptor internalization. Our work demonstrates that differential subcellular signaling is critical to the overall biased response observed at CXCR3, which has important implications for drugs targeting chemokine receptors and other GPCRs.

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 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 Pituitary adenylate cyclase-activating polypeptide-induced PAC1 receptor internalization and recruitment of MEK/ERK signaling enhance excitability of dentate gyrus granule cells

2020 ◽  
Vol 318 (5) ◽  
pp. C870-C878 ◽  
Author(s):  
Gregory C. Johnson ◽  
Rodney L. Parsons ◽  
Victor May ◽  
Sayamwong E. Hammack
Keyword(s):  
Adenylate Cyclase ◽  
Dentate Gyrus ◽  
Granule Cells ◽  
Sodium Current ◽  
Brain Slices ◽  
Receptor Activation ◽  
Receptor Internalization ◽  
Erk Signaling ◽  
Pac1 Receptor ◽  
Pituitary Adenylate Cyclase

Pituitary adenylate cyclase activating polypeptide (PACAP; ADCYAP1) is a pleiotropic neuropeptide widely distributed in both the peripheral and central nervous systems. PACAP and its specific cognate PAC1 receptor ( ADCYAP1R1) play critical roles in the homeostatic maintenance of multiple physiological and behavioral systems. Notably, maladaptations in the PACAPergic system have been associated with several psychopathologies related to fear and anxiety. PAC1 receptor transcripts are highly expressed in granule cells of the dentate gyrus (DG). Here, we examined the direct effects of PACAP on DG granule cells in brain slices using whole cell patch recordings in current clamp mode. PACAP significantly increased the intrinsic excitability of DG granule cells via PAC1 receptor activation. This increased excitability was not mediated by adenylyl cyclase/cAMP or phospholipase C/PKC activation, but instead via activation of an extracellular signal-regulated kinase (ERK) signaling pathway initiated through PAC1 receptor endocytosis/endosomal signaling. PACAP failed to increase excitability in DG granule cells pretreated with the persistent sodium current blocker riluzole, suggesting that the observed PACAP effects required this component of the inward sodium current.

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