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

May, Víctor; Buttolph, Thomas R.; Girard, Beatrice M.; Clason, Todd A.; Parsons, Rodney L.

doi:10.1152/ajpcell.00001.2014

Cited by 50 publications

(98 citation statements)

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“…T-type calcium currents can be modulated by multiple intracellular signaling cascades including PKA and PKC (8,21,24,27,39). PAC 1 receptor activation can stimulate both the adenylyl cyclase/cAMP/PKA and PLC/DAG/ PKC transduction pathways (4,54) and through PAC 1 receptor internalization/endosome formation can recruit the MEK/ERK signaling cascade (33). Although MAPK phosphorylation of Ca v has not been identified (23), modulation of T-type calcium function and expression requiring activation of MEK/ERK signaling has been demonstrated in a variety of cell types (11,16,38).…”

Section: Discussionmentioning

confidence: 99%

Nickel suppresses the PACAP-induced increase in guinea pig cardiac neuron excitability

Tompkins

Merriam

Girard

et al. 2015

American Journal of Physiology-Cell Physiology

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Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent intercellular signaling molecule involved in multiple homeostatic functions. PACAP/PAC1 receptor signaling increases excitability of neurons within the guinea pig cardiac ganglia, making them a unique system to establish mechanisms underlying PACAP modulation of neuronal function. Calcium influx is required for the PACAP-increased cardiac neuron excitability, although the pathway is unknown. This study tested whether PACAP enhancement of calcium influx through either T-type or R-type channels contributed to the modulation of excitability. Real-time quantitative polymerase chain reaction analyses indicated transcripts for Cav3.1, Cav3.2, and Cav3.3 T-type isoforms and R-type Cav2.3 in cardiac neurons. These neurons often exhibit a hyperpolarization-induced rebound depolarization that remains when cesium is present to block hyperpolarization-activated nonselective cationic currents (Ih). The T-type calcium channel inhibitors, nickel (Ni(2+)) or mibefradil, suppressed the rebound depolarization, and treatment with both drugs hyperpolarized cardiac neurons by 2-4 mV. Together, these results are consistent with the presence of functional T-type channels, potentially along with R-type channels, in these cardiac neurons. Fifty micromolar Ni(2+), a concentration that suppresses currents in both T-type and R-type channels, blunted the PACAP-initiated increase in excitability. Ni(2+) also blunted PACAP enhancement of the hyperpolarization-induced rebound depolarization and reversed the PACAP-mediated increase in excitability, after being initiated, in a subset of cells. Lastly, low voltage-activated currents, measured under perforated patch whole cell recording conditions and potentially flowing through T-type or R-type channels, were enhanced by PACAP. Together, our results suggest that a PACAP-enhanced, Ni(2+)-sensitive current contributes to PACAP-induced modulation of neuronal excitability.

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Section: Discussionmentioning

confidence: 99%

Nickel suppresses the PACAP-induced increase in guinea pig cardiac neuron excitability

Tompkins

Merriam

Girard

et al. 2015

American Journal of Physiology-Cell Physiology

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“…All experiments were performed using cultured HEK 293 cells stably expressing the GFP-tagged PAC1Hop1 receptor as described previously (May et al, 2010; May et al, 2014). HEK 293 cells were transfected using Mirus TransIT-LT1 transfection reagent (Mirus Bio, Madison, WI) and cultured in Dulbecco’s modified Eagle’s medium/F-12 containing 8% fetal bovine serum and 500 μg/ml Geneticin for stable cell selection.…”

Section: Methodsmentioning

confidence: 99%

“…Control and treated cultures were extracted as previously described (May et al, 2010; May et al, 2014) with 75 μl RIPA buffer (50 mM Tris-HCl, pH 8.0, 120 mM NaCl, 5 mM EDTA, 1% NP-40, 0.1% SDS) containing 0.3 mg/ml phenylmethylsulfonylfluoride, protease inhibitors (16 μg/ml benzamidine, 2 μg/ml leupeptin, 50 μg/ml lima bean trypsin inhibitor, 2 μg/ml pepstatin A) and phosphatase inhibitor mix (5 mM EDTA, 5 mM EGTA, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 50 mM sodium fluoride) (May et al, 2002; Girard et al, 2004; May et al, 2010). Total sample proteins were determined using the Coomassie Plus reagent (Pierce Biotechnology, Rockford, IL).…”

Section: Methodsmentioning

confidence: 99%

“…The calcium-sensitive dye fura-2AM (Life Technologies Corporation, Carlsbad, CA) was used to measure PACAP-induced changes in intracellular Ca 2+ ([Ca 2+ ] i ) (May et al, 2014). The cells were maintained in either the Ca 2+ -containing or the Ca 2+ -deficient physiological solution, incubated in 4 μM fura-2AM with 0.08% pluronic acid (Life Technologies Corporation, Carlsbad, CA) for 30 min at room temperature, and washed in dye-free physiological solution for ~30 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…Using a stably expressing GFP-tagged PAC1 Hop1 receptor HEK cell line, we have shown recently that PACAP/PAC1 receptor signaling activates ERK through two parallel, temperature-dependent pathways (May et al, 2014). One mechanism involves internalization of the PACAP/PAC1 complex and the resulting formation of signaling endosomes for scaffold recruitment of adaptor proteins important for MEK/ERK signal transduction.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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In HEK cells expressing GFP-tagged PAC1Hop1 receptors, PACAP augments ERK phosphorylation through two parallel pathways; one through PACAP/PAC1 receptor internalization/endosome MEK/ERK signaling, the other through PLC/DAG/PKC activation. We examined whether elevation of intracellular calcium ([Ca2+]i) was required for either of the PACAP/PAC1 receptor-mediated ERK activation mechanisms. The PACAP (25 nM)-induced elevation of [Ca2+]i was greater with cells maintained in Ca2+-containing than in Ca2+-deficient solution, suggesting that both calcium release from internal stores and calcium influx contributed to the rise in [Ca2+]i. A thapsigargin-induced increase in [Ca2+]i also was greater with calcium in the external solution. OAG, the cell permeable analogue of DAG, increased [Ca2+]i, but only in Ca2+-containing solution. Decreasing external calcium or depleting internal calcium stores did not block PACAP-induced PAC1 receptor internalization. Omission of calcium from the external solution, but not thapsigargin pretreatment, significantly blunted PACAP-stimulated ERK phosphorylation. The PKC inhibitor BimI decreased PACAP-mediated ERK activation in both Ca2+-containing or Ca2+-deficient solutions. In contrast, following Pitstop 2 pretreatment to block endocytic mechanisms, PACAP activated ERK only when calcium was present in the external solution. We conclude that the endosome signaling pathway is largely calcium-independent whereas calcium influx appears necessary for the PLC/DAG/PKC component of PACAP-induced ERK activation.

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G Protein‐Coupled Receptor Endosomal Signaling and Regulation of Neuronal Excitability and Stress Responses: Signaling Options and Lessons From the PAC1 Receptor

May

Parsons

2016

Journal Cellular Physiology

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Our understanding of G protein coupled receptor (GPCR) mechanisms and functions have evolved considerably. Among the many conceptual realignments, GPCRs can exist in an ensemble of active microstates that have the potential to differentially engage specific downstream signaling events. Furthermore, among GPCR dynamics, GPCR internalization and vesicular trafficking are no longer solely mechanisms for desensitization, but now appreciated to form intricate endosomal signaling complexes that can potentially target second messengers to intracellular compartments with high temporal and spatial resolution. The PACAPergic system is important in the maintenance of physiological homeostasis in the central and peripheral nervous systems and activation of the PACAP-selective PAC1 receptor can generate differential but coordinate plasma membrane and endosomal signals for cellular responses. The integration of these signals can modulate PACAP-induced changes in ionic conductances that gate neuronal excitability. PACAP/PAC1 receptor generation of endosomal ERK signals participate in chronic pain and anxiety-like responses which can be attenuated with endocytosis inhibitors. From the abilities of ligands to stabilize the different GPCR microstates for biased downstream signaling, the development of biased PAC1 receptor agonists and antagonists may provide opportunities to dissociate the homeostatic regulatory signals of PACAP from the maladaptive effects. In particular, the development of biased antagonists to PAC1 receptor-mediated endosomal signaling may offer therapeutic options for chronic pain and stress-related disorders. J. Cell. Physiol. 232: 698-706, 2017. © 2016 Wiley Periodicals, Inc.

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

Cited by 50 publications

References 51 publications

Nickel suppresses the PACAP-induced increase in guinea pig cardiac neuron excitability

Nickel suppresses the PACAP-induced increase in guinea pig cardiac neuron excitability

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

G Protein‐Coupled Receptor Endosomal Signaling and Regulation of Neuronal Excitability and Stress Responses: Signaling Options and Lessons From the PAC1 Receptor

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