Calcium influx, but not intracellular calcium release, supports PACAP-mediated ERK activation in HEK PAC1 receptor cells.
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 and the other through PLC/DAG/PKC activation. We examined whether elevation of intracellular calcium ([Ca(2+)]i) was required for either of the PACAP/PAC1 receptor-mediated ERK activation mechanisms. The PACAP (25 nM)-induced elevation of [Ca(2+)]i was greater with cells maintained in Ca(2+)-containing than in Ca(2+)-deficient solution, suggesting that both calcium release from internal stores and calcium influx contributed to the rise in [Ca(2+)]i. A thapsigargin-induced increase in [Ca(2+)]i also was greater with calcium in the external solution. OAG, the cell permeable analogue of DAG, increased [Ca(2+)]i, but only in Ca(2+)-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 Ca(2+)-containing or Ca(2+)-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.
Department of Neurological Sciences, College of Medicine, University of Vermont, Burlington, VT, 05405, USA.
Victor
V
May
Todd A
TA
Clason
Thomas R
TR
Buttolph
Beatrice M
BM
Girard
Rodney L
RL
Parsons
D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D020935:MAP Kinase Signaling System; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide / Q000378:metabolism*; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I / Q000378:metabolism*
D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D048049:Extracellular Signal-Regulated MAP Kinases; D002118:Calcium