Merriam, L.A, Baran, C.N. ('08), Girard, B., Hardwick, J.C., May, V., and Parsons, R.L. (2013). PAC1 receptor internalization and endosomal signaling mediate the PACAP-induced increase in guinea pig neuron excitability. Journal of Neuroscience, 6 March 2013, 33(10): 4614-4622. View the article.
Following G protein-coupled receptor activation and signaling at the plasma membrane, the receptor complex often is rapidly internalized via endocytic vesicles for trafficking into various intracellular compartments and pathways. Increasingly, the translocation of the receptor complex into signaling endosomes is recognized to be a key mechanism to produce refined and sustained intracellular signals, which may be distinct from those generated at the cell surface, for many cellular responses including growth, differentiation and survival. Pituitary adenylate cyclase activating polypeptide (PACAP; Adcyap1) is a potent neurotransmitter/neurotrophic peptide and mediates its diverse cellular functions in part through internalization of its cognate G protein-coupled PAC1 receptor (Adcyap1r1). In the current studies, we examined whether PAC1 receptor endocytosis participates in regulation of neuronal excitability. While PACAP increased excitability in 90% of guinea pig cardiac neurons, whole mount preparation pretreatment with Pitstop 2 or dynasore to inhibit clathrin and dynaminI/II, respectively, suppressed the PACAP-induced increase in excitability. Further, after the PACAP- induced action potential generation had increased, subsequent addition of inhibitor gradually attenuated excitability with no apparent changes in action potential properties. Likewise, the PACAP-induced neuronal excitability was markedly decreased at ambient temperature. Receptor trafficking studies with GFP-PAC1 cell lines demonstrated the efficacy of Pitstop 2 and dynasore and low temperature to suppress PAC1 receptor endocytosis. In contrast, brefeldin A pretreatments to disrupt Golgi vesicle trafficking had no effects on PACAP-induced excitability, and PACAP/PAC1 receptor signaling still maximally increased neuronal cAMP production even with endocytic blockade. In aggregate, these studies demonstrate that PACAP/PAC1 receptor complex endocytosis is a key step for the PACAP modulation of cardiac neuron excitability.