Date of Completion
College of Arts and Science Honors
Sayamwong Hammack, PhD.
Victor May, PhD.
PACAP, VPAC receptors, Extracellular signal-dependent kinase (ERK), Receptor endocytosis, Protein Kinase A (PKA), Protein Kinase C (PKC)
Pituitary adenylate-cyclase activating polypeptide (PACAP) is highly conserved signaling molecule in eukaryotes known to regulate a myriad of metabolic processes within the brain as well as the body (Sherwood, Krueckl, & McRory, 2000; Vaudry et al., 2009). The mechanism of action underlying PACAP signaling is known to occur through the PAC1, VPAC1 and VPAC2 G protein-coupled receptors (GPCRs) to initiate signaling cascades that can phosphorylate extracellular signal-related kinase (ERK) proteins to influence many intracellular events including gene expression, long-term potentiation, or induce growth factors, all of which are a specific response to extracellular events (Chang, & Karin, 2001). In the current study, HEK cells expressing VPAC1 or VPAC2 receptors were exposed to PACAP27 (P27) to induce receptor activation and ERK phosphorylation (pERK). The VPAC expressing cells were treated with P27 for varying durations to assess the temporal changes in pERK; further, the cells were pre-treated with signaling pathway inhibitors to determine the predominant mechanisms of P27-mediated ERK phosphorylation. From previous work, it was hypothesized that PACAP-mediated ERK activation of VPAC-type receptors would be elicited via receptor endocytosis and the PKA pathway. Experiments were conducted in triplicate, and the pathway inhibitors included two receptor endocytosis blockers (Pitstop2 and Dynasore), a PKA inhibitor (KT5720), and a PKC inhibitor (Bim1). Relative expression of ERK1/2 and pERK proteins were visualized and compared by quantitative Western analyses. Unlike, PAC1 receptors, PACAP activation of VPAC receptors resulted in transient ERK activation. Contrary to expectations, the experimental results also showed that receptor endocytosis blocker Pitstop2 and PKC inhibitor Bim1 reduced ERK phosphorylation. These inhibitor results are similar to previous experiments conducted on HEK-expressing PAC1 receptors treated with the same pathway inhibitors (May, Buttolph, Girard, Clason, & Parsons, 2014), providing further sight into how PACAP-mediated mechanisms of ERK phosphorylation may be implicated physiological conditions.
Linden, Anne K., "PACAP-induced ERK phosphorylation in VPAC1 and VPAC2-expressing HEK cells is mediated by receptor endocytosis and PKC signaling" (2017). UVM College of Arts and Sciences College Honors Theses. 45.
Available for download on Monday, May 11, 2020