Computational protocol: Muscarinic receptors stimulate AC2 by novel phosphorylation sites, whereas Gβγ subunits exert opposing effects depending on the G-protein source

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Protocol publication

[…] Epac2-camps was from Dr Martin Lohse (University of Würzburg, Würzburg, Germany) [,]. Rat HA–M3 muscarinic receptor was kindly provided by Dr Jürgen Wess [NIDDK (National Institute of Diabetes and Digestive and Kidney Diseases), Bethesda, MD, U.S.A.). Gtα (transducin) was a gift from Professor Heidi Hamm (Vanderbilt University, Nashville, TN, U.S.A.). Wild-type rat AC2 was subcloned into pcDNA3 vector with an N-terminal HA tag. The potential PKC phosphorylation sites of AC2 were identified using NetPhos 2.0 [], GPS 2.1 [], PPSP 1.0 [] and PhoScan []. Any potential PKC phosphorylation site residues in AC2 were substituted to alanine or aspartic acid residues using the Phusion® high-fidelity polymerase kit (Finnzymes) following the manufacturer's instructions, and by site-specific mutagenesis according to the QuikChange protocol (Stratagene). All of the AC2 constructs were verified by DNA sequencing. […]

Pipeline specifications

Software tools NetPhos, PPSP, PhoScan
Application PTM analysis
Diseases Protein C Deficiency
Chemicals Cyclic AMP, Carbachol, Somatostatin