Direct involvement of G protein alpha(q/11) subunit in regulation of muscarinic receptor-mediated sAPP alpha release

Abstract

The G(q/11) protein-coupled receptors, such as muscarinic (M1 & M3) receptors, have been shown to regulate the release of a soluble amyloid precursor protein (sAPP alpha) produced from a-secretase processing. However, there is no direct evidence for the precise characteristics of G proteins, and the signaling mechanism for the regulation of G(q/11) protein-coupled receptor-mediated sAPP alpha release is not clearly understood. This study examined whether the muscarinic receptor-mediated release of sAPP alpha is directly regulated by G alpha(q/11) proteins. The HEK293 cells were transiently cotransfected with muscarinic M3 receptors and a dominant-negative minigene construct of the G protein alpha subunit. The sAPP alpha release in the media was measured using an antibody specific for sAPP. The sAPP alpha release enhancement induced by muscarinic receptor stimulation was decreased by a G(q/11) minigene construct, whereas it was not blocked by a control minigene construct (the G alpha carboxy peptide in random order, G alpha(q)R) or G alpha constructs. This indicated a direct role of the G alpha(q/11) protein in the regulation of muscarinic M3 receptor-mediated sAPP alpha release. We also investigated whether the transactivation of the epidermal growth factor receptor (EGFR) by a muscarinic agonist could regulate the sAPP alpha release in SH-SY5Y cells. Pretreatment of a specific EGFR kinase inhibitor, tyrophostin AG1478 (250 nM), blocked the EGIF-stimulated sAPP alpha release, but did not block the oxoM-stimulated sAPP alpha release. This demonstrated that the transactivation of the EGFR by muscarinic receptor activation was not involved in the muscarinic receptor-mediated sAPP alpha release.