Visualization of the binding between gintonin, a Panax ginseng-derived LPA receptor ligand, and the LPA receptor subtypes and transactivation of the EGF receptor

Abstract

Gintonin is a ginseng-derived exogenous G-protein-coupled lysophosphatidic acid (LPA)receptor ligand. Gintonin exerts its neuronal and non-neuronal in vitro and in vivo effects through LPAreceptor subtypes. However, it is unknown whether gintonin can bind to the plasma membrane of cellsand can transactivate the epidermal growth factor (EGF) receptor. In the present study, we examinedwhether gintonin-biotin conjugates directly bound to LPA receptors and transactivated the EGF receptor. Methods: We designed gintonin-biotin conjugates through gintonin biotinylation and examined whethergintonin-biotin conjugate binding sites co-localized with the LPA receptor subtype binding sites. Wefurther examined whether gintonin-biotin transactivated the EGF receptor via LPA receptor regulationvia phosphor-EGF and cell migration assays. Results: Gintonin-biotin conjugates elicit [Ca2þ]i transient similar to that observed with unbiotinylatedgintonin in cultured PC3 cells, suggesting that biotinylation does not affect physiological activity ofgintonin. We proved that gintonin-biotin conjugate binding sites co-localized with the LPA1/6 receptorbinding sites. Gintonin-biotin binding to the LPA1 receptor transactivates the epidermal growth factor(EGF) receptor through phosphorylation, while the LPA1/3 receptor antagonist, Ki16425, blocked phos phorylation of the EGF receptor. Additionally, an EGF receptor inhibitor AG1478 blocked gintonin-biotinconjugate-mediated cell migration. Conclusions: We observed the binding between ginseng-derived gintonin and the plasma membranetarget proteins corresponding to the LPA1/6 receptor subtypes. Moreover, gintonin transactivated EGFreceptors via LPA receptor regulation. Our results suggest that gintonin directly binds to the LPA receptorsubtypes and transactivates the EGF receptor. It may explain the molecular basis of ginseng physiology/pharmacology in biological systems.