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

Sun-Hye Choi; Ra Mi Lee; Han-Sung Cho; Sung Hee Hwang; Hong-Ik Hwang; Hyewhon Rhim; Hyoung-Chun Kim; Do-Geun Kim; Ik-Hyun Cho; Seung-Yeol Nah

doi:10.1016/j.jgr.2021.10.004

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

J Ginseng Res. 2022 May;46(3):348-356. doi: 10.1016/j.jgr.2021.10.004. Epub 2021 Oct 15.

Authors

Affiliations

¹ Ginsentology Research Laboratory and Department of Physiology, College of Veterinary Medicine, Konkuk University, Seoul, Republic of Korea.
² Department of Pharmaceutical Engineering, College of Health Sciences, Sangji University, Wonju, Republic of Korea.
³ Center for Neuroscience, Korea Institute of Science and Technology, Seoul, Republic of Korea.
⁴ Neuropsychopharmacology and Toxicology Program, College of Pharmacy, Kangwon National University, Chunchon, Republic of Korea.
⁵ Dementia Research Group, Korea Brain Research Institute, Daegu, Republic of Korea.
⁶ Department of Science in Korean Medicine, Brain Korea 21 Plus Program, Department of Conversions Medical Science, Graduate School, Kyung Hee University, Seoul, Republic of Korea.

Abstract

Background: 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 LPA receptor subtypes. However, it is unknown whether gintonin can bind to the plasma membrane of cells and can transactivate the epidermal growth factor (EGF) receptor. In the present study, we examined whether gintonin-biotin conjugates directly bound to LPA receptors and transactivated the EGF receptor.

Methods: We designed gintonin-biotin conjugates through gintonin biotinylation and examined whether gintonin-biotin conjugate binding sites co-localized with the LPA receptor subtype binding sites. We further examined whether gintonin-biotin transactivated the EGF receptor via LPA receptor regulation via phosphor-EGF and cell migration assays.

Results: Gintonin-biotin conjugates elicit [Ca²⁺]_i transient similar to that observed with unbiotinylated gintonin in cultured PC3 cells, suggesting that biotinylation does not affect physiological activity of gintonin. We proved that gintonin-biotin conjugate binding sites co-localized with the LPA1/6 receptor binding 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 phosphorylation of the EGF receptor. Additionally, an EGF receptor inhibitor AG1478 blocked gintonin-biotin conjugate-mediated cell migration.

Conclusions: We observed the binding between ginseng-derived gintonin and the plasma membrane target proteins corresponding to the LPA1/6 receptor subtypes. Moreover, gintonin transactivated EGF receptors via LPA receptor regulation. Our results suggest that gintonin directly binds to the LPA receptor subtypes and transactivates the EGF receptor. It may explain the molecular basis of ginseng physiology/pharmacology in biological systems.

Keywords: EGF receptor; Gintonin; Gintonin-biotin conjugates; LPA receptor co-localization; Transactivation.