Neuropilin-1 is a glial cell line-derived neurotrophic factor receptor in glioblastoma

// Shen Sun 1, 2, 3, * , Yu Lei 2, 4, * , Qi Li 2 , Yue Wu 2 , Lin Zhang 2 , Pei-Pei Mu 2 , Guang-Quan Ji 2 , Chuan-Xi Tang 2 , Yu-Qian Wang 2 , Jian Gao 5 , Jin Gao 2 , Li Li 6 , Lang Zhuo 7 , Yun-Qing Li 1 and Dian-Shuai Gao 1, 2 1 Department of Anatomy and Histology, The Fourth Military Medical University, Xi’an, Shanxi, China 2 Department of Neurobiology and Anatomy, Xuzhou Key Laboratory of Neurobiology, Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, Xuzhou, Jiangsu, China 3 Department of Histology and Embryology, Xuzhou Medical University, Xuzhou, Jiangsu, China 4 Department of Neurobiology, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Shanghai, China 5 Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, Xuzhou, Jiangsu, China 6 Department of Pathophysiology, Xuzhou Medical University, Xuzhou, Jiangsu, China 7 Department of Epidemiology, School of Public Health, Xuzhou Medical University, Xuzhou, Jiangsu, China * These authors have contributed equally to this work Correspondence to: Dian-Shuai Gao, email: gds@xzhmu.edu.cn Keywords: glial cell line-derived neurotrophic factor, glioblastoma, membrane receptor, neuropilin-1, cell proliferation Received: February 24, 2017      Accepted: May 12, 2017      Published: June 27, 2017 ABSTRACT The aim of this study was to identify the receptor for glial cell line-derived neurotrophic factor (GDNF) in glioblastoma multiforme (GBM). After GST pull-down assays, membrane proteins purified from C6 rat glioma cells were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS). The differentially expressed proteins were annotated using Gene Ontology, and neuropilin-1 (NRP1) was identified as the putative GDNF receptor in glioma. NRP1 was more highly expressed in human GBM brains and C6 rat glioma cells than in normal human brains or primary rat astrocytes. Immunofluorescence staining showed that NRP1 was recruited to the membrane by GDNF, and NRP1 co-immunoprecipitated with GDNF. Using the NRP1 and GDNF protein structures to assess molecular docking in the ZDOCK server and visualization with the PyMOL Molecular Graphics System revealed 8 H-bonds and stable positive and negative electrostatic interactions between NRP1 and GDNF. RNAi knockdown of NRP1 reduced proliferation of C6 glioma cells when stimulated with GDNF. NRP1 was an independent risk factor for both survival and recurrence in GBM patients. High NRP1 mRNA expression correlated with shorter OS and DFS (OS: χ 2 =4.6720, P =0.0307; DFS: χ 2 =11.013, P=0.0009). NRP1 is thus a GDNF receptor in glioma cells and a potential therapeutic target.