Tracellular N- and C-terminal tails, 2 ProDom domains I11-L161 (N-PD1131) and V492-V640 (C-PD1131) situated in the N-terminal and C-terminal of PiT2 respectively14,15. Corresponding towards the protein functions of PiT2, loop regions in PD domain, for example 671, 10741, 51730 amino acid residues are necessary for amphotropic murine Ninhydrin Autophagy leukemia virus (A-MuLV) binding16,17, and PD domains also play a crucial role in sustaining transport function18. In IBGC families, 23 missense variants happen to be identified in SLC20A2, and these missense variants are mainly located in two PD domains of PiT219. The PiT2 also consists of a 246-aa (about 38 % amino acids of PiT2) big intracellular loop7 domain in between N-PD1131 and C-PD113120. B tger and Pedersen had reported that the PiT2 with deleted loop7 had typical retroviral recognition, and transport functions15. So far, there is certainly no definite proof that missense variants in loop7 impact the transport function of PiT2 which result in IBGC19. Consequently, it remains an intriguing question concerning the function of loop7 domain inside the nervous system.Important Laboratory of Molecular Biophysics on the Ministry of Education, Center for Human Genome Research, College of Life Science and Technology, Huazhong 7α-Hydroxy-4-cholesten-3-one Metabolic Enzyme/Protease University of Science and Technology (HUST), Wuhan, 430074, China. 2 College of Life Sciences, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei University, Wuhan, 430062, China. 3Department of Neurology, Xiangya Hospital, Central South University, Changsha, Hunan, 410008, China. 4College of Life Sciences, Northeast Regular University, Changchun, 130024, China. Xi-Xiang Ma, Xiangyang Li and Ping Yi contributed equally to this work. Correspondence and requests for supplies should be addressed to S.J. (e mail: [email protected]) or J.-Y.L. (e mail: [email protected])Received: 27 February 2017 Accepted: 4 December 2017 Published: xx xx xxxxSCIENTIfIC RepoRts | (2017) 7:17850 | DOI:10.1038s41598-017-17953-www.nature.comscientificreportsFigure 1. The loop7 domain of PiT2 impacts neurite outgrowth in Neuro2A cells. (a,b) Differentiated Neuro2A cells stained with anti-HAAlexa Flour488 (green) and DAPI (blue) with overexpression of HA-tagged wild sort PiT2 (a) or HA-tagged PiT2-loop7 (b). Scale bar, 20 m. (c) Neuro2A cells were transiently transfected with pSIH-PiT2 or pSIH-scramble. Cell lysates were immunoblotted with anti-PiT2 and anti-actin antibodies. Full length blots are shown in Supplementary Fig. S2. (d,e) Differentiated Neuro2A cells stained with DAPI (blue) with transfection of pSIH-scramble (d) or pSIH-PiT2 (e). Scale bar, 20 m. (f) Quantification of neurite length of differentiated Neuro2A cells transfected with wild variety PiT2 or PiT2-loop7 plasmids. (g) Average length from the longest neurite of Neuro2A cells transfected with scramble and shRNA-PiT2 were statistically analyzed. Error bars show the mean s.e.m. of one hundred randomly chosen cells from every single group in 3 independent experiments. suggests P 0.001.To investigate achievable functions of loop7 domain of PiT2 inside the nervous method, we performed immunofluorescence assays of Neuro2A cells transfected with PiT2 that lack loop7, and discovered that loop7 deletion affected the subcellular localization of PiT2 protein and neurite outgrowth in Neuro2A cells. To reveal the function of loop7 in PiT2, we performed yeast two-hybrid screening and identified microtubule-associated protein 1B (MAP1B) as a novel interactor of loop7 in PiT2. MAP1B i.
