Multiple sequences orchestrate subcellular trafficking of neuronal PAS domain-containing protein 4 (NPAS4).

Neuronal Per-Arnt-Sim (PAS) domain-containing protein 4 (NPAS4) is a basic helix-loop-helix (bHLH)-PAS transcription factor first discovered in neurons in the neuronal layer of the mammalian hippocampus and later discovered in pancreatic β-cells. NPAS4 has been proposed as a therapeutic target not only for depression and neurodegenerative diseases associated with synaptic dysfunction but also for type 2 diabetes and pancreas transplantation. The ability of bHLH-PAS proteins to fulfil their function depends on their intracellular trafficking, which is regulated by specific sequences, i.e. the nuclear localization signal (NLS) and the nuclear export signal (NES). However, until now, no study examining the subcellular localization signals of NPAS4 has been published. We show here that Rattus norvegicus NPAS4 was not uniformly localized in the nuclei of COS-7 and N2a cells 24 h after transfection. Additionally, cytoplasmic localization of NPAS4 was leptomycin B-sensitive. We demonstrate that NPAS4 possesses a unique arrangement of localization signals. Its bHLH domain contains an overlapping NLS and NES. We observed that its PAS-2 domain contains an NLS, an NES, and a second, proximally located, putative NLS. Moreover, the C terminus of NPAS4 contains two active NESs that overlap with a putative NLS. Our data indicate that glucose concentration could be one of the factors influencing NPAS4 localization. The presence of multiple localization signals and the differentiated localization of NPAS4 suggest a precise, multifactor-dependent regulation of NPAS4 trafficking, potentially crucial for its ability to act as a cellular stress sensor and transcription factor.