Neurotensin-induced miR-133α expression regulates neurotensin receptor 1 recycling through its downstream target aftiphilin

Neurotensin (NT) triggers signaling in human colonic epithelial cells by activating the G protein-coupled receptor, the neurotensin receptor 1 (NTR1). Activated NTR1 traffics from the plasma membrane to early endosomes, and then recycles. Although sustained NT/NTR1 signaling requires efficient NTR1 recycling, little is known about the regulation of NTR1 recycling. We recently showed that NT/NTR1 signaling increases expression of miR-133 alpha. Herein, we studied the mechanism of NT-regulated miR-133 alpha expression and examined the role of miR-133a in intracellular NTR1 trafficking in human NCM460 colonocytes. We found that NT-induced miR-133 alpha upregulation involves the negative transcription regulator, zinc finger E-box binding homeobox 1. Silencing of miR-133 alpha or overexpression of aftiphilin (AFTPH), a binding target of miR-133 alpha, attenuated NTR1 trafficking to plasma membrane in human colonocytes, without affecting NTR1 internalization. We localized AFTPH to early endosomes and the trans-Golgi network (TGN) in unstimulated human colonic epithelial cells. AFTPH overexpression reduced NTR1 localization in early endosomes and increased expression of proteins related to endosomes and the TGN trafficking pathway. AFTPH overexpression and de-acidification of intracellular vesicles increased NTR1 expression. Our results suggest a novel mechanism of GPCR trafficking in human colonic epithelial cells by which a microRNA, miR-133 alpha regulates NTR1 trafficking through its downstream target AFTPH.