NIS is basolateraly sorted by AP-1A and AP-1B clathrin adaptors in MDCK cells

Abstract

Sodium Iodide Symporter (NIS) is a transmembrame protein located in the basal membrane of thyrocytes through a mechanism that is regulated by the thyroid stimulating hormone (TSH) at transcriptional and posttranscriptional levels. Despite the physiological and clinical relevance of NIS membrane localization for thyroid hormone biosynthesis and cancer treatment respectively, little is known concerning the molecular mechanisms and the traffic routes implicated in NIS basolateral sorting and plasma membrane targeting. For this reason, we decided to study NIS basolateral traffic in the rat follicular thyroid PCCl3 cells and in the MDCK-human NIS stably transfected cell line (MDCKhNIS). First, we performed time course kinetics of NIS plasma membrane targeting and intracellular compartment co-localization analysis after TSH stimulation in PCCl3 cells. Through membrane biotinylation we determined that NIS reaches the plasma membrane in 24 hours post-stimulation. By confocal microscopy we observed that NIS after exiting the ER passes through the Golgi apparatus where co-localizes with g-adaptin. This protein consists in a subunit of the clathrin adaptor proteins AP-1A and AP-1B that sort basolateral proteins at the trans-Golgi network and at common recycling endosomes, respectively. In order to characterize the role of AP-1 proteins in NIS basolateral traffic we used MDCKhNIS cells depleted for AP-1A, AP-1B or both, and we evaluated NIS polarized distribution through confocal microscopy and domain-selective biotinylation. We observed that knockdown of AP-1B caused a loss of NIS basolateral polarity compared to that in wildtype cells. In contrast, NIS was normally distributed after depletion of AP-1A. Strikingly, the decrease in NIS basolateral localization was more prominent in the case of the double knockdown than in AP-1B knockdown cells, indicating that AP-1A also participates in NIS basolateral sorting. In conclusion, both AP-1A and AP-1B mediate NIS basolateral sorting; nevertheless, the interaction between NIS and the clathrin-coated machinery is still under study.