Investigations into the Plasma Membrane Trafficking of Multidrug Resistance Protein 4 (ABCC4/MRP4)

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Date
2015-09-10
Authors
Miah, Mohammad Fahad
Keyword
Multidrug Resistance Protein 4 , Membrane Protein Trafficking
Abstract
Multidrug resistance protein 4 (MRP4) is a member of subfamily C of the ATP-binding cassette superfamily of membrane transport proteins. In polarized cells, MRP4 localizes to the apical or basolateral plasma membrane depending on the tissue type. To gain insight into mechanisms regulating MRP4 plasma membrane trafficking, its interactions with the PDZ domain-containing CortBP1, a member of Shank2 family of adapter proteins, were investigated. Since CortBP1 is known to recruit proteins involved in endocytosis, it was hypothesized that CortBP1 plays a role in MRP4 internalization from the plasma membrane. Ectopic expression of CortBP1 led to decreased total MRP4 levels in HEK293T cells and cell surface biotinylation experiments and confocal microscopy showed that this decrease was at the plasma membrane. Pull-down experiments indicated that the interaction between CortBP1 and MRP4 requires the last four amino acids 1322ETAL of the transporter. They also suggested that MRP4 may exist in a complex with CortBP1 and endocytic proteins. In SH-SY5Y and BE(2)-C human neuroblastoma cells, RNAi-mediated knockdown of endogenous SHANK2 isoforms (including CORTBP1) resulted in increased total MRP4 levels. Confocal microscopy of SHANK2-depleted BE(2)-C cells suggested that this increase occurred at the plasma membrane. These observations demonstrate that the internalization of plasma membrane MRP4 is regulated by CortBP1, and possibly other SHANK2 family members. In addition to PDZ domain-containing adapter proteins, N-glycans can regulate the apical membrane localization of some proteins. Whether this is true for MRP4 is unknown. Based on in silico analyses and topological models of human MRP4, it was hypothesized that it is glycosylated at Asn746 and Asn754. Single substitutions of these Asn residues with Gln (N746Q, N754Q) increased the electrophoretic mobility of MRP4; double substitutions (N746/754Q) increased its mobility further such that it was comparable to wild-type MRP4 deglycosylated with PNGase F. Importantly, confocal microscopy indicated that the apical membrane localization of the single and double Asn mutants in polarized LLC-PK1 cells was comparable to wild-type MRP4. This suggests that N-glycans are not required for apical membrane localization of MRP4, at least in LLC-PK1 cells. Overall, the results of these studies provide novel insights into the membrane trafficking of MRP4.
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