Abstract 897: Role of intracellular loops 1 and 3 in folding and cell surface expression of human P-glycoprotein (ABCB1).

P-glycoprotein (ABCB1) is an ATP-Binding Cassette (ABC) transporter that effluxes a variety of structurally diverse compounds including anticancer drugs out of the cell. Known crystal structures of ABC transporters show that the transmembrane domain (TMD) is connected to the nucleotide-binding domain (NBD) through a ball and socket joint. Also, the intracellular loops (ICLs) 1 and 3 are shown to be in close proximity to the NBDs, creating an extensive interaction surface between the TMDs and NBDs. Computational models of human P-gp in the apo and nucleotide-bound conformation have been developed based on structures of mouse mdr1a, C. elegans P-gp, Sav1866 and MsbA transporters. In these models, the adenine group of ATP forms hydrogen bonds with conserved D164 and D805 on ICLs 1 and 3, respectively, which are located at the interface between the NBDs and TMDs. We investigated the role of D164 and D805 by substituting these residues with cysteine in a cys-less background. The D164C/D805C mutant P-gp was expressed in High-Five insect cells as well as in HeLa cells using the BacMam baculovirus expression system. In the insect cells grown at 27°C, the expression of the mutant P-gp was comparable to that of cysless-WT protein and exhibited the same ATP-binding affinity but a lower V max for ATP hydrolysis. However, when the mutant protein was expressed in HeLa cells at 37°C, we found that the D164C/D805C P-gp was mislocalized and failed to transport almost all tested drug substrates. HeLa cells transduced with D164C/D805C mutant baculovirus were grown for 16-18 hrs in the presence of cyclosporine A (CsA), a substrate that is also known to function as a chemical chaperone. This treatment resulted in expression of mutant P-gp at the cell surface to the same extent as that of cysless-WT protein; similarly, the transport was also restored to the level of cysless-WT P-gp. A partial rescue of the mutant P-gp expression and function was observed in the presence of the CFTR correctors VRT-325 and compound-4a. In addition, when BacMam baculovirus-transduced HeLa cells were cultured at 27°C for 40 hrs, the mutant protein rescued to the cell surface with transport activity to the same extent as cysless-WT P-gp. Studies of each mutant form separately revealed that the D164C mutant had intermediate cell surface expression and was rescued completely to the plasma membrane in the presence of CsA. On the other hand, the expression of D805C at the cell surface was drastically decreased and even when HeLa cells were grown in the presence of CsA, only 10-15% of mutant protein was expressed at the cell surface compared to cysless-WT protein. These findings in aggregate illustrate the role of residues in ICLs 1 and 3 in the proper folding of P-gp and specifically show that the D164 and D805 residues are not critical for ATP binding or hydrolysis as proposed earlier, but are instead important for inter-domain interactions and assembly of a functional transporter. Citation Format: Khyati Kapoor, Jaya Bhatnagar, Eduardo E. Chufan, Suresh V. Ambudkar. Role of intracellular loops 1 and 3 in folding and cell surface expression of human P-glycoprotein (ABCB1). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 897. doi:10.1158/1538-7445.AM2013-897