Evidence for the Locations of Distinct Steroid and Vinca Alkaloid Interaction Domains within the Murine mdr1b P-Glycoprotein

Abstract

P-glycoproteins (P-gp) cause the efflux of a wide variety of unrelated hydrophobic compounds out of cells. However, the locations of the sites at which different classes of molecules initially interact with the protein are not well defined. A unique system was developed to search for P-gp drug-interaction domains using mutational analysis. The strategy is based upon identifying mutations that cause a decrease in the activity of P-gp inhibitors, which are structurally related to chemotherapeutic drugs transported by P-gps. Evidence of distinct steroid and taxane interaction domains has already been presented. The work reported here extends the study of the steroid interaction domain and presents evidence for a separate vinblastine interaction domain. A total of 10 steroid-related mutations, involving seven amino acids that are confined within transmembrane segments (TMS) 4 to 6, have been characterized. The location of these mutations indicates that steroids interact with the transporter within the inner leaflet of the plasma membrane. Four previously unidentified, Vinca-related mutations, involving three amino acids, have also been found. Unexpectedly, these mutations are clustered within an eight-amino acid segment proximal to the TMS-4 region. This portion of the protein is thought to be within the cytoplasmic compartment of the cell. Thus, the results suggest that at least part of the initial interaction between P-gp and Vinca alkaloids occurs in the cytoplasm. The steroid interaction domain does not extend into this region of the protein. However, this cytoplasmic section of the protein is likely to play an important role in promoting steroid transport.

Identification of Putative Binding Sites of P‐glycoprotein Based on its Homology Model

“…33 Mutations in an eight amino-acid segment of murine IC2 238-245 (239-246 in human P-gp) have been shown to cause reduction in the resistance to vincristine and paclitaxel and to have no influence on daunorubicin and colchicines. [34,35] Hana et al [36] mutated a number of residues in TM11 of P-gp by alanine and found that the mutation in positions G935 and I936 (G939, I940 in human P-gp) displayed an overall decrease in resistance to actinomycin, doxorubicin, vinblastine, and colchicine. These amino acids have not been found in the pocket, however, close to them F938 has been identified.…”

“…[39] Mutations of murine W231, A301, S308 in TM4 and TM5 (human W232, A302, S309, Table 7) influence the resistance profile of the P-gp mutants in relation to steroids and are distinct from those related to Vinca alkaloids (human F239, K242, and A246 binding region 1) located within the cytoplasmic portion of the protein. [34] Figure 8 shows binding region 2 as generated by Site Finder. The front views from the membrane (Figure 8 A), the pore (Figure 8 B), and the top view from outside the cell (Figure 8 C) clearly show that the region is composed of pockets that face the membrane (identified by the gray spheres representing hydrophobic atoms) as well as the pore (identified by the red spheres representing hydrophilic atoms).…”

Identification and Characterization of the Binding Sites of P-Glycoprotein for Multidrug Resistance-Related Drugs and Modulators

“…Considerable effort has been invested in characterizing drug resistance changes caused by Pglycoprotein mutations [52,53,[127][128][129][130][131][132][133][134][135][136][137][138][139][140][141][142][143][144][145]. P-glycoprotein mutations that have appeared in cultured cells selected for resistance to particular drugs [130][131][132], and mutations incorporated by site-directed mutagenesis have been evaluated [52,53,127,129,[134][135][136][137][138][139][140][141][142][143][144][145].…”

“…P-glycoprotein mutations that have appeared in cultured cells selected for resistance to particular drugs [130][131][132], and mutations incorporated by site-directed mutagenesis have been evaluated [52,53,127,129,[134][135][136][137][138][139][140][141][142][143][144][145]. In many instances, as a result of a particular mutation, resistance to one drug may increase, while the resistance to other drugs may either remain the same or change.…”

“…Thus, TM segment 9 represents a site that cooperates with TM segment 6 to mediate drug resistance and [ 125 I]IAAP labeling. Mutations in mouse mdr1b P-glycoprotein that alter the ability of steroids to act as either substrates or inhibitors have been shown to be confined to a small portion of the protein within TM segments 4, 5 and 6 [144]. However, the steroid interaction domain does not appear to overlap that of the Taxol interaction domain [145].…”