Evidence for the locations of distinct steroid and Vinca alkaloid interaction domains within the murine mdr1b P-glycoprotein.
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.
Gruol
Donald J
DJ
The Sidney Kimmel Cancer Center, San Diego, California 92121, USA. dgruol@skcc.org
King
Miranda N
MN
Kuehne
Martin E
ME
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013256:Steroids / Q000378:metabolism*; D014747:Vinblastine / Q000494:pharmacology*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000972:Antineoplastic Agents, Phytogenic; D013256:Steroids; D014747:Vinblastine