Study of membrane orientation and glycosylated extracellular loops of mouse P-glycoprotein by in vitro translation.
Increased expression of P-glycoprotein (Pgp) has been demonstrated to cause multidrug resistance (MDR) in vitro, and it may be responsible for chemotherapy failure in a number of human cancers. Pgp is a plasma membrane protein thought to function as an energy-dependent drug transporter. From its deduced protein sequence the topology of Pgp was proposed to contain 12 transmembrane domains with six extracellular loops and two cytoplasmic ATP-binding sites. To investigate further the membrane orientation of Pgp, we have expressed a full length cDNA of mouse mdr1, as well as its truncated forms, in a cell-free system supplemented with dog pancreatic microsomal membranes (RM). We determined which domains of the in vitro-synthesized Pgp had transversed the RM membranes by analyzing their resistance to protease digestion and their glycosylation state. To our surprise, this system revealed that a significant portion of in vitro-synthesized Pgp molecules has an additional glycosylated domain in the C-terminal half. Previously, only the first predicted extracellular loop near the N terminus had been thought to be glycosylated. Furthermore, we discovered that Pgp has at least two functional signal recognition particle/docking protein dependent signal sequences, one at the N-terminal half and the other at the C-terminal half. These findings suggest a new topological model for in vitro synthesized P-glycoprotein which may be relevant to its in vivo topology.
Zhang
J T
JT
Ontario Cancer Institute, Princess Margaret Hospital, Toronto, Canada.
Ling
V
V
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000818:Animals; D015153:Blotting, Western; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006868:Hydrolysis; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D015183:Restriction Mapping; D014158:Transcription, Genetic
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D004247:DNA