Study of membrane orientation and glycosylated extracellular loops of mouse P-glycoprotein by in vitro translation

Jian-Ting Zhang, Victor Ling

Research output: Contribution to journalArticle

105 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)18224-18232
Number of pages9
JournalJournal of Biological Chemistry
Volume266
Issue number27
StatePublished - Sep 25 1991
Externally publishedYes

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P-Glycoprotein
Membranes
Signal Recognition Particle
Topology
Glycosylation
Chemotherapy
Cell-Free System
In Vitro Techniques
Multiple Drug Resistance
Cell membranes
Protein Sorting Signals
Blood Proteins
Digestion
Membrane Proteins
Proteins
Peptide Hydrolases
Complementary DNA
Adenosine Triphosphate
Binding Sites
Cell Membrane

ASJC Scopus subject areas

  • Biochemistry

Cite this

Study of membrane orientation and glycosylated extracellular loops of mouse P-glycoprotein by in vitro translation. / Zhang, Jian-Ting; Ling, Victor.

In: Journal of Biological Chemistry, Vol. 266, No. 27, 25.09.1991, p. 18224-18232.

Research output: Contribution to journalArticle

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