Functional analysis of chimeric genes obtained by exchanging homologous domains of the mouse mdr1 and mdr2 genes.

Abstract

A fufl-length cDNA clone for the mouse mdrl gene can confer multidrug resistance when introduced by transfection into otherwise drug-sensitive cells. In the same assay, a full-length cDNA clone for a closely related member of the mouse mdr gene family, mdr2, fails to confer multidrug resistance. To identify the domains of mdrl which are essential for multidrug resistance and which may be functionally distinct in mdr2, we have constructed chimeric cDNA molecules in which discrete domains of mdr2 have been introduced into the homologous region of mdrl and analyzed these chimeras for their capacity to transfer drug resistance. The two predicted ATP-binding domains of mdr2 were found to be functional, as either could complement the biological activity of mdrl. Likewise, a chimeric molecule in which the highly sequence divergent linker domain of mdr2 had been introduced in mdrl could also confer drug resistance. However, the replacement of either the aminoor carboxy-terminus transmembrane (TM) domain regions of mdrl by the homologous segments of mdr2 resulted in inactive chimeras. The replacement of as few as two TM domains from either the amino ( or the carboxy (TM7-8) half of mdrl by the homologous mdr2 regions was sufficient to destroy the activity of mdrl. These results suggest that the functional differences detected between mdrl and mdr2 in our transfection assay reside within the predicted TM domains.The emergence of multidrug resistance in cultured cells is linked to the overexpression of a membrane phosphoglycoprotein termed P-glycoprotein, believed to act as an ATPdependent efflux pump to reduce the intracellular accumulation of antitumor drugs. P-glycoprotein has been shown to interact directly with a remarkably broad range of substrates; most are small hydrophobic lipid-soluble molecules of natural origin such as anthracyclines, vinca alkaloids, and colchicine (14, 22). P-glycoproteins are encoded by a small gene family, termed mdr or pgp, which comprises three members in rodents and two members in humans (40). Analysis of the nucleotide and predicted amino acid sequences of full-length cDNA clones corresponding to the three mouse (13,24,25,29) (30) origins. Both mdrl and mdr3 can confer drug resistance in transfection experiments, but the two proteins appear to have overlapping but distinct substrate specificities (13). In transfection experiments, mouse mdr2 fails to confer multidrug resistance (25).Analysis of the mouse mdr gene transcripts in normal tissues shows that expression of the three genes is controlled in a tissue-specific manner (11). mdrl is constitutively expressed in adrenal glands and kidney and is induced at high levels in the endometrial glands of the pregnant uterus (1, 2). mdr2 is primarily expressed in liver and muscle, and mdr3 is primarily expressed in intestine and lung.

A missense mutation in ABCB4 gene involved in progressive familial intrahepatic cholestasis type 3 leads to a folding defect that can be rescued by low temperature

“…These domains are highly conserved between ABCB1 and ABCB4 and have been shown to be interchangeable. 27 Furthermore, ABCB1 can be expressed as a GFP-fusion protein that retains drug-transporting activity. Because many ABCB4 missense mutations are found in the first NBD domain, ABCB1-GFP may be a good model to understand the effect of these mutations, to screen for means of rescuing the mutants, and to test whether they are active.…”

Characterization of Phosphorylation-defective Mutants of Human P-glycoprotein Expressed in Mammalian Cells

“…A similar conclusion was implied in earlier work by Buschman and Gros (51), who showed that a chimeric mouse mdr1 P-glycoprotein containing the mouse mdr2 linker region was functional as a drug transporter. Since the mouse mdr2 linker region lacks the classic RRXS recognition sequence that is present in the mdr1 gene product, the argument was made that phosphorylation of this site was dispensable, at least for the multidrug transport activity of mouse P-glycoprotein (51).…”

Protein kinases and multidrug resistance

“…The general conclusions from such studies are that phosphorylation (and dephosphorylation) are not essential for basal drug efflux activity of Pgp and development of the MDR phenotype. This was also implied by a preliminary study of Buschman and Gros (1991) using a chimeric mdr1 Pgp containing the mdr2 linker region which lacks -R-R-X-S-phosphorylation sites. The mutant Pgp forms described above can be used to answer further questions, for example, whether similar findings occur when they are transfected into other cell types, if the turnover time of mutant Pgp is similar to the normal form and how phosphorylation of individual serine residues in the linker region influences drug transport.…”