STRUCTURE/FUNCTION ANALYSIS OF DRUG EFFLUX PUMPS ENCODED 
BY THE MULTIDRUG-RESISTANCE GENE FAMILY 
Philippe Gros, Ph.D., International Research Scholar 
Multidrug resistance (MDR) , the phenomenon by 
which tumor cells develop resistance to a wide 
range of structurally unrelated chemotherapeutic 
drugs, is caused by the overexpression of P- 
glycoprotein (P-gp). P-gp binds photoactivatable an- 
alogues of ATP and cytotoxic drugs and is thought to 
function as a membrane-associated ATP-driven drug 
efflux pump. P-gp is encoded by a small family of 
closely related genes, termed mdrov pgp, that share 
considerable sequence homology and common an- 
cestral origins. There are three mdr genes in rodents 
(mdrl, mdr 2, mdr 3) and two in humans {MDRl, 
MDR2). 
Full-length cDNA clones corresponding to the 
three mouse genes have been cloned and character- 
ized. The three P-gps are highly homologous (80- 
85% sequence identity), with identical predicted 
structural features that include 12 transmembrane 
(TM) domains and two nucleotide binding sites. 
Each P-gp is formed by two homologous halves that 
show sequence conservation with a large group of 
bacterial transport proteins participating in the im- 
port and export of specific substrates in Escherichia 
coli. 
The mdr gene family is itself part of a larger gene 
family that includes the STE6 gene of the yeast Sac- 
charomyces cerevisiae, the gene responsible for the 
transmembrane transport of the "a" mating phero- 
mone; the pfmdrl gene of the malarial parasite 
Plasmodium falciparum, associated with chloro- 
quine (CLQ) efflux in this parasite; and in humans, 
the CFTR chloride channel gene, in which muta- 
tions cause cystic fibrosis, and the TAP- 1 /TAP- 2 
family, encoding peptide pumps participating in 
antigen presentation by T lymphocytes. Therefore 
the mdr supergene family codes for transport pro- 
teins that act by the same mechanism on different 
types of substrates. Transfection and overexpression 
of full-length mdr cDNAs into otherwise drug- 
sensitive cells show that mdrl and mdrS can di- 
rectly convey MDR, while mdr2 cannot. 
The mechanism by which P-gp mediates drug ef- 
flux remains unknown. So do the discrete protein 
domains and amino acid residues implicated in rec- 
ognition of structurally heterogeneous MDR drugs. 
Answering these questions is a necessary prerequi- 
site to the rational design of new cytotoxic drugs or 
experimental strategies aimed at blocking or by- 
passing the action of this pump. In the absence of a 
three-dimensional structure, the structure/function 
analysis of P-gp has focused on the creation and char- 
acterization of chimeric and mutant P-gps. 
Identification of Protein Domains 
Implicated in Drug Recognition 
P-gps encoded by mouse mdrl and mdr3 confer 
distinct drug-resistance profiles to transfected cells. 
Both clones confer comparable levels of resistance 
to vinblastine (VBL) , while mdr 5 confers preferen- 
tial resistance to actinomycin D (ACT), and mdrl to 
colchicine (COL). To identify protein domains im- 
plicated in the preferential drug resistance encoded 
by either parental mdr clone, homologous protein 
domains were exchanged in a series of 16 hybrid 
cDNA clones. While all chimeric clones conferred 
similar levels of VBL resistance, the levels of ACT 
and COL resistance conferred by the various chi- 
meras were heterogeneous, being either similar to 
the parental mdrl or mdr3 clones or, in many cases, 
intermediate between the two. Only those chimeric 
proteins carrying segments overlapping both the 
amino and carboxyl sets of TM domains of the re- 
spective parent conveyed the full preferential drug- 
resistance profile of this parent. 
These results suggest that 1) both parental pro- 
teins transport VBL by the same mechanism, which 
involves protein determinants that are conserved in 
both parents and that can be interchanged in chi- 
meric proteins, and 2) the preferential drug- 
resistance profiles encoded by parental mdrl or 
mdrS involve several determinants associated with 
TM domains from both homologous halves of P-gp. 
A simple Ser Phe substitution at position 941 
(mdrl) or 939 (mdr3), within predicted TMll, 
was shown to have a dramatic effect on the overall 
activity and substrate specificity of the two pumps. 
The modulating effect of this mutation was very 
strong for COL and adriamycin (ADR) but only mod- 
erate for VBL. For mdrl, the Ser Phe replacement 
produced a unique mutant protein that retained the 
capacity to confer VBL resistance but lost the ability 
to confer ADR or COL resistance. 
These results suggest that ADR/COL and VBL may 
have distinct binding sites on P-gp and that the ser- 
ine residue within TMl 1 plays a key role in the rec- 
ognition and transport of the former drugs by P-gp. 
The same residue has also been found mutated in the 
pfmdrl gene from CLQ-resistant isolates of the hu- 
man malarial parasite Plasmodium falciparum. To- 
gether these studies indicate that the TMl 1 domain 
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