Genetic Basis of Multidrug Resistance 
of structurally and functionally unrelated com- 
pounds. The identification of P-gp segments and 
residues implicated in drug recognition and trans- 
port is a necessary prerequisite to the rational de- 
sign of new cytotoxic compounds capable of 
blocking or bypassing the action of P-gp in drug- 
resistant tumor cells. We have exploited the high 
degree of sequence similarity and striking func- 
tional differences detected among members of 
the mouse mdr family to identify, in chimeric 
and mutant proteins, segments and residues im- 
portant for drug recognition. To identify the do- 
mains of mdrl that are essential for MDR and that 
may be functionally distinct in the biologically 
inactive mdr2, we have constructed 1 1 chimeric 
molecules in which discrete domains of mdr2 
have been introduced into the homologous re- 
gion of mdrl. We have analyzed these chimeras 
for their capacity to confer MDR. 
The two predicted ATP-binding sites of mdr2 
were found to be functional, as either could com- 
plement the biological activity of mdrl. How- 
ever, the replacement of either the amino- or the 
carboxyl-terminus transmembrane (TM) domain 
region of mdrl by the homologous segment of 
mdr2 resulted in inactive chimeras. Replace- 
ment of as few as two TM domains of mdrl from 
either the amino- or carboxyl-terminal halves by 
the corresponding segment of mdr2 was suffi- 
cient to destroy mdrVs activity. These observa- 
tions suggest that the functional differences de- 
tected between mdrl and mrfr2 reside within the 
TM domains of the two proteins. 
P-gps encoded by mouse mdrl and mdr3 
confer distinct drug resistance profiles. While 
both clones confer comparable levels of resis- 
tance to vinblastine (VBL), mdr 3 confers prefer- 
ential resistance to actinomycin D (ACT), and 
mdrl to colchicine (COL). 
To identify protein domains implicated in the 
preferential drug resistance encoded by either pa- 
rental mdr clone, homologous protein domains 
were exchanged in a series of 16 hybrid cDNA 
clones, and the drug resistance profiles encoded 
by the corresponding chimeric proteins were an- 
alyzed. While all chimeric clones conferred simi- 
lar levels of VBL resistance, the levels of ACT and 
COL resistance conferred by the various clones 
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 seg- 
ments that overlapped both the amino and car- 
boxyl sets of TM domains of the respective parent 
conveyed the parent's full preferential drug re- 
sistance profile. These results suggest that 
the resistance profiles encoded by mdrl or 
mdr 3, possibly representing sites of drug- protein 
interactions, involve several determinants asso- 
ciated with TM domains from both homologous 
halves of P-gp. Recently we have tentatively iden- 
tified one of these sites. We have observed that a 
simple serine-to-phenylalanine substitution at 
position 941 (mdrl) or 939 (m<^r3), within pre- 
dicted TMl 1 , had a dramatic effect on the overall 
activity of the two pumps. 
The modulating effect of this mutation on 
mdrl and mdr 3 varied for the drugs tested. It was 
very strong for COL and adriamycin (ADR) but 
only moderate for VBL. For mdrl, the serine-to- 
phenylalanine 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 serine residue within TMl 1 
plays a key role in P-gp's recognition and trans- 
port of the former drugs. We are currently prob- 
ing drug-P-gp interactions at this residue, using 
additional mutants together with COL and ADR 
analogues modified at key positions on their re- 
spective backbone. 
Other investigators have found the same resi- 
due mutated in the pfmdrl gene from isolates of 
the human malarial parasite Plasmodium falci- 
parum that are resistant to chloroquine (CLQ). 
Such resistance is caused by an increased ATP- 
dependent CLQ efflux and is associated with mu- 
tant alleles of the mdr homologue pfmdrl, map- 
ping near TMl or within TMl 1 . Taken together, 
these and our studies indicate that the TMl 1 do- 
main of mdr and mdr-like genes is critical for 
drug recognition and transport. Besides their 
high degree of hydrophobicity, no significant ho- 
mology is detected between TMll domains of 
mdrl-3 and pfmdrl proteins. Both, however, 
have the potential of forming amphipathic 
helices. 
The mdr Ser^'^"^^' residues and the mutated 
residues in pfmdrl fall within the hydrophilic 
side of this helix, and the mutant residues map 
near what appears to be the boundary of the hy- 
drophilic side. These amphipathic helices may 
be important for the recognition of hydrophobic 
compounds, such as MDR drugs, that readily par- 
tition within the cell's lipid bilayer. 
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