Specific protein domains and amino acids responsible for the apparent capacity of P-glycoprotein (mdr) to recognize and transport a large group of structurally unrelated drugs have not been identified. We have introduced a single Ser -* Phe substitution within the predicted TM11 domain of mdrl (position 941) and mdr3 (position 939) and analyzed the effect of these substitutions on the drug-resistance profiles of these two proteins. Mutations at this residue drastically altered the overall degree of drug resistance conveyed by mdrl and mdr3. The modulating effect of this mutation on mdrl and mdr3 varied for the drugs tested: it was very strong for colchicine and adriamycin and moderate for vinbLastne. For mdrl, the Ser'4' -+ Phe'4' substitution produced a unique mutant protein that retained the capacity to confer vinblastine resistance but lost the ability to confer adriamycin and colchicine resistance. These results strongly suggest that the predicted TM11 domain of proteins encoded by mdr and mdrlike genes plays an important role in the recognition and transport of their specific substrates.Multidrug resistance is caused by the amplification and overexpression ofa small gene family, designated mdr orpgp (1), which is composed of two members in humans, MDR] and MDR2 (2, 3), and three members in rodents, mdrl (mdrlb), mdr2, and mdr3 (mdrla) (4)(5)(6)(7), that code for membrane P-glycoproteins (P-gps). P-gp has been shown to bind ATP (8) and drug analogs (9, 10) and has ATPase activity (11). It is believed to function as an ATP-dependent drug efflux pump to reduce intracellular drug accumulation in resistant cells (1,12). Sequence analyses of mdr gene cDNA clones predict polypeptides composed of 12 transmembrane (TM) domains and two nucleotide binding (NB) sites. P-gps are formed by two symmetrical and sequence homologous halves that share a common ancestral origin with a large group of bacterial transport proteins (13). The mdr gene family is part of a larger family of mdr-like genes encoding sequencehomologous proteins sharing similar predicted secondary structures and proposed membrane-associated transport functions. The pfmdrl gene of Plasmodium falciparum (14) associated with chloroquine (CLQ) resistance, the yeast STE-6 gene responsible for export of the a mating pheromone in Saccharomyces cerevisiae (15), and the CFTR gene in which mutations cause cystic fibrosis in humans (16) form part of this family. The regions of strongest sequence homology among these proteins overlap the predicted NB sites, which are believed to underlie a common functional aspect of transport (17). The protein segments and residues implicated in substrate binding and transport have not been precisely identified. Despite a very high degree of sequence homology (between 75% and 85% identity), striking functional differences have been detected between individual mdr genes. Mouse mdrl (18) and mdr3 (6, 19) and human MDR] (20) can confer multidrug resistance in transfection experiments, whereas mouse mdr2 (5) and human MDR2 (3...

The serine residue located at position 939 and 941 in the predicted transmembrane segment 11 of P-glycoprotein (P-gp) encoded by mouse mdr3 and mdr1, respectively, appears to be important for interaction of chemotherapeutic drugs and reversal agents with P-gp. To further understand the role of this residue in this process and to identify the structural requirements involved, we have replaced this serine residue by alanine, cysteine, threonine, tyrosine, tryptophan, and aspartic acid and tested the effect of these mutations on the overall activity and substrate specificity of mdr1 and mdr3. All mutant proteins could be expressed at high levels in the membrane fractions of LR73 Chinese hamster cells transfected with the corresponding mutant cDNAs. All introduced mutations had limited effect on the capacity of mdr1 and mdr3 to confer resistance to vinblastine. The modulatory effect of mutations on resistance to colchicine, adriamycin, and actinomycin D was more dramatic. The hydroxyl group of serine did not seem essential for interaction with these drugs since mutant mdr1 and mdr3 bearing alanine or cysteine at that position behaved essentially as wild type, while threonine-bearing mutants showed significantly reduced resistance to these drugs. The insertion at that site of residues with bulkier side chains had more complex effects on P-gp function. While introducing tyrosine, tryptophan, or aspartic acid caused an almost complete loss of colchicine and adriamycin resistance in both mdr1 and mdr3, the replacement to tyrosine or tryptophan had the opposite effect on mdr1 and mdr3 for actinomycin D resistance, causing either a 3-fold increase or a 4-8-fold decrease in resistance to this drug, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

P-Glycoproteins (P-gps) encoded by the mouse mdr1 and mdr3 (Phe939, mdr3F) genes confer distinct drug resistance profiles. While the mdr1 and mdr3F clones confer comparable levels of vinblastine (VBL) resistance, mdr3F confers actinomycin D (ACT) resistance levels 2-fold greater than mdr1, while mdr1 confers resistance to colchicine at levels 7-fold greater than mdr3F. We wished to identify in chimeric proteins discrete protein domains responsible for the distinct drug resistance profiles of mdr1 and mdr3F. Homologous protein domains were exchanged in hybrid cDNA clones, and the specific drug resistance profiles conferred by chimeric proteins were determined in stably transfected cell clones expressing comparable amounts of wild-type or chimeric P-gps. Immunoblotting experiments showed that all chimeras were found expressed in membrane-enriched fractions of transfected cell clones and all conveyed cellular drug resistance at levels above the background of nontransfected drug-sensitive LR73 cells. For VBL, all chimeric constructs were found to convey similar levels of resistance. For COL and ACT, the levels of resistance conferred by the various chimeras were heterogeneous, being similar to either the parental mdr1 or the parental mdr3F clones, or in many cases being intermediate between the two. The preferential COL and ACT resistance phenotypes of mdr1 and mdr3F, respectively, did not segregate in chimeric proteins with any specific protein segment. Taken together, our results suggest that the preferential drug resistance phenotypes encoded by the mdr1 and mdr3F clones implicate complex interactions between the two homologous halves of the respective P-gp.

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