MODULATORY EFFECTS ON SUBSTRATE-SPECIFICITY OF INDEPENDENT MUTATIONS AT THE SERINE(939 941) POSITION IN PREDICTED TRANSMEMBRANE DOMAIN-11 OF P-GLYCOPROTEINS/

The serine residue located at position 939 and 941 in the predicted tr ansmembrane segment 11 of P-glycoprotein (P-gp) encoded by mouse mdr3 and mdr1, respectively, appears to be important for interaction of che motherapeutic drugs and reversal agents with P-gp. To further understa nd the role of this residue in this process and to identify the struct ural requirements involved, we have replaced this serine residue by al anine, cysteine, threonine, tyrosine, tryptophan, and aspartic acid an d tested the effect of these mutations on the overall activity and sub strate specificity of mdr1 and mdr3. All mutant proteins could be expr essed 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 conf er resistance to vinblastine. The modulatory effect of mutations on re sistance to colchicine, adriamycin, and actinomycin D was more dramati c. The hydroxyl group of serine did not seem essential for interaction with these drugs since mutant mdr1 and mdr3 bearing alanine or cystei ne 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, try ptophan, or aspartic acid caused an almost complete loss of colchicine and adriamycin resistance in both mdr1 and mdr3, the replacement to t yrosine or tryptophan had the opposite effect on mdr1 and mdr3 for act inomycin D resistance, causing either a 3-fold increase or a 4-8-fold decrease in resistance to this drug, respectively. Drug accumulation a nd efflux studies indicated that the modulatory effect of the mutation s detected in cell survival assays were paralleled by a concomitant mo dulation of drug transport by mutant proteins. These results indicate that Ser939/941 plays a key role in the interaction of colchicine and adriamycin with P-gp and that the size of the carbon side chain is the primary structural determinant at that site. Interaction of P-gp with actinomycin D seems to involve additional nonoverlapping determinants , distinct in mdr1 and mdr3.