Modulatory effects on substrate specificity of independent mutations at the serine939/941 position in predicted transmembrane domain 11 of P-glycoproteins.
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)
Dhir
R
R
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Grizzuti
K
K
Kajiji
S
S
Gros
P
P
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002470:Cell Survival; D006224:Cricetinae; D004277:DNA, Single-Stranded; D004351:Drug Resistance; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011499:Protein Processing, Post-Translational; D012694:Serine / Q000378:metabolism*; D013379:Substrate Specificity
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002352:Carrier Proteins; D004277:DNA, Single-Stranded; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D012694:Serine