Functional analysis of P-glycoprotein mutants identifies predicted transmembrane domain 11 as a putative drug binding site

Shama Kajiji, France Talbot, Kart Grizzuti, Valerie Van Dyke-Phillips, Michael Agresti, Ahmad Safa, Philippe Gros

Research output: Contribution to journalArticle

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Abstract

The substitution of a single serine to phenylalanine residue within the predicted transmembrane domain 11 of P-glycoproteins (P-gps) encoded by mouse mdr1 (Ser941, 1S; Phe941, 1F) or mdr3 (Ser939, 3S; Phe939, 3F) strongly modulates both the overall activity and substrate specificity of the two P-gps. In cell clones expressing either wild-type (1S, 3S) or mutant P-gps (1F, 3F), we show that the modulating effect of the mutation on the levels of adriamycin (ADM) resistance detected in drug cytotoxicity assays is paralleled by a similar modulation of the intracellular accumulation and extracellular efflux of radiolabeled adriamycin ([14C]ADM) from preloaded cells. Cytofluorescence studies with ADM on live cells produce similar results and demonstrate strong nuclear ADM accumulation only in drug-senfeitive LR cells and in the IF expressing cells, with little if any accumulation in 1S, 3S, or 3F expressing cells. Drug cytotoxicity and drug transport assays carried out in the presence of verapamil or progesterone suggest that the Ser to Phe substitution also reduces the capacity of these two reversal agents to modulate P-gp activity. Labeling experiments with the photoactivatable P-gp ligands iodoarylazidoprazosin and azidopine indicate a strong reduction in binding of these photoactivatable probes to the mutant P-gps (1F, 3F) as compared to their wild-type counterparts (1S, 3S). These results indicate that the studied mutations in TM11 reduce drug transport by decreasing initial drug binding to P-gp. This phenotype is opposite to that of a mutation near TM3 in human MDR1 (pst 185), where decreased drug transport is associated with increased drug binding and decreased drug release from P-gp [Safa, A. R., Stern, R. K., Choi, K., Agresti, M., Tamai, I., Metha, N. D., & Roninson, I. B. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7225-7229].

Original languageEnglish (US)
Pages (from-to)4185-4194
Number of pages10
JournalBiochemistry
Volume32
Issue number16
StatePublished - 1993
Externally publishedYes

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Functional analysis
P-Glycoprotein
Binding Sites
P-Glycoproteins
Doxorubicin
Pharmaceutical Preparations
Cytotoxicity
Mutation
Assays
Clone cells
Substitution reactions
Verapamil
Substrate Specificity
Phenylalanine
Serine
Progesterone
Labeling
Clone Cells
Ligands
Phenotype

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kajiji, S., Talbot, F., Grizzuti, K., Van Dyke-Phillips, V., Agresti, M., Safa, A., & Gros, P. (1993). Functional analysis of P-glycoprotein mutants identifies predicted transmembrane domain 11 as a putative drug binding site. Biochemistry, 32(16), 4185-4194.

Functional analysis of P-glycoprotein mutants identifies predicted transmembrane domain 11 as a putative drug binding site. / Kajiji, Shama; Talbot, France; Grizzuti, Kart; Van Dyke-Phillips, Valerie; Agresti, Michael; Safa, Ahmad; Gros, Philippe.

In: Biochemistry, Vol. 32, No. 16, 1993, p. 4185-4194.

Research output: Contribution to journalArticle

Kajiji, S, Talbot, F, Grizzuti, K, Van Dyke-Phillips, V, Agresti, M, Safa, A & Gros, P 1993, 'Functional analysis of P-glycoprotein mutants identifies predicted transmembrane domain 11 as a putative drug binding site', Biochemistry, vol. 32, no. 16, pp. 4185-4194.
Kajiji S, Talbot F, Grizzuti K, Van Dyke-Phillips V, Agresti M, Safa A et al. Functional analysis of P-glycoprotein mutants identifies predicted transmembrane domain 11 as a putative drug binding site. Biochemistry. 1993;32(16):4185-4194.
Kajiji, Shama ; Talbot, France ; Grizzuti, Kart ; Van Dyke-Phillips, Valerie ; Agresti, Michael ; Safa, Ahmad ; Gros, Philippe. / Functional analysis of P-glycoprotein mutants identifies predicted transmembrane domain 11 as a putative drug binding site. In: Biochemistry. 1993 ; Vol. 32, No. 16. pp. 4185-4194.
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T1 - Functional analysis of P-glycoprotein mutants identifies predicted transmembrane domain 11 as a putative drug binding site

AU - Kajiji, Shama

AU - Talbot, France

AU - Grizzuti, Kart

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AU - Agresti, Michael

AU - Safa, Ahmad

AU - Gros, Philippe

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N2 - The substitution of a single serine to phenylalanine residue within the predicted transmembrane domain 11 of P-glycoproteins (P-gps) encoded by mouse mdr1 (Ser941, 1S; Phe941, 1F) or mdr3 (Ser939, 3S; Phe939, 3F) strongly modulates both the overall activity and substrate specificity of the two P-gps. In cell clones expressing either wild-type (1S, 3S) or mutant P-gps (1F, 3F), we show that the modulating effect of the mutation on the levels of adriamycin (ADM) resistance detected in drug cytotoxicity assays is paralleled by a similar modulation of the intracellular accumulation and extracellular efflux of radiolabeled adriamycin ([14C]ADM) from preloaded cells. Cytofluorescence studies with ADM on live cells produce similar results and demonstrate strong nuclear ADM accumulation only in drug-senfeitive LR cells and in the IF expressing cells, with little if any accumulation in 1S, 3S, or 3F expressing cells. Drug cytotoxicity and drug transport assays carried out in the presence of verapamil or progesterone suggest that the Ser to Phe substitution also reduces the capacity of these two reversal agents to modulate P-gp activity. Labeling experiments with the photoactivatable P-gp ligands iodoarylazidoprazosin and azidopine indicate a strong reduction in binding of these photoactivatable probes to the mutant P-gps (1F, 3F) as compared to their wild-type counterparts (1S, 3S). These results indicate that the studied mutations in TM11 reduce drug transport by decreasing initial drug binding to P-gp. This phenotype is opposite to that of a mutation near TM3 in human MDR1 (pst 185), where decreased drug transport is associated with increased drug binding and decreased drug release from P-gp [Safa, A. R., Stern, R. K., Choi, K., Agresti, M., Tamai, I., Metha, N. D., & Roninson, I. B. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7225-7229].

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