P-glycoprotein function involves conformational transitions detectable by differential immunoreactivity

Abstract

The MDR1 P-glycoprotein (Pgp), a member of the ATP-binding cassette family of transporters, is a transmembrane ATPase efflux pump for various lipophilic compounds, including many anti-cancer drugs. mAb UIC2, reactive with the extracellular moiety of Pgp, inhibits Pgp-mediated efflux. UIC2 reactivity with Pgp was increased by the addition of several Pgp-transported compounds or ATP-depleting agents, and by mutational inactivation of both nucleotide-binding domains (NBDs) of Pgp. UIC2 binding to Pgp mutated in both NBDs was unaffected in the presence of Pgp transport substrates or in ATP-depleted cells, whereas the reactivities of the wild-type Pgp and Pgps mutated in a single NBD were increased by these treatments to the level of the double mutant. These results indicate the existence of different Pgp conformations associated with different stages of transport-associated ATP hydrolysis and suggest trapping in a transient conformation as a mechanism for antibody-mediated inhibition of Pgp.P-glycoprotein (Pgp), the product of the human MDR1 gene, acts as a broad specificity plasma membrane efflux pump for many hydrophobic compounds (1, 2) and recently was shown to function as a short chain lipid translocase (3). Pgp is a member of a superfamily of ATP-binding cassette (ABC) transporters, characterized by the presence of conserved ABC domains containing consensus nucleotide-binding domain (NBD) sequence motifs (4). ABC transporters of a subgroup that includes the MDR1 Pgp, a closely related MDR2 gene product that acts as a phospholipid translocase (5, 6), the yeast STE6 protein that transports the a pheromone (7), and the cystic fibrosis transmembrane conductance regulator (8), are characterized by a common architecture. These proteins are composed of two halves separated by a ''linker'' region; each half comprises a hydrophobic region with six predicted membrane-spanning segments and the ABC domain.Expression of the MDR1 Pgp in tumor cells is associated with a clinically important phenotype of crossresistance to many structurally diverse anti-cancer drugs, which are pumped out by Pgp. Pgp was shown to bind its transport substrates (9), an event that most probably occurs in the lipid bilayer of the plasma membrane (10), and to hydrolyze ATP (11). The ATPase activity of Pgp is strongly stimulated by the addition of Pgp transport substrates (12). The stoichiometry, temporal sequence, and structural transitions linking the binding and transport of a Pgp substrate with the binding and hydrolysis of ATP are as yet unknown.We previously have developed a mouse mAb UIC2, specific for the extracellular moiety of the human MDR1 Pgp (13). In contrast to several other mAbs that react with Pgp on the surface of intact cells, the addition of UIC2 to tissue culture media decreases the activity of Pgp toward all the tested Pgp transport substrates (13-16). The conformational epitope that is recognized by UIC2 is distinct from the epitopes of the other mAbs, because only UIC2 fails to react with a mutant Pgp that carries a d...

Allosteric Modulation of Human P-glycoprotein

“…Competitive Inhibitors-Substrates and competitive inhibitors induce a conformational change in Pgp, which causes an increased reactivity to UIC2, a monoclonal antibody specific for a conformation-sensitive Pgp external epitope (32). A similar increase in reactivity was observed with Pgp-expressing NIH-MDR1 cells in the presence of the competitive inhibitor cyclosporin A (32) (Fig.…”

P-glycoprotein inhibits caspase-8 activation but not formation of the death inducing signal complex (disc) following Fas ligation

“…This notion is supported by evidence that mAb UIC2, which reversed Pgp mediated resistance to Fas, traps P-gp in a transient conformation to inhibit drug efflux function. 16 To date, viral and cellular FLIPs, cow pox virus CrmA, adenovirus protein E3-14.7 kDa, activation of Protein Kinase C (PKC) and depletion of glutathione have been implicated in resistance to the Fas-mediated cell death pathway by inhibiting activation of caspase-8 through various mechanisms. 13,17 ± 21 Although the Fas-death receptor pathway is perhaps the best-characterized cell death pathway, all of the components of the Fas-DISC may not be entirely known.…”

“…The mutant P-gp used in the following studies contained double lysine to alanine substitutions at amino acids 233 (K433M) and 1076 (K1076M) within the Walker A motifs, which disrupted nucleotide binding, and ATPase and drug-efflux activity. 4,16 Mutant constructs containing double lysine to alanine substitutions at 433(K433M) and 1076(K1076M) amino acids were generated by excising 4.1 kB Eag1 ± Eag1 fragment from clone pUCFVXMDH3 ± Sal(DAUG)-MDR/Neo (provided by Igor Roninson, Chicago University, Chicago, IL) and cloned into MSCV vector. To generate high titre virus supernatant, plasmids were co-transfected with an amphotropic`helper' packaging plasmid pEQ-Pam3(7E) into 293T cells by calcium phosphate precipitation.…”

Structural and Functional Consequences of Mutating Cysteine Residues in the Amino Terminus of Human Multidrug Resistance-associated Protein 1

“…Previously, FACS analysis with monoclonal antibody as a probe has been used to determine conformational changes of P-glycoprotein (34). Recently, we developed a monoclonal antibody, IU2H10, directed against the amino terminus of human MRP1 (18).…”