P-GLYCOPROTEIN FUNCTION INVOLVES CONFORMATIONAL TRANSITIONS DETECTABLE BY DIFFERENTIAL IMMUNOREACTIVITY

The MDR1 P-glycoprotein (Pgp), a member of the ATP-binding cassette fa mily of transporters, is a transmembrane ATPase efflux pump for variou s lipophilic com pounds, including many anti-cancer drugs, mAb UIC2, r eactive with the extracellular moiety of Pgp, inhibits Pgp-mediated ef flux. UIC2 reactivity with Pgp was increased by the addition of severa l Pgp-transported compounds or ATP-depleting agents, and by mutational inactivation of both nucleotide-binding domains (NBDs) of Pgp. UIC2 b inding to Pgp mutated in both NBDs was unaffected in the presence of P gp transport substrates or in ATP-depleted cells, whereas the reactivi ties of the wild-type Pgp and Pgps mutated in a single NBD were increa sed by these treatments to the level of the double mutant. These resul ts indicate the existence of different Pgp conformations associated wi th different stages of transport-associated ATP hydrolysis and suggest trapping in a transient conformation as a mechanism for antibody-medi ated inhibition of Pgp.