Functional analysis of chimeric proteins constructed by exchanging homologous domains of two P-glycoproteins conferring distinct drug resistance profiles

Abstract

P-Glycoproteins (P-gps) encoded by the mouse mdr1 and mdr3 (Phe939, mdr3F) genes confer distinct drug resistance profiles. While the mdr1 and mdr3F clones confer comparable levels of vinblastine (VBL) resistance, mdr3F confers actinomycin D (ACT) resistance levels 2-fold greater than mdr1, while mdr1 confers resistance to colchicine at levels 7-fold greater than mdr3F. We wished to identify in chimeric proteins discrete protein domains responsible for the distinct drug resistance profiles of mdr1 and mdr3F. Homologous protein domains were exchanged in hybrid cDNA clones, and the specific drug resistance profiles conferred by chimeric proteins were determined in stably transfected cell clones expressing comparable amounts of wild-type or chimeric P-gps. Immunoblotting experiments showed that all chimeras were found expressed in membrane-enriched fractions of transfected cell clones and all conveyed cellular drug resistance at levels above the background of nontransfected drug-sensitive LR73 cells. For VBL, all chimeric constructs were found to convey similar levels of resistance. For COL and ACT, the levels of resistance conferred by the various chimeras were heterogeneous, being similar to either the parental mdr1 or the parental mdr3F clones, or in many cases being intermediate between the two. The preferential COL and ACT resistance phenotypes of mdr1 and mdr3F, respectively, did not segregate in chimeric proteins with any specific protein segment. Taken together, our results suggest that the preferential drug resistance phenotypes encoded by the mdr1 and mdr3F clones implicate complex interactions between the two homologous halves of the respective P-gp.

ATP-dependent transport systems for organic anions

“…In humans, at least two P-glycoprotein genes (MDRl and MDR2) lead to the expression of proteins with distinct transport specificities. Studies with heterologously expressed chi-genes) established that the discrimination between substrates is due to complex interactions between domains of the protein (9). The presence of P-glycoproteins, or multiple drug resistance gene products, in liver CPM has been demonstrated (10,11); the pumps are responsible for the transport of mostly hydrophobic, neutral or positively charged compounds into bile.…”

Identification of a Cryptic Bacterial Promoter in Mouse (mdr1a) P-Glycoprotein cDNA

“…Several early papers used cloning as a method for the propagation and amplification of plasmids containing mdr1a [ 12 , 44 , 45 ]. Unfortunately, published literature often did not report the exact cloning conditions (bacterial host strain used, bacterial growth conditions, etc.)…”

Basic Mechanisms of Antibiotic Resistance: Molecular Properties of Multidrug Transporters

“…A limited set of chimeric Pgp proteins has been constructed using mammalian P-glycoproteins, such as human MDR2, as partner protein to identify proteins segments and amino acids in Pgp implicated in drug recognition (81)(82)(83). Human MDR2 (often called MDR3), a phosphatidylcholine-specific translocase in the bile canicular membrane of hepatocytes, shares about 77% identity with Pgp.…”