The structure, expression, and regulation of themdr (P-glycoprotein) multigene family

Abstract

A hallmark of the multidrug resistant phenotype is the overproduction of a family of 130-180 kDa integral membrane phosphoglycoproteins collectively called P-glycoprotein. Gene-specific hybridization probes were derived from three classes of mouse P-glycoprotein cDNAs. These probes revealed the differential amplification and/or transcriptional activation of three distinct but closely related mdr genes (mdr1a, mdr1b, and mdr2) in independently-selected multidrug resistant J774.2 mouse cell lines. Overexpression of mdr1a and mdr1b was found to correlate, in general, with the differential overproduction of either a 120 or 125 kDa P-glycoprotein precursor, respectively. This same correlation was observed in a single cell line during the course of stepwise selection for resistance to vinblastine in which a switch in gene expression from mdr1b to mdr1a resulted in a switch from the 125- to 120-kDa P-glycoprotein precursor. These findings suggest that differential overexpression of distinct mdr genes which encode unique P-glycoprotein isoforms is a possible mechanism for generating diversity in the multidrug resistant phenotype.;The mdr1a gene is a complex transcriptional unit whose expression is associated with multiple transcript sizes. Independently-selected multidrug-resistant J774.2 cell lines differentially overexpress either 4.6- and 5.0-kb, or 4.7- and 5.1-kb mdr1a transcripts. However, abundant overproduction of the mdr1a gene product was observed only in cell lines which overexpressed the 4.6- and 5.0-kb mRNAs. In order to determine the basis for mdr1a transcript heterogeneity and the relationship between transcript size and translational efficiency, genomic and cDNA sequence analysis of the 5{dollar}\sp\prime{dollar} and 3{dollar}\sp\prime{dollar} ends of the mdr1a gene was carried out. Promoter sequence analysis and primer extension mapping indicated that mdr1a transcripts were differentially initiated from either an upstream or a downstream promoter, to generate 5.1- and 4.7-kb transcripts or 5.0- and 4.6-kb transcripts, respectively, in four multidrug-resistant J774.2 cell lines. The addition of unique 5{dollar}\sp\prime{dollar}-untranslated sequences by transcript initiation from the upstream promoter correlated with a 70-85% decrease in translational efficiency. Sequence analysis of 3{dollar}\sp\prime{dollar} cDNA variants and a 3{dollar}\sp\prime{dollar} genomic fragment revealed that the 5.1- and 5.0-kb mRNAs had identical 3{dollar}\sp\prime{dollar}-untranslated regions which differed from those of the 4.7- and 4.6-kb mRNAs as a result of the utilization of a more downstream alternative poly(A) addition signal. In addition, the identification of putative AP-1 and AP-2 promoter elements suggests a possible role for protein kinase A and protein kinase C in the regulation of mdr1a. The implications of these findings for mdr gene expression and regulation are discussed.