THE MOUSE PLASMA-MEMBRANE CA2+ PUMP ISOFORM-1 PROMOTER - CLONING AND CHARACTERIZATION

The expression of plasma membrane Ca2+ pump (PMCA) is regulated by various hormones or agonists via multiple second messenger pathways, Two different 5' segments of the PMCA1 gene (isoform 1) were cloned from a mouse genomic library. While one segment contained the 3' end of intron 1 and exon 2, the other segment was found to encompass the 5'-flanking region of the gene, exon 1, and the 5' portion of intron 1. Sequence analysis of the 5'-flanking region suggested the presence of the putative promoter. Pour sites for initiation of transcription (spanning 64 bp) were identified by RNase protection assay and primer extension analysis, The promoter region was very CC-rich, contained no ''TATA box,'' but had a ''CAAT box'' at -51, Comparison of sequence with known cis-regulatory motifs disclosed that the 5'-flanking region has a number of potential regulatory elements including an AP-1 site at -354, AP-2 binding sites at -267 and -123, Sp1 binding sites at -127, -111, and +3, and a cyclic AMP response element binding protein site at -67, To demonstrate promoter activity, a segment containing 611 bp of the promoter region (from -442 to +169) was subcloned in front of a promoterless chloramphenicol acetyltransferase (CAT) gene, This segment was able to drive the expression of chloramphenicol acetyltransferase in transient transfections of mouse (or human) neuroblastoma cells as well as rat aortic endothelial cells. Deletion analysis demonstrated that a fragment from -256 to +169 showed strong promoter activity, while a fragment from -117 to +168 had CAT activity that was not different from the vector control, The promoter was stimulated threefold by phorbol ester and twofold by cyclic AMP, These results provide further proof indicating up-regulation of the PMCA1 gene by multiple second messenger pathways. (C) 1995 Academic Press, Inc.