Membrane cofactor protein (MCP; CD46) is a widely expressed C regulatory protein that inhibits C activation on self-tissue. MCP binds C3b and C4b deposited on autologous cells and then serves as a cofactor for their inactivation by limited proteolytic cleavage. To characterize the DNA sequence elements responsible for controlling MCP expression, the 5' flanking region of the human MCP gene was cloned. Sequencing of 1350 nucleotides upstream from the ATG codon revealed a GC-rich region in the initial 500 nucleotides that is especially rich in the CpG dinucleotide. A CAAT box in reverse orientation, surrounded by four putative SP1 binding sites but lacking a typical TATA element, was within the first 200 nucleotides of this GC-rich region. The major transcriptional initiation site for HeLa cells, determined by primer extension and S1 nuclease protection analyses, was located 105 nucleotides from the translational start site. This overall orientation of the promoter region is characteristic of "housekeeping" genes. The MCP promoter region was further examined in HEp-2 cells by the chloramphenicol acetyltransferase (CAT) reporter gene assay, using various constructs derived from the 5' region of the MCP gene. The MCP promoter activity was confined to the GC-rich region from -624 to +96 (start site of transcription being +1). Inclusion of an AT-rich sequence from -624 to -1204 resulted in a 42% reduction in CAT activity suggesting that an inhibitor is present among the AT-rich sequences. The 5' flanking region of a highly homologous partial duplication of the MCP gene was also cloned and sequenced, and various constructs were assessed in the CAT reporter system. Many of the functionally relevant sequences seen in MCP are also found in the MCP-like 5' UT region, which is 85% homologous to MCP. The most striking difference was a 224 nucleotide deletion that was upstream from the corresponding MCP region harboring most of the promoter activity. Although expression of an MCP-like protein has not been reported, the MCP-like promoter region produced promoter activity comparable with that of MCP. These results serve as a basis for subsequent analyses of the expression of MCP in various cells and tissues and for understanding the mechanism of its modulation in inflammatory conditions. Also, through a comparison of the 5' region of MCP with other genes in the regulators of C activation gene cluster (at 1 q32), we propose a model for the evolution of the promoters in this tight linkage group.(ABSTRACT TRUNCATED AT 400 WORDS)