Our long term goal is to understand the regulation of complement by cell surface proteins. In pursuing this goal we have isolated an 18 kDa protein from normal human erythrocytes that regulates complement activation. He refer to this protein as membrane inhibitor of reactive lysis or MIRL. This protein has been characterized structurally and functionally and has been shown to be involved in the pathogenesis of a clinical disorder, paroxysmal nocturnal hemoglobinuria. Recent data suggesting that MIRL expression may be increased in response to complement activation has led to investigation of potential mechanisms regulating MIRL expression. We have demonstrated MIRL expression is enhanced by exposure to PMA. Furthermore, this enhancement occurs at the transcriptional level and may be modulated at the post-translational level as well. Our immediate goal is to characterize the mechanism by which MIRL expression is regulated.
Specific Aim I : To determine if complement activation can induce MIRL expression. K562 cells will be exposed to activated complement and assayed for MIRL RNA and protein expression.
Specific Aim II : To determine if protein kinase C mediates PMA induced MIRL expression. K562 cells will be incubated in the presence of PMA or PMA with protein kinase C inhibitors and MIRL expression assayed.
Specific Aim III : To explain the discrepancy between PMA induced MIRL RNA and protein levels. The fate of the increased MIRL RNA will be determined by metabolically labeling cells and immunoprecipitating MIRL with antibody.
Specific Aim I V: To determine the cis-DNA sequence responsible for conferring PMA responsiveness. The 5' flanking region of the MIRL gene will be cloned. The specific sequences responsible for conferring PMA responsiveness will be determined by their ability to confer PMA responsiveness to a heterologous promoter and DNase I footprinting studies.
Specific Aim V : To isolate the trans-acting protein(s) which bind to PMA responsive element of the MIRL gene. A cDNA expression library will be constructed from K562 RNA. The transcriptional regulatory protein will be cloned using the PMA responsive element as probe.
| Endo, M; Ware, R E; Vreeke, T M et al. (1996) Molecular basis of the heterogeneity of expression of glycosyl phosphatidylinositol anchored proteins in paroxysmal nocturnal hemoglobinuria. Blood 87:2546-57 |
| Holguin, M H; Martin, C B; Eggett, T et al. (1996) Analysis of the gene that encodes the complement regulatory protein, membrane inhibitor of reactive lysis (CD59). Identification of an alternatively spliced exon and characterization of the transcriptional regulatory regions of the promoter. J Immunol 157:1659-68 |
| Holguin, M H; Martin, C B; Weis, J H et al. (1993) Enhanced expression of the complement regulatory protein, membrane inhibitor of reactive lysis (CD59), is regulated at the level of transcription. Blood 82:968-77 |
