For project 1, site directed mutants of the phosphodiesterase 3A will be expressed in yeast and in insect (Sf9) cells. The cells required for the transformation of the constructs and the recombinant cells will be grown in the cell culture core. For Project 2, various megakaryocyte cell lines including Meg-01 cells, human erythroleukemia (HEL) cells, and CHRF288 cells, all of which must be cultured for the purpose of preparing mRNA in order to carry out in vitro transcription/translation studies as detailed under Specific Aim 1b. In addition (Specific Aim 3), will utilize Chinese hamster ovary (CHO) cells and L-cells to co-express the various subunits of the GPIb-IX complex including GPIbalpha, GBIbbeta, and GPIX. These cells will be co-transfected with platelet FXI cDNA to determine whether the expression of platelet FXI by these cells requires co-expression with GPIb-IX. For Project 3, deletion mutants of PLCbeta2 and Galphaq upstream region from the normal and the patient gene will expressed fused to a luciferase reporter gene in NIH-3T3, HEK- 293, CHO, or CV-1 cells. For Project 4, A5 cells, CHO cells transfected with fibrinogen receptor, will be used to develop a cell model for fibrinogen receptor activation. These cell lines stably expressing alpha2A and P2Y1 receptors will be maintained and tested for agonist-induced fibrinogen receptor activation. One such cell line used for transient transfections by constitutively active or dominant negative protein kinase C isoform constructs. As part of Specific Aim 3, C6-2B cells will be grown for transfections with candidate P2T/AC receptor cDNA clones, and for functional analysis. 132N1 cells will also be grown for transfection with the pools of cDNAs in expression cloning studies. As part of Specific Aim 4, 1321N1 astrocytoma will be grown and transfected with P2Y1 and/or P2T/AC receptor mutants generated by site- directed mutagenesis and will be tested for functional coupling.
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