FIG. 9.

Calcium and cGMP synergistically increase the transactivation potential of full-length Gal4-CREB but not of Gal4-CREB-Δbzip; effect of A-C/EBP. (A) UMR106 cells were transfected with the reporter plasmid pGAL4-Luc, pRSV-βGal, and G-kinase II; cells were cotransfected with a vector encoding either full-length Gal4-CREB or Gal4-CREB-Δbzip. Cells were treated with A23187 (Ca⁺⁺) and/or CPT-cGMP (cGMP), and reporter gene activities were measured as described in the legend to Fig. 3. The luciferase/β-galactosidase activity ratio of untreated cells transfected with full-length Gal4-CREB was assigned a value of 1. We transfected different amounts of vector encoding full-length Gal4-CREB (15 ng) or Gal4-CREB-Δbzip (5 ng) to produce similar reporter gene activities in untreated cells. When the same amount of each vector was transfected, the activity of Gal4-CREB-Δbzip was 2.8- ± 0.4-fold higher than the activity of full-length Gal4-CREB (not shown). (B) In parallel experiments, cells were transfected with 0.1 μg, 0.3 μg, or 1 μg of full-length Gal4-CREB or Gal4-CREB-Δbzip, as indicated, to examine the expression levels of both constructs by Western blotting with an antibody specific for the Gal4 DNA-binding domain. (C) UMR106 cells were transfected with full-length Gal4-CREB, pGAL4-Luc, pRSV-βGal, and G-kinase II as described for panel A; cells were cotransfected with either empty vector or 40 ng of expression vector encoding A-CREB, A-C/EBP, or A-Fos. Cells were treated, and luciferase/β-galactosidase activity ratios were determined as described for panel A.