CAMP REGULATES G-PROTEIN-ALPHA(I-2) SUBUNIT GENE-TRANSCRIPTION IN POLARIZED LLC-PK(1) CELLS BY INDUCTION OF A CCAAT BOX NUCLEAR-BINDING FACTOR

Heterotrimeric G-proteins function as signal transducers for a variety of hormone-coupled enzyme systems in eukaryotic cells. In LLC-PK1 ren al cells, vasopressin-stimulated adenylylcyclase activity is regulated in part, by the counterbalancing activity of stimulatory G-proteins ( G(s)) and inhibitory pertussis toxin-sensitive G-proteins (G(i)). Two G(i) genes encoding the G(i) isoforms Galpha(i-2) and Galpha(i-3) are expressed in LLC-PK1 cells. In polarized cells, these isoforms are top ographically segregated to different membranes, which allows for the s elective inhibition of adenylylcyclase by Galpha(i-2). The genes encod ing these isoforms are similarly regulated in these cells during growt h and differentiation but differ in response to steroid hormone signal s (Holtzman, E. J., Kinane, T. B., West, K., Soper, B. W., Karga, H., Ausiello, D. A., and Ercolani, L. (1993) J. Biol. Chem. 268, 3964-3975 ). We now demonstrate after stimulating polarized LLC-PK1 cells with f orskolin, which raises intracellular cAMP levels 50-fold, Galpha(i-2) but not Galpha(i-3) protein is increased 3-fold at 12 h and remains el evated above control values by 24 h. In cells stably transfected with Galpha(i-2) or Galpha(i-3) gene 5'-flanking sequences fused to firefly luciferase cDNA reporter gene, forskolin treatment increased Galpha(i -2) transcription 3-fold but inhibited Galpha(i-3) transcription by 50 % at 12 h. In vivo footprinting of forskolin-treated cells was perform ed to examine the molecular basis for activation of the Galpha(i-2) ge ne. Protected guanosines were identified in a 135-base pair (bp) area previously associated with enhancer activity of this gene in non-polar ized cells. This DNA segment did not contain the classical cAMP respon se element 5'-TGACGTCA-3'. Utilizing the 135-bp DNA segment as a probe in mobility shift assays, which compared nuclear extracts from cells before and after forskolin treatment, an induced nuclear protein compl ex was identified. Following systematic reduction and mutation of this DNA segment, a ''CCAAT'' box motif was identified that bound the indu ced nuclear protein complex during forskolin-induced Galpha(i-2) gene transcriptional activation. Induction of this nuclear protein complex was prevented in forskolin-treated cells by cycloheximide. To demonstr ate functional activity of the CCAAT box motif, cells were transiently transfected with plasmids encoding either the minimal 135-bp segment or a multimerized CCAAT box segment fused to a Rous sarcoma minimal pr omoter/firefly luciferase reporter gene. The transcriptional response of these plasmids in polarized forskolin-treated cells was similar in magnitude but retarded temporally to that observed in cells stably tra nsfected with the Galpha(i-2) firefly luciferase gene. These studies d emonstrate that chronic cAMP elevation initiates a counter-regulatory response in fully polarized renal cells that includes increased transc ription of the Galpha(i-2) gene. In contrast to other cAMP-responsive genes, increased Galpha(i-2) gene transcription appears to be mediated by a cAMP pathway that requires induction of a member of the CCAAT bo x family of DNA-binding proteins.