Transcriptional regulation of G-protein alpha i subunit genes in LLC-PK1 renal cells and characterization of the porcine G alpha 1-3 gene promoter.
Heterotrimeric guanine nucleotide-binding proteins (G-proteins) function as signal transducers for a variety of hormone-coupled enzyme and ion transport systems in eukaryotic cells. The expression of pertussis toxin-sensitive G-proteins (Gi) which couple their cognate receptors and effectors are regulated by cell cycle-dependent events in porcine LLC-PK1 renal epithelial cells. G alpha i-2 and G alpha i-3 isoforms are detected in these cells, and like G alpha i-2 (Holtzman, E. J., Soper, B. W., Stow, L. L., Ausiello, D. A., and Ercolani, L. (1991) J. Biol. Chem. 266, 1763-1771), we now demonstrate that G alpha i-3 mRNA and protein is coordinately expressed in these cells during differentiation. To gain further insights into these events, the porcine G alpha i-3 gene minimal promoter was characterized and found 67 base pairs upstream from the major transcription start site. The 56-base pair minimal promoter lacked TATAAA and GC boxes but did contain a sequence GGAAGTG conserved in both the human and porcine gene that could potentially bind an adenovirus E4TF1 transcription factor. In cells stably transfected with G alpha i-2 or G alpha i-3 gene 5'-flanking sequences fused to firefly luciferase cDNA reporter, temporal 10-15-fold transcriptional activation of both genes occurred before cellular polarization. Utilizing mobility shift assays which compared nuclear extracts from cells before and after cell polarization, a motif in the 5' region of the gene promoter GTACTTCCGCT was identified that bound an induced nuclear protein complex during transcriptional activation. In polarized cells complemented with the human glucocorticoid receptor, dexamethasone decreased G alpha i-2 but increased G alpha i-3 basal transcription and mRNA content 3-fold. These studies demonstrate that both G alpha i genes are dynamically regulated in LLC-PK1 cells by both growth, differentiation, and hormone signals.
Holtzman
E J
EJ
Renal Unit, Massachusetts General Hospital, Charlestown 02129.
Kinane
T B
TB
West
K
K
Soper
B W
BW
Karga
H
H
Ausiello
D A
DA
Ercolani
L
L
D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation; D002460:Cell Line; D004247:DNA; D003907:Dexamethasone / Q000494:pharmacology; D004848:Epithelium / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics*; D005786:Gene Expression Regulation; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013552:Swine; D014158:Transcription, Genetic
D012333:RNA, Messenger; D003907:Dexamethasone; D004247:DNA; D019204:GTP-Binding Proteins