Regulation of the G-protein alpha i-2 subunit gene in LLC-PK1 renal cells and isolation of porcine genomic clones encoding the gene promoter.
Heterotrimeric G-proteins function as signal transducers for a variety of hormone-coupled enzyme and ion transport systems in eukaryotic cells. We have studied G-protein-coupled processes that appear to be developmentally regulated in polarized pig kidney cells (LLC-PK1). Following trypsinization, LLC-PK1 cells differentiate from a rounded cell type to a fully polarized epithelium by 7 days of culture. During this differentiation, the expression of G-protein alpha i-2 subunit mRNA was not detected until day 4 of culture, it peaked at day 6, and declined thereafter. In contrast, G-protein alpha s subunit mRNA which peaked on day 4 was easily detected on all culture days. The presence of the alpha i-2 protein on epithelial cell basolateral membranes followed the same pattern of mRNA expression during culture. To understand the developmental expression of the alpha i-2 subunit in non-polarized cells and its potential regulation by hormones and second messengers in polarized cells at the transcriptional level, genomic DNA segments encoding the alpha i-2 gene promoter were isolated from an EMBL-3 porcine genomic library. S1 nuclease analysis of LLC-PK1 mRNA with cRNA probes derived from these DNA segments revealed major and a minor transcriptional start sites 131 and 171 base pairs upstream of the translation initiation site. The porcine and human alpha i-2 subunit genes shared a 78% sequence identity in their 5' flanks which suggested an evolutionary conservation of cis elements required to influence their transcription. The porcine alpha i-2 gene promoter was identified by fusing DNA segments encoding putative 5'-flanking areas of the gene to a plasmid that contained a firefly luciferase reporter gene but lacked a promoter. The minimal promoter was found between -130 and -60 base pairs from the major transcription start site. No typical "TATA-like" sequences were found. However, a "GC" box and a "TGTGG" sequence were two potential cis elements required for basal transcription of the porcine gene promoter which shared a 76% sequence identity to the promoter of another GTP-binding protein, the human c-Ha-ras proto-oncogene. Transcription of the gene was inhibited following treatment of renal cells with 10(-8) M dexamethasone.(ABSTRACT TRUNCATED AT 400 WORDS)
Holtzman
E J
EJ
Renal Unit, Massachusetts General Hospital, Boston, Massachusetts.
Soper
B W
BW
Stow
J L
JL
Ausiello
D A
DA
Ercolani
L
L
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002454:Cell Differentiation; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D007668:Kidney; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D000090063:Proto-Oncogene Mas; D012333:RNA, Messenger / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D013552:Swine
C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D012333:RNA, Messenger; D019204:GTP-Binding Proteins