Regulation of the G-protein αi-2 subunit gene in LLC-PK1 renal cells and isolation of porcine genomic clones encoding the gene promoter

Holtzman, Eliezer J., Soper, Brian W., Stow, Jennifer L., Ausiello, Dennis A. and Ercolani, Louis (1991) Regulation of the G-protein αi-2 subunit gene in LLC-PK1 renal cells and isolation of porcine genomic clones encoding the gene promoter. Journal of Biological Chemistry, 266 3: 1763-1771.

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Author Holtzman, Eliezer J.
Soper, Brian W.
Stow, Jennifer L.
Ausiello, Dennis A.
Ercolani, Louis
Title Regulation of the G-protein αi-2 subunit gene in LLC-PK1 renal cells and isolation of porcine genomic clones encoding the gene promoter
Formatted title
Regulation of the G-protein αi-2 subunit gene in LLC-PK1 renal cells and isolation of porcine genomic clones encoding the gene promoter
Journal name Journal of Biological Chemistry   Check publisher's open access policy
ISSN 0021-9258
1083-351X
Publication date 1991-01-25
Sub-type Article (original research)
Open Access Status File (Publisher version)
Volume 266
Issue 3
Start page 1763
End page 1771
Total pages 9
Place of publication Bethesda, MD, United States
Publisher American Society for Biochemistry and Molecular Biology
Language eng
Formatted abstract
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 αi-2 subunit mRNA was not detected until day 4 of culture, it peaked at day 6, and declined thereafter. In contrast, G-protein αs subunit mRNA which peaked on day 4 was easily detected on all culture days. The presence of the αi-2 protein on epithelial cell basolateral membranes followed the same pattern of mRNA expression during culture. To understand the developmental expression of the αi-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 αi-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 αi-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 αi-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. These studies suggest that αi-2 gene expression is regulated in LLC-PK1 cells. Identification of the gene’s promoter and 5’-flanking sequences provide a basis for elucidating ’cis-acting” DNA sequences and “trans-acting” protein factors that act in concert to control the transcriptional regulation of the αi-2 gene which may modulate G-protein coupled effector responses in vasopressin-sensitive renal epithelia.
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: Institute for Molecular Bioscience - Publications
 
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