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 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)
Highlights
From the Renal Unit and Deoartmentsof Medicine and $Pathology, Massachusetts General Hospital and Haruard Medical SchoolB, ostonM, assachuset& 02114 ’
S I 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
We have previously described a model for vasopressin-sensitive adenylylcyclase activation in the pigkidneycell lineLLC-PK,where Gi servesas a tonic inhibitor of vasopressin activation of adenylyl cyclase
Summary
Renal Unit, 8th crease in the transcriptioonf a subunit genes [17]. Hormones and lymphokines that modulate transcriptioin target genes, such as corticosterone, thyroxine, and interleukin 1also differentially alteraiand a, subunit mRNAlevels in ratcerebral cortex, rat fat, rat cardiac heart, and human endothecleilalsl, respectively [18,19,20,21]. Three positive clones (10-4,13-2, and 19-1)were obtained segments encoding the ai.*gene and characterized its promoter in renal epithelial cells utilizing a sensitive firefly luciferase reporter gene assay These studies provide a basis for elucidating cis-acting DNA sequences and “trans-acting” protein factors that act in concert to control the transcripand were subjected to restriction endonuclease mapping and Southern blot analysis [31]. The nucleic acids in this solution were ethanol precipitated and Nested deletions of the 5'-flanking regions in this plasmid were made resuspended in 25% formamide 5 mM EDTA, 0.1% bromphenol blue, by exonuclease 111-mungbean nuclease digestion (Stratagene) utiliz- 0.1%xylene cyanol These samples and a "P-end-labeled HaeIII ing unique AatII and Sal sites or by excision of discrete sequences ladder of @X174 DNA(New England Biolabs) were electrophoresed with site-specific restriction enzymes.
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