IntroductionMuscle is an important target tissue for insulin-like growth factor (IGF) action.

Insulin regulates cell function by first binding to the insulin receptor (IR) localized on the cell surface. With the cloning of IR cDNA and the IR-gene promoter, the regulation of the IR gene during differentiation and by various hormones can be studied. Muscle is a major target tissue for insulin action. BC3H1 cells, a mouse muscle cell line in culture, are a model cell type for studying insulin action. Differentiation in these cells results in a 5- to 10-fold increase in IR binding and a 5- to 10-fold increase in IR content. Studies of IR mRNA by Northern and slot-blot analyses reveal a 10-fold increase in IR mRNA after differentiation. These studies indicate that there is a selective increase in IR-gene expression during muscle differentiation. A similar increase in IR-gene expression is observed for the IR during pancreatic acinar cell differentiation. Glucocorticoids increase IR content in several target tissues. Studies in cultured IM-9 lymphocytes indicate that glucocorticoids induce a 5-fold increase in IR mRNA levels. Studies of IR mRNA half-life indicate that glucocorticoids do not alter IR mRNA stability. When the transcription of the IR is measured by elongation assays, glucocorticoids directly stimulate IR transcription 5- to 10-fold. The effect is detectable within 30 min of glucocorticoid treatment and is maximal within 2 h. Therefore, these studies demonstrate that the IR gene is under the direct regulation of glucocorticoids. Insulin downregulates the IR in various target tissues. Prior studies indicate that this downregulation was partly because of accelerated IR degradation. Studying AR42J pancreatic acinar cells, we also found that insulin accelerates IR degradation. Moreover, in these cells, insulin decreases IR biosynthesis by approximately 50%. Studies of IR mRNA indicate there is a concomitant decrease in IR mRNA levels after insulin treatment. Thus, insulin decreases IR-gene expression. The genomic structure of the IR promoter has been elucidated. Primer extension and nuclease S1 analysis indicate that IR mRNA has multiple start sites. The promoter fragment was ligated to a promoterless "reporter" plasmid containing the bacterial gene chloramphenicol acetyltransferase (CAT). When this plasmid is transfected into cultured cells, CAT activity is detected, indicating promoter activity. Various portions of a genomic fragment were ligated to a promoter to study glucocorticoid regulation of the IR promoter. These studies indicate that IR-gene expression is regulated by differentiation and hormonal agents.(ABSTRACT TRUNCATED AT 400 WORDS)

The promoter region of the human insulin-receptor (HINSR) gene was isolated from a human chromosome 19 bacteriophage library. With S1 nuclease mapping and primer-extension analysis, we identified multiple transcription-initiation sites. Dexamethasone, a known inducer of HINSR transcription, enhanced transcription of all major transcription-initiation sites. DNA sequence analysis indicated that the HINSR promoter has neither a TATA box nor a CAAT box. The HINSR promoter region contains six GGGCGG sequences that may be binding sites for the transcription factor Sp1. In addition, there were three TCCC sequences that were putative promoter regulatory regions. The HINSR gene promoter has structural similarity to the epidermal growth factor receptor gene promoter and has some features of the promoter of the meglutol (hydroxymethylglutaryl, HMG) CoA reductase gene and the early promoter of simian virus 40.

The insulin-like growth factor-I receptor (IGFIR) is a membrane-bound glycoprotein that mediates the action of insulin-like growth factors. The cDNAs for the human IGFIR have been cloned and expressed, but the structures of the gene and its promoter have not been elucidated. In this study, we isolated an IGFIR promoter clone from a human chromosome 15 library. This clone contained the promoter, first exon, and a portion of the first intron. Sequence analysis of the 5' region that contained the promoter revealed that it lacked both TATA and CAAT boxes. The promoter contained binding sites for the transcription factors Sp1, AP-2, and the epidermal growth factor receptor transcription factor (ETF). Primer extension analysis of IGFIR mRNA indicated the presence of a single transcription start site 1,012 bp upstream from the ATG. When the putative promoter was ligated into a promoterless CAT vector and transfected mto HEPG2 cells, CAT activity was expressed, indicating that promoter activity was contained in this fragment. Other constructs containing the promoter and portions of the 5' untranslated region were used in transfection studies, and indicated that the 5' untranslated regions may play a role in promoter activity. Comparison of the human IGFIR promoter with that of the rat IGFIR promoter revealed significant sequence homology. Comparison of the IGFIR promoter with that of the human insulin receptor (IR) revealed structural similarities, although the arrangement of promoter elements differed.

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