Abstract
Prolactin gene expression is affected by numerous signals, but many of the promoter elements required for these responses have not been clearly identified. This report identifies sequences within the prolactin gene promoter that are required for the responses to cAMP and insulin. The cAMP response element, -101 to -92 shares a 6 of 8 base pair homology with previously identified cAMP response elements. Mutation of this element also results in a > 100-fold decrease in basal prolactin gene expression. This is characteristic of cAMP response elements, but the importance of this element to basal prolactin gene transcription was previously unrecognized. The insulin response element, -97 to -67, is not homologous to previously reported insulin response elements and mediates the 10-fold increases in prolactin gene expression due to insulin observed in GH cells. These elements also function to mediate insulin and cAMP responses from the heterologous delta MTV-CAT reporter plasmid. Together, insulin and cAMP increase prolactin gene expression additively. The clustering of these elements may provide clues to the independent and possible coordinate regulation by these effectors.
Highlights
Prolactin gene expressioins affectedby numeroussig- protein kinase A
We report the localization of the insulin and CAMP reshares a6 of 8 base pair homology with previously idens-ponse elements of the prolactin promoter
Internal deletions tified CAMPresponse elements. Mutation of this elemenoftthe prolactin promoter have demonstrated that the insulin results in a >100-fold decrease in basal prolactin a n d CAMPresponse elements overlap but are not identical
Summary
Pit-1 binding site between -66 and -36 where replacement of wild type sequences with linker results ain5040% decrease in insulin stimulation. The second is the linker replacementa t -76 t o -67 With this construct, the insulin-stimulated increase is approximat4e-lfyold. Analysis of this sequence revealed that it was a 7 of 10 match with -96 to -87 (Fig. 2 A ). -47, and -46 to -37 (LS-67/-56, LS-57/-46, and LS-47/-36 respectively) in the Pit-1 bindirnegion, potentiate theeffect of lactin-CAT plasmid and 5 pg of an human insulin receptor expression CAMPby approximately 1.5-fold.As with the insulin response, vector (pRT3HIR2)(81, and incubated withouetffectors for 24 h to allow this potentiationprobably results from the linker-scanning muthe cells to begin expressing CAT. Responses were separate, but linker-scanning mutants were unable to completely eliminate the insulin response without
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