Cloning of the mouse somatostatin receptor subtype 5 gene: Promoter structure and function

Somatostatin is a peptide hormone whose actions are mediated by five somato statin receptor subtypes (sst1-5). In the pituitary, somatostatin inhibits TSH release from thyrotropes and GH release from somatotropes. me have show n that sst5 transcripts and protein are induced by thyroid hormone in TtT-9 7 thyrotropic tumors. To map sequences responsible for promoter activity in pituitary cells, we cloned the mouse sst5 coding region of 362 amino acids and 12 kb of upstream DNA. Initial transfection studies in TtT-97 or GH3 c ells mapped high levels of basal promoter activity to a 5.6-kb fragment ups tream of the translational start, whereas shorter genomic fragments had low activity. To identify the transcriptional start site we used 5' RACE with TtT-97 poly A+ RNA and a sst5 antisense coding region primer. Sequence comp arison between the complementary DNA and the gene revealed that the mouse s st5 gene contains 3 exons and 2 introns. The entire coding region was conta ined in exon 3. Two differently sized RACE products demonstrated alternate exon splicing of two untranslated exons in TtT-97 cells. A promoter fragmen t from -290/+48 linked to a luciferase reporter demonstrated 600- and 900-f old higher activity over a promoterless control in GH3 mammosomatotropes an d TtT-97 thyrotropes, respectively, whereas a larger fragment extending to -6400 exhibited no additional promoter activity. Cloning of the sst5 gene w ill facilitate the mapping of basal and regulated responses at the transcri ptional level.