Cloning of the mouse somatostatin receptor subtype 5 gene: promoter structure and function.
Somatostatin is a peptide hormone whose actions are mediated by five somatostatin receptor subtypes (sstl-5). In the pituitary, somatostatin inhibits TSH release from thyrotropes and GH release from somatotropes. We have shown that sst5 transcripts and protein are induced by thyroid hormone in TtT-97 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 cells mapped high levels of basal promoter activity to a 5.6-kb fragment upstream 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 comparison between the complementary DNA and the gene revealed that the mouse sst5 gene contains 3 exons and 2 introns. The entire coding region was contained in exon 3. Two differently sized RACE products demonstrated alternate exon splicing of two untranslated exons in TtT-97 cells. A promoter fragment from -290/+48 linked to a luciferase reporter demonstrated 600- and 900-fold higher activity over a promoterless control in GH3 mammosomatotropes and TtT-97 thyrotropes, respectively, whereas a larger fragment extending to -6400 exhibited no additional promoter activity. Cloning of the sst5 gene will facilitate the mapping of basal and regulated responses at the transcriptional level.
Gordon
D F
DF
Division of Endocrinology, Metabolism, and Diabetes, University of Colorado Health Sciences Center, Denver 80262, USA. david.gordon@uchsc.edu
Woodmansee
W W
WW
Lewis
S R
SR
James
R A
RA
Wood
W M
WM
Ridgway
E C
EC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D005091:Exons; D015870:Gene Expression; D007438:Introns; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010902:Pituitary Gland / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017481:Receptors, Somatostatin / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D015183:Restriction Mapping; D014162:Transfection
D017481:Receptors, Somatostatin; C094573:somatostatin receptor 5; D004247:DNA