Characterization of a cGMP-response element in the guanylyl cyclase/natriuretic peptide receptor A gene promoter.
Previous studies have shown that atrial natriuretic peptide (ANP) can inhibit transcription of its receptor, guanylyl cyclase A, by a mechanism dependent on cGMP and have suggested the presence of a putative cGMP-response element (cGMP-RE) in the Npr1 gene promoter. To localize and characterize the putative cis-acting element, we have subcloned a 1520-bp fragment of the rat Npr1 promoter in an expression vector containing the luciferase reporter gene. Several fragments, generated by exonuclease III-directed deletions, were transiently transfected into cells to measure their promoter activity. Deletion from -1520 to -1396 of a 1520-bp-long Npr1 promoter led to a 5-fold increase in luciferase activity. Subsequent deletion to the position -1307 resulted in a decrease of luciferase activity by 90%. Preincubation of cells with 100 nM of ANP or 100 microM 8-bromo-cGMP inhibited luciferase activity of the 1520-bp and 1396-bp-long fragments, but not the activity of the 1307-bp fragment, suggesting that the cGMP-RE is localized between positions -1396 and -1307. The cGMP regulatory region was narrowed by gel shift assays and footprinting to position -1372 to -1354 from the transcription start site of Npr1 and indicated its interaction with transcriptional factor(s). Cross-competition experiments with mutated oligonucleotides led to the definition of a consensus sequence (-1372 AaAtRKaNTTCaAcAKTY -1354) for the novel cGMP-RE, which is conserved in the human (75% identity) and mouse (95% identity) Npr1 promoters.
Hum
David
D
Centre de recherche, Centre hospitalier de l'Université de Montréal-Hôtel-Dieu, Montréal, Québec, Canada.
Besnard
Sandrine
S
Sanchez
Rocio
R
Devost
Dominic
D
Gossard
Francis
F
Hamet
Pavel
P
Tremblay
Johanne
J
D000818:Animals; D009320:Atrial Natriuretic Factor / Q000494:pharmacology; D003001:Cloning, Molecular; D016384:Consensus Sequence; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology / Q000502:physiology*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006162:Guanylate Cyclase / Q000235:genetics*; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D051381:Rats; D017461:Receptors, Atrial Natriuretic Factor / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D020218:Response Elements / Q000187:drug effects / Q000235:genetics*
D011993:Recombinant Fusion Proteins; C016276:8-bromocyclic GMP; D009320:Atrial Natriuretic Factor; D008156:Luciferases; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; C091715:atrial natriuretic factor receptor A; D006152:Cyclic GMP