title: Decreased expression of the GHRH receptor gene due to a mutation in a Pit-1 binding site.
abstract: A variety of mutations in the gene encoding the GHRH receptor (GHRHR) that are predicted to alter protein structure or function have been recently described in patients with isolated GH deficiency type IB. In the present report we describe a patient with isolated GH deficiency type IB who was heterozygous for two novel mutations in this gene: a missense mutation in codon 329 that replaces lysine with glutamic acid (K329E) and an A-->C transversion (position -124) in one of the two sites of the promoter region that binds the pituitary-specific transcription factor Pit-1, which is required for GHRHR expression. Chinese hamster ovary cells that were transfected with a cDNA encoding the K329E GHRHR expressed the receptor but failed to show a cAMP response after treatment with GHRH, confirming the lack of functionality. To test the effect of the A-->C mutation at position -124 of the promoter, we transfected rat GH3 pituitary cells, which express endogenous Pit-1, with plasmids in which the luciferase reporter gene was under the control of either the wild-type or the mutant promoter. GH3 cells expressing the mutant promoter showed significantly less luciferase activity than cells expressing the wild-type promoter. DNA-binding studies confirmed that the A-->C base change markedly reduces DNA binding to the Pit-1 protein. These results demonstrate that mutations in the GHRHR are not limited to the coding sequence and that promoter mutations that impair Pit-1 binding can reduce expression of the GHRHR gene.
authors.affiliation: Division of Endocrinology, and the Ilyssa Center for Molecular and Cellular Endocrinology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA. salvator@jhmi.edu
authors.forename: Roberto
authors.initials: R
authors.lastname: Salvatori
authors.forename: Xiaoguang
authors.initials: X
authors.lastname: Fan
authors.forename: Primus E
authors.initials: PE
authors.lastname: Mullis
authors.forename: Azeb
authors.initials: A
authors.lastname: Haile
authors.forename: Michael A
authors.initials: MA
authors.lastname: Levine
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D002460:Cell Line; D002675:Child, Preschool; D006224:Cricetinae; D000242:Cyclic AMP / Q000494:pharmacology; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015870:Gene Expression; D018698:Glutamic Acid; D013007:Growth Hormone-Releasing Hormone / Q000494:pharmacology; D006579:Heterozygote; D019382:Human Growth Hormone / Q000172:deficiency*; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008239:Lysine; D008297:Male; D009154:Mutation; D020125:Mutation, Missense; D010902:Pituitary Gland / Q000378:metabolism; D011401:Promoter Regions, Genetic; D051381:Rats; D018013:Receptors, Neuropeptide / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018017:Receptors, Pituitary Hormone-Regulating Hormone / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection
chemical_list: D004268:DNA-Binding Proteins; C494187:POU1F1 protein, human; C494186:Pou1f1 protein, rat; D018013:Receptors, Neuropeptide; D018017:Receptors, Pituitary Hormone-Regulating Hormone; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; D019382:Human Growth Hormone; D018698:Glutamic Acid; D013007:Growth Hormone-Releasing Hormone; D000242:Cyclic AMP; D008156:Luciferases; C048250:somatotropin releasing hormone receptor; D008239:Lysine