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(Hypertension. 2008;52:737.)
© 2008 American Heart Association, Inc.

Original Articles

Characterization and Functional Analyses of the Human G Protein–Coupled Receptor Kinase 4 Gene Promoter

Sandra Hasenkamp; Ralph Telgmann; Jan A. Staessen; Claudia Hagedorn; Corinna Dördelmann; Martin Bek; Stefan-Martin Brand-Herrmann; Eva Brand

From the University Hospital Muenster, Department of Internal Medicine D (S.H., M.B., E.B.), and Leibniz-Institute for Arteriosclerosis Research, Department of Molecular Genetics of Cardiovascular Disease (R.T., C.H., C.D., S.-M.B-H.), University of Muenster, Muenster, Germany; Campus Gasthuisberg (J.A.S.), Department of Cardiovascular Diseases, Division of Hypertension and Cardiovascular Rehabilitation, University of Leuven, Leuven, Belgium.

Correspondence to Eva Brand, University Hospital Muenster, Department of Internal Medicine D, Albert-Schweitzer-Strasse 33, D-48149 Muenster, Germany. E-mail eva.brand{at}ukmuenster.de

The G protein–coupled receptor kinase 4 is involved in renal sodium handling and blood pressure regulation. Missense variants have already been tested functionally and are associated with hypertension, but no data on promoter analyses are yet available. We scanned 94 hypertensive white subjects for genetic variation and performed promoter reporter gene analyses in HEK293T, COS7, and SaOs-2 cells. Transient transfections with various full lengths and wild-type deletion constructs revealed that 1851 bp of the flanking region and 275 bp of the 5'-untranslated region were sufficient for transcriptional activities and composed a powerful cis-active element in the distal 293 bp. The –1702T and +2T alleles resulted in drastic general reductions of promoter function, whereas an activity increasing effect of +268C was cell type specific. Electrophoretic mobility-shift assay, supershift, and cotransfection analyses of transcription factor binding sites predicted in silico (Alibaba2.1/Transfac7) resulted in allele-specific binding patterns of nuclear proteins and identified the participation of CCAAT/enhancer-binding protein transcription factor family members. The G protein–coupled receptor kinase 4 core promoter resides in the first 1851 bp upstream of its transcription start site. The 4 identified genetic variants within this region exert allele-specific impact on both cell type– and stimulation-dependent transcription and may affect the expression balance of renal G protein–coupled receptor kinase 4.

Key Words: GRK4 • genetic variants • functional promoter analyses • deletion constructs • regulatory regions

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