Rat β1-adrenergic receptor regulatory region containing consensus AP-2 elements recognizes novel transactivator proteins

Philbert Kirigiti, Yong Feng Yang, Xiaorong Li, Biao Li, Clare N. Midson, Curtis Machida

Research output: Contribution to journalArticle

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Abstract

β1-Adrenergic receptors (β1-ARs) serve as important regulators of central nervous system (CNS)-mediated behavior and several neural functions, including mood, memory, neuroendocrine control, and stimulation of autonomic function. Using β1-AR-luciferase reporter recombinants, we have previously determined that important β1-AR genetic elements controlling expression within the C6 glioma cell line are contained within the region -396 to -299, relative to the translational start site. By conducting progressive internal deletions of the rat β1-AR 5' flanking region and with the use of β1-AR- luciferase recombinants, we have verified that this region contains the primary β1-AR promoter and/or major regulatory elements. To begin the identification of protein factors involved in β1-AR transcriptional activity conferred by this β1-AR region and flanking sequences, we conducted electrophoretic mobility shift assays using defined β1-AR DNA subregion probes. One probe (GS-1), encompassing the region -396 to -367, was found to produce two major and two minor mobility shift complexes when bound to nuclear extracts from the β1-AR expresser C6 cell line. UV-crosslinking of DNA-protein complexes, coupled with DNase I digestion, indicated that this β1-AR region interacts with one major protein of approximately 117 kDa molecular weight and additional minor proteins. GS-1 DNA-protein complexes were observed using β1-AR expresser tissues in the CNS, including cortex, hippocampus, and olfactory bulb. No DNA-protein complexes were observed when using nuclear extracts from β1-AR nonexpresser tissues; in some cases, using L6 cells, previously characterized to express little or no β1-ARs, a reduction in intensities of the DNA-protein complexes was observed. Competition experiments indicate that nuclear protein binds to one of two subregions within the GS-1 sequence that contain AP-2-like consensus elements. Recombinant AP-2 protein will bind to both the β1-AR GS-1 promoter fragment and commercially available AP-2 consensus element control probes. Interestingly, using antibody supershift and immunoblotting experiments, no supershifts were observed and the major 117-kDa protein was not immunoreactive to antibodies recognizing either AP-2α or AP-2β. These results support our contention that this β1-AR regulatory region contains AP-2 consensus elements that recognize novel transactivator proteins. (C) 2000 Academic Press.

Original languageEnglish (US)
Pages (from-to)181-192
Number of pages12
JournalMolecular Cell Biology Research Communications
Volume3
Issue number3
DOIs
StatePublished - Mar 2000

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Trans-Activators
Nucleic Acid Regulatory Sequences
Adrenergic Receptors
Consensus
Proteins
DNA
Luciferases
Central Nervous System
Cell Line
Antibodies
5' Flanking Region
Olfactory Bulb
Deoxyribonuclease I
DNA Probes
Electrophoretic Mobility Shift Assay
Nuclear Proteins
Immunoblotting
Glioma

ASJC Scopus subject areas

  • Molecular Biology

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Rat β1-adrenergic receptor regulatory region containing consensus AP-2 elements recognizes novel transactivator proteins. / Kirigiti, Philbert; Yang, Yong Feng; Li, Xiaorong; Li, Biao; Midson, Clare N.; Machida, Curtis.

In: Molecular Cell Biology Research Communications, Vol. 3, No. 3, 03.2000, p. 181-192.

Research output: Contribution to journalArticle

Kirigiti, Philbert ; Yang, Yong Feng ; Li, Xiaorong ; Li, Biao ; Midson, Clare N. ; Machida, Curtis. / Rat β1-adrenergic receptor regulatory region containing consensus AP-2 elements recognizes novel transactivator proteins. In: Molecular Cell Biology Research Communications. 2000 ; Vol. 3, No. 3. pp. 181-192.
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