Rat beta 1-adrenergic receptor regulatory region containing consensus AP-2 elements recognizes novel transactivator proteins.
beta 1-Adrenergic receptors (beta1-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 beta 1-AR-luciferase reporter recombinants, we have previously determined that important beta 1-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 beta 1-AR 5' flanking region and with the use of beta 1-AR-luciferase recombinants, we have verified that this region contains the primary beta 1-AR promoter and/or major regulatory elements. To begin the identification of protein factors involved in beta 1-AR transcriptional activity conferred by this beta 1-AR region and flanking sequences, we conducted electrophoretic mobility shift assays using defined beta 1-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 beta 1-AR expresser C6 cell line. UV-crosslinking of DNA-protein complexes, coupled with DNase I digestion, indicated that this beta 1-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 beta 1-AR expresser tissues in the CNS, including cortex, hippocampus, and olfactory bulb. No DNA-protein complexes were observed when using nuclear extracts from beta 1-AR nonexpresser tissues; in some cases, using L6 cells, previously characterized to express little or no beta1-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 beta 1-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-2 alpha or AP-2 beta. These results support our contention that this beta 1-AR regulatory region contains AP-2 consensus elements that recognize novel transactivator proteins.
Kirigiti
P
P
Division of Neuroscience, Oregon Regional Primate Research Center, Beaverton, Oregon, 97006, USA.
Yang
Y F
YF
Li
X
X
Li
B
B
Midson
C N
CN
Machida
C A
CA
D000818:Animals; D001483:Base Sequence; D002490:Central Nervous System / Q000378:metabolism; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004586:Electrophoresis; D016133:Polymerase Chain Reaction; D051381:Rats; D018342:Receptors, Adrenergic, beta-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D015534:Trans-Activators / Q000378:metabolism*; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D004268:DNA-Binding Proteins; D018342:Receptors, Adrenergic, beta-1; D011994:Recombinant Proteins; D015534:Trans-Activators; D050656:Transcription Factor AP-2; D014157:Transcription Factors