TY - JOUR
T1 - Rat β1-adrenergic receptor regulatory region containing consensus AP-2 elements recognizes novel transactivator proteins
AU - Kirigiti, Philbert
AU - Yang, Yong Feng
AU - Li, Xiaorong
AU - Li, Biao
AU - Midson, Clare N.
AU - Machida, Curtis A.
N1 - Funding Information:
P.K. and Y.-F.Y. contributed equally to this work. The authors thank Dr. Guibao Gu for his guidance in conducting the rat brain dissections described in this article. We thank Dr. Steven Nordeen (University of Colorado) for the use of the luciferase expression vectors pXP1 and control pT109luc, and Dr. Kim Neve for the acquisition of the C6 glioma cell line. We acknowledge the help of Yibing Jia and Kalama Taylor of the ORPRC Molecular Biology and Cell Culture Core Facilities, respectively, for their assistance in automated DNA sequencing and cell culture propagation. The authors also acknowledge the artistic support of Joel Ito, and the secretarial assistance of Carol Houser. We also thank Dr. Robert Searles for his help in the design of the progressive internal deletion recombinants constructed during early phases of this project. We also thank other current members of the Machida Laboratory, specifically Ying Bai, Susan Nguyen, and Matt Palmer for their help and support during the development of this article. C.M.N. is now at Pacific University School of Optometry in Forest Grove, Oregon. C.A.M. is supported by NIH Grants RR00163, HL 42358, and DK 53462 and was a former recipient of an American Heart Association Established Investiga-torship. Y.-F.Y. was the recipient of a 1997 American Heart Associ- ation, Oregon Affiliate, Postdoctoral Fellowship. B.L. was a 1997 Leukemia Research Foundation Postdoctoral Fellow.
PY - 2000/3
Y1 - 2000/3
N2 - 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.
AB - 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.
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U2 - 10.1006/mcbr.2000.0212
DO - 10.1006/mcbr.2000.0212
M3 - Article
C2 - 10860867
AN - SCOPUS:0033921969
SN - 1522-4724
VL - 3
SP - 181
EP - 192
JO - Molecular Cell Biology Research Communications
JF - Molecular Cell Biology Research Communications
IS - 3
ER -