Rat β1-Adrenergic Receptor Regulatory Region Containing Consensus AP-2 Elements Recognizes Novel Transactivator Proteins

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.