Transcriptional regulation of neuronal nicotinic acetylcholine receptor genes. A possible role for the DNA-binding protein Puralpha.
Nicotinic acetylcholine receptors constitute a multigene family (alpha2-alpha9, beta2-beta4) expressed in discrete temporal and spatial patterns within the nervous system. The receptors are critical for proper signal transmission between neurons and their targets. The molecular mechanisms underlying receptor gene expression have not been completely elucidated but clearly involve regulation at the level of transcription. We previously identified a novel 19-base pair (bp) transcriptional regulatory element in the promoter region of the rat beta4 subunit gene. This 19-bp element interacts specifically with DNA-binding proteins enriched in nuclear extracts prepared from adult rat brain. Using a combination of cellulose-phosphate, DNA-cellulose, and DNA sequence-specific affinity chromatographies, we purified the 19-bp element binding activity approximately 19,000-fold. Analysis by denaturing gel electrophoresis revealed the presence of four polypeptides in the most purified fraction, ranging in molecular masses between 31 and 114 kDa. Peptide sequence analysis revealed that one of the polypeptides is the bovine homologue of the transcriptional regulatory factor, Puralpha. Electrophoretic mobility shift assays indicated that Puralpha interacts directly and specifically with the 19-bp element. In addition, mobility shift assays using an anti-Puralpha monoclonal antibody revealed the presence of Puralpha, or an immunologically related protein, in nuclear extracts prepared from brain tissue. We hypothesize that the interaction between Puralpha and the 19-bp element is critical for proper expression of the beta4 subunit gene.
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Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center, San Antonio, Texas 78245-3207, USA.
Tomkinson
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Gardner
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D017124:Conserved Sequence; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D051381:Rats; D011978:Receptors, Nicotinic / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D014157:Transcription Factors; D014158:Transcription, Genetic
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors