The long term objective of this project is to relate adrenergic receptor (AR) molecular structure to hormone/drug interaction and evoked physiologic responses. Within this context, the overall focus of this project is transcriptional regulation of the human alpha1aAR. alpha1ARs are an important component in benign prostatic hypertrophy (BPH), a disease occurring in elderly men. Three alpha1ARs have been identified using molecular and ligand binding approaches. The PI recently demonstrated that the predominant subtype of alpha1AR mRNA in human prostate is the alpha1aAR, specifically localized to prostate stroma; this finding has recently been confirmed at a protein level with ligand binding and contraction studies. Because of its link to BPH, it is important to understand the function of the human alpha1aAR. Therefore we plan to test the hypothesis that increases in prostate alpha1AR transcription are important in the development of BPH.
Specific aims i nclude cloning of 4-5 kb 5' untranslated region (UTR) of the human alpha1aAR followed by determination of the site of initiation of alpha1aAR transcription in human cells (using primer extension analysis and RNase protection assays). Studies characterizing regulation of alpha1aAR transcription in human cells will include designing, creating, and testing luciferase reporter constructs containing varying amounts of 5, UTR from the alpha1aAR, and confirming these results using mutagenesis approaches. Identification and location of binding sites for proteins involved in alpha1aAR transcription will be accomplished using DNase I footprinting and gel shift mobility assays. Comparison of alpha1aAR mRNA regulation in different human cell lines will follow. Finally, characterization of human prostate primary cultures with cell-type specific antibodies, alpha1AR ligand binding, and RNase protection assays will occur. If possible, alpha1AR mRNA regulation experiments will be performed in human smooth muscle primary cultures. Extensive experience and expertise in the molecular pharmacology of alpha1ARs, availability of human alpha1AR cDNAs, stable cell lines expressing each human alpha1AR subtype, and recent cloning of 1.5kb of human alpha1a 5' UTR, places us in a unique position to evaluate transcriptional regulation of human alpha1aARs. Information derived from these studies should ultimately prove useful in understanding the mechanism of human diseases such as benign prostatic hypertrophy.
| Schwinn, Debra A; Roehrborn, Claus G (2008) Alpha1-adrenoceptor subtypes and lower urinary tract symptoms. Int J Urol 15:193-9 |
