RUI: Developmental Regulation of Hepatic Beta2-Adrenergic Receptor Gene Expression

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9728212 Baeyens Many hormones and drugs initiate their actions on cells by binding to specific cell surface receptors located in the plasma membrane. For the catecholamines, epinephrine and norepinephrine, the receptors are known as adrenergic receptors (ARs) and include two major types termed alpha1- and beta-ARs. Epinephrine and norepinephrine have important effects on liver metabolism including glycogenolysis and gluconeogenesis. Liver cells express both alpha- and beta2-AR subtypes that work by different second messenger systems to transduce catecholamine stimulation into biological effects. Both alpha1- and beta2-ARs regulate liver metabolism by promoting glycogenolysis through increased phosphorylase activity. Catecholamine control of glucose metabolism is age-dependent in male rat liver. In fetal and young male rats (less than 14 days old), epinephrine works through beta2-ARs to activate phosphorylase, while in adult male rats epinephrine acts almost exclusively through alpha1-ARs to increase phosphorylase activity. The changes in receptor levels with development are mirrored by changes in beta2-AR mRNA levels. There appears to be a multilevel regulatory control of beta2-AR gene expression in the developing rat liver since both the transcription rate of the beta2-AR gene and the stability of beta2-AR mRNA decline during early postnatal development in male rat hepatocytes. The working hypothesis upon which this research is based is that beta2-AR gene expression is dynamically regulated during early liver development at both the transcriptional and post-transcriptional levels. The goal of this study is to determine the nature of the mechanisms responsible for controlling beta2-AR gene expression during early hepatic development. To examine the basic transcriptional control mechanisms regulating beta2-AR gene expression in male rat hepatocytes during early development, key cis-element(s) will be located on the beta2-AR gene by transfecting hepatocytes of different ages with constructs cons isting of various parts of the regulatory region of the beta2-AR gene. Further characterization of key cis-elements and interacting trans-factors present in fetal and early postnatal hepatic nuclear extracts will be done using standard molecular techniques including gel mobility shift and DNase footprinting assays. To investigate the post-transcriptional control of beta2-AR gene expression during development, proteins that interact with and alter beta2-AR mRNA stability will be examined. One such protein, designated p85, is found only in postnatal hepatic cytosol and may serve as a destabilizing element. Using cross-linking techniques the binding site for p85 on the beta2-AR transcript will be precisely identified. Mechanisms by which p85 regulates transcript stability will be investigated by mutating bases on the beta2-AR gene which give rise to the binding site of p85 on beta2-AR mRNA. Following transfection of hepatocytes of different developmental ages with the mutated gene, total mRNA and beta2-AR receptor numbers will be determined. The primary purpose of this study is to determine why the liver beta2-AR gene switches off during early postnatal development. The rate at which the information is copied from the DNA into messenger RNA may decrease after birth or the mRNA may be less stable after birth. This study should lead to a better understanding of the mechanisms underlying the changes in gene expression that occur during mammalian development.