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| Daniel K Yee Ph.D. |
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| Research Associate Professor Department of Animal Biology |
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| Qualifications |
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1986 B.A. University of Pennsylvania, Philadelphia, PA 1991 Ph.D. Cornell University, Ithaca, NY, Biochemistry |
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| Research Interests |
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My longstanding interest in neuropeptides and their actions developed from my doctoral research, which involved the isolation and characterization of a potent neuroactive peptide from mammalian brain. Specifically, my thesis work involved the isolation of this novel neuroactive factor, which we named neurocatin, as well as extensive characterization of its effects on catecholamine and serotonin metabolism in rat brain synaptosomes and in human neuroblastoma cells. The peptide altered metabolism of these monoamine neurotransmitters by increasing the activity of both tyrosine hydroxylase and tryptophan hydroxylase, the rate-limiting steps in the synthesis of catecholamines and serotonin, respectively. In addition, neurocatin inhibited monoamine oxidase activity, the main catabolic enzyme for both neurotransmitter systems. The dramatic changes in monoamine metabolism suggested that this peptide may play a major role in the central nervous system. Following my thesis training, I sought to complement my previous research on the actions of neuropeptides through a thorough investigation of the cell surface receptors that mediate their actions. In developing my research program, I have utilized a broad range of methodologies involving molecular biology, pharmacology, and biochemistry. My current research is primarily focused on understanding the molecular properties of receptors for the peptide hormone angiotensin II (AngII), an important regulator of body fluid homeostasis and cardiovascular function. At least two main subtypes of AngII receptors, referred to as type 1 (AT1) and type 2 (AT2), have been identified. While the role of the AT1 receptor has been firmly established in the regulation of blood pressure and body fluid composition, the physiological role of the AT2 receptor has only recently begun to be understood. The extensive distribution of the AT2 receptor in fetal tissues and the more recent discovery that the AT2 receptor is capable of mediating programmed cell death point towards a role for it in controlling the development of various tissues. This role may even continue into the adult animal, as the AT2 receptor has been demonstrated to suppress coronary cell proliferation and neointima formation in carotid artery following balloon-injury. The ability to manipulate these processes via the AT2 receptor may hold some clinical benefit in controlling wound healing and the treatment of heart disease. Both AngII receptor subtypes have been cloned and conform to the seven transmembrane structural motif of G-protein coupled receptors (GPCRs). Interestingly, although the two subtypes bind AngII with identical affinities (KD of 3-5 nM), they share a relatively low 34% amino acid homology. Use of molecular biological techniques to introduce specific mutations into proteins has proven to be an invaluable research strategy in the analysis of protein structure and function. With respect to GPCRs, this strategy has been extensively used to map the structural elements that define ligand binding, G-protein coupling, and receptor activation of several receptor systems. In the study of AngII receptors, mutagenesis experiments have been conducted primarily on the AT1 subtype, yielding information about AT1 binding epitopes and structural elements that are important for receptor activation and G-protein coupling. These mutational data have led to several computer models of the AT1 receptor. In contrast, analogous information on the AT2 subtype has been lacking. Moreover, since the two subtypes share a relatively low level of homology, the extent to which the AT1 mutagenesis and modeling data are applicable to the AT2 subtype remains equivocal. Thus, my primary research focus has been on using mutational approaches, i.e. systematically generating point mutants and chimeric AT1/AT2 receptors, to study the AT2 receptor. My goal is to integrate the growing AT2 mutational data into an AT2 computer model. While my research has begun to identify critical structural elements that define AT2 receptor binding and function, the AT2 mutational data also provides further insights into the AT1 subtype. By comparing and contrasting AT1 and future AT2 models, a greater understanding of the molecular properties of the entire AngII receptor family may be established. Additional research projects that are ongoing include identifying and investigating possible AT2-mediated signal transduction pathways, which has been elusive and controversial for this subtype. In addition, because some neuronal cells coexpress both receptor subtypes, I am interested in studying the possible functional implications of AT1 and AT2 receptors “cross-talk,” either through direct receptor-receptor interactions or through post-receptor events. Finally, receptor mutagenesis and the resultant computer models are largely indirect assessments of a receptor’s structural architecture. More direct use of biochemical and biophysical approaches of protein analysis are precluded due to limitations of protein expression for receptors, i.e. relatively low protein yields in currently used expression systems. Accordingly, I have been experimenting with a variety of heterologous expression systems for the overproduction of AngII receptor proteins. Such a system could potentially allow for more direct biophysical and biochemical measurements of receptor-ligand interactions than are currently possible. The approaches that I use in my research of AngII receptors are clearly applicable to the study of other peptide receptor systems. Thus, I frequently interact with my colleagues here at the University of Pennsylvania who study the actions of peptides that control feeding and satiety, such as melanocortins and neuropeptide Y. |
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