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    Functional expression of alpha-2 adrenergic receptor subtypes in cultured mammalian cells

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    Author
    Pepperl, David John.
    Issue Date
    1994
    Committee Chair
    Regan, John W.
    
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    Publisher
    The University of Arizona.
    Rights
    Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
    Abstract
    The ɑ₂ adrenergic receptors are among the most extensively studied members of the G-protein coupled receptor superfamily. They have been purified from native tissue and cloned from a number of species. Presently, three pharmacologically distinct subtypes of ɑ₂ adrenergic receptors have been identified, termed the ɑ₂-C10, ɑ₂-C2 and ɑ₂-C4. Although stable expression of these proteins in suitable host cells is commonly used for studying the pharmacology and 2nd messenger coupling of these proteins, stable expression systems are extremely time-consuming. Therefore, one focus of this work was to develop a more efficient approach for studying ɑ₂ adrenergic receptor-2nd messenger coupling. A transient gene expression system should dramatically decrease the time required for studying receptor function. Using a cAMP-dependent reporter plasmid and a responsive cell system, we have demonstrated transient functional expression of ɑ₂ adrenergic receptor subtypes. Agonist activation of these receptor subtypes produces unique intracellular responses, suggesting specific receptor-effector interactions within the transfected cells. To directly address these interactions, stable cell lines expressing the ɑ₂ receptor subtypes were developed. Both the ɑ₂-C4 and ɑ₂-C10 receptor subtypes can be stably-expressed at relatively high levels in these cells. All three subtypes expressed in this cell line exhibited the pharmacology appropriate for their respective subtypes. Moreover, agonist activation of both ɑ₂-C4 and ɑ₂-C10 receptors in these cells produced identical dose-dependent inhibition of cAMP production. These studies have demonstrated that ɑ₂ adrenergic receptors can be expressed in human choriocarcinoma cells, and that agonist activation of these subtypes produces unique intracellular responses. This approach has also demonstrated the potential for regulation of gene expression by ɑ₂ adrenergic receptors. Most importantly however, development of a more rapid functional expression system has dramatically increased our ability to study ɑ₂ adrenergic receptor function. In the future, transient expression in JEG-3 cells should provide a useful tool for examining the effect of mutation on ɑ₂ adrenergic receptor function. Further studies should address functional expression of other G-protein coupled receptors as well as help define the structural basis for ɑ₂adrenergic receptor activity.
    Type
    text
    Dissertation-Reproduction (electronic)
    Degree Name
    Ph.D.
    Degree Level
    doctoral
    Degree Program
    Pharmacology & Toxicology
    Graduate College
    Degree Grantor
    University of Arizona
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