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

Persistent Link:
http://hdl.handle.net/10150/186772
Title:
Functional expression of alpha-2 adrenergic receptor subtypes in cultured mammalian cells.
Author:
Pepperl, David John.
Issue Date:
1994
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 and Toxicology; Graduate College
Degree Grantor:
University of Arizona
Committee Chair:
Regan, John W.

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleFunctional expression of alpha-2 adrenergic receptor subtypes in cultured mammalian cells.en_US
dc.creatorPepperl, David John.en_US
dc.contributor.authorPepperl, David John.en_US
dc.date.issued1994en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © 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.en_US
dc.description.abstractThe 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.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplinePharmacology and Toxicologyen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.chairRegan, John W.en_US
dc.contributor.committeememberHalpert, Jamesen_US
dc.contributor.committeememberLai, Josephineen_US
dc.contributor.committeememberLindell, Thomasen_US
dc.contributor.committeememberMiesfeld, Rogeren_US
dc.identifier.proquest9432841en_US
All Items in UA Campus Repository are protected by copyright, with all rights reserved, unless otherwise indicated.