The Use of Synthetic Peptides to Probe the Interactions and Interrelations of the G-protein/ α2-adrenergic receptor/effector mechanism
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Authors
McEwen, Dyke P.
Issue Date
1997
Type
Thesis
Language
en_US
Keywords
Alternative Title
Abstract
Over the past 10-15 years, extensive research has been completed on the structure and function of G-proteins within the cell. G-proteins are guanine nucleotide binding proteins whose main operation is to relay a signal from the activated receptor to the downstream effector. In this study, the adrenergic receptor, specifically, the a2-adrenergic receptor was the targeted receptor family. The G-protein family is able to couple to the α2-adrenergic receptor, changing its conformation and allowing agonist drugs to bind. The purposes of this project were to investigate the specific interactions of the G-proteins coupling to the adrenergic receptors, as well as to investigate the
interactions within the G-protein α, ��, and �� subunits.
Radioactive ligand binding assays were used to test the inhibition of G-protein/
receptor coupling by synthetic pep tides, synthesized from either type 2 adenylyl cyclase or from the blades of the ����-subunit propeller. From these binding assays, the results showed that two of the peptides did show inhibition of G-protein/ receptor coupling. The site of inhibition could not be determined from the assay, but it was concluded that the peptides were acting on the system to inhibit G-protein interactions with the a α2-adrenergic receptor. To further analyze the site of interaction of the peptides, the flow
cytometry technique was used to determine the site of the specific interactions
between the α and ����-subunits by the synthetic peptides. This would indicate
whether the site of interaction for the peptides was on the G-protein heterotrimer or it was located on the receptor. However, due to time constraints, no definite sites have been identified as positive sites of interaction for the synthetic peptides.
The formation of the �� �� dimer, also investigated in this project, was tested by cell treatments with an ansamycin drug, namely geldanamycin, followed by immunoblotting. The results show that ���� did not decrease in concentration within the cell after being exposed to the drug for various time periods. Therefore, the conclusion was made that the ���� dimer did not benefit from the proposed chaperone protein in the aid of protein folding. However, this conclusion is still under review. At the end of the project, the
specific mechanism of G-protein/ receptor coupling inhibition had not been
determined. However, various synthetic peptides were successful in
inhibiting the G-protein from coupling to the receptor.
Description
vii, 53 p.
Citation
Publisher
Kalamazoo College
License
U.S. copyright laws protect this material. Commercial use or distribution of this material is not permitted without prior written permission of the copyright holder.