The Use of Synthetic Peptides to Probe the Interactions and Interrelations of the G-protein/ α2-adrenergic receptor/effector mechanism
McEwen, Dyke P.
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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, 𝛽, and 𝛾 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 𝛽𝛾-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 𝛽𝛾-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 𝛽 𝛾 dimer, also investigated in this project, was tested by cell treatments with an ansamycin drug, namely geldanamycin, followed by immunoblotting. The results show that 𝛽𝛾 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 𝛽𝛾 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.