In this thesis I developed an assay based on BRET, in which receptor is tagged at the C-terminal with a luminescent donor and the G subunit of G protein is tagged at the N-terminal with a fluorescent acceptor. The extent of receptor-G protein coupling can be measured as change of the RET signal in membranes prepared from cells expressing the two tagged proteins. In this assay, the addition of GDP to the membranes can inhibit ligand-induced activity and abolish spontaneous receptor activity. Thus, the BRET signal in the presence of GDP marks the level of “zero coupling” between receptor and G protein, and the net difference between absence and presence of the guanine nucleotide is a precise measure of the constitutive activation. Ligands that enhance the BRET signal above the basal level are positive agonists, whereas those inhibiting the basal signal are inverse agonists
The mechanism of constitutive activity in delta and mu opioid receptors / Vezzi, Vanessa. - (2013 Mar 01).
The mechanism of constitutive activity in delta and mu opioid receptors
VEZZI, VANESSA
01/03/2013
Abstract
In this thesis I developed an assay based on BRET, in which receptor is tagged at the C-terminal with a luminescent donor and the G subunit of G protein is tagged at the N-terminal with a fluorescent acceptor. The extent of receptor-G protein coupling can be measured as change of the RET signal in membranes prepared from cells expressing the two tagged proteins. In this assay, the addition of GDP to the membranes can inhibit ligand-induced activity and abolish spontaneous receptor activity. Thus, the BRET signal in the presence of GDP marks the level of “zero coupling” between receptor and G protein, and the net difference between absence and presence of the guanine nucleotide is a precise measure of the constitutive activation. Ligands that enhance the BRET signal above the basal level are positive agonists, whereas those inhibiting the basal signal are inverse agonistsFile | Dimensione | Formato | |
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Tesi_Vanessa.Vezzi.pdf
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Note: PhD thesis
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