RT Journal Article
SR Electronic
T1 Correlation between Activity and Domain Complementation in Adenylyl Cyclase Demonstrated with a Novel FRET Sensor
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP mol.115.101626
DO 10.1124/mol.115.101626
A1 Michael Ritt
A1 Sivaraj Sivaramakrishnan
YR 2016
UL http://molpharm.aspetjournals.org/content/early/2016/01/21/mol.115.101626.abstract
AB Adenylyl cyclase (AC) activity relies on multiple effectors acting through distinct binding sites. While crystal structures have revealed the location of these sites and biochemical studies have explored the kinetics of ACs, the interplay between conformation and activity remains incompletely understood. Here, we describe a novel FRET sensor that functions both as a soluble cyclase and a reporter of complementation within the catalytic domain. We report a strong linear correlation between catalytic domain complementation and cyclase activity upon stimulation with forskolin and GαS. Exploiting this, we dissect the mechanism of action of a series of forskolin analogues and a P-site inhibitor, 2'-d3'-AMP. Lastly, we demonstrate that this sensor is functional in live cells, wherein it reports forskolin-stimulated activity of AC.