PT  - JOURNAL ARTICLE
AU  - Michael Ritt
AU  - Sivaraj Sivaramakrishnan
TI  - Correlation between Activity and Domain Complementation in Adenylyl Cyclase Demonstrated with a Novel Fluorescence Resonance Energy Transfer Sensor
AID  - 10.1124/mol.115.101626
DP  - 2016 Apr 01
TA  - Molecular Pharmacology
PG  - 407--412
VI  - 89
IP  - 4
4099  - http://molpharm.aspetjournals.org/content/89/4/407.short
4100  - http://molpharm.aspetjournals.org/content/89/4/407.full
SO  - Mol Pharmacol2016 Apr 01; 89
AB  - Adenylyl cyclase (AC) activity relies on multiple effectors acting through distinct binding sites. Crystal structures have revealed the location of these sites, and biochemical studies have explored the kinetics of ACs, but the interplay between conformation and activity remains incompletely understood. Here, we describe a novel fluorescence resonance energy transfer (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 analogs and a P-site inhibitor, 2′-d3′-AMP. Finally, we demonstrate that this sensor is functional in live cells, wherein it reports forskolin-stimulated activity of AC.