Real‐time examination of cAMP activity at relaxin family peptide receptors using a BRET‐based biosensor

摘要

Relaxin family peptide (RXFPs) 1‐4 receptors modulate the activity of cyclic adenosine monophosphate (cAMP) to produce a range of physiological functions. RXFP1 and RXFP2 increase cAMP via Gαs, whereas RXFP3 and RXFP4 inhibit cAMP via Gαi/o. RXFP1 also shows a delayed increase in cAMP downstream of Gαi3. In this study we have assessed whether the bioluminescence resonance energy transfer (BRET)‐based biosensor CAMYEL (cAMP sensor using YFP‐Epac‐Rluc), which allows real‐time measurement of cAMP activity in live cells, will aid in understanding ligand‐ and cell‐specific RXFP signaling. style="fixed-case">CAMYEL detected concentration‐dependent changes in style="fixed-case">cAMP activity at style="fixed-case">RXFP1‐4 in recombinant cell lines, using a variety of ligands with potencies comparable to those seen in conventional style="fixed-case">cAMP assays. We used style="fixed-case">RXFP2 and style="fixed-case">RXFP3 antagonists to demonstrate that style="fixed-case">CAMYEL detects dynamic changes in style="fixed-case">cAMP by reversing style="fixed-case">cAMP activation or inhibition respectively, with real‐time addition of antagonist after agonist stimulation. To demonstrate the utility of style="fixed-case">CAMYEL to detect style="fixed-case">cAMP activation in native cells expressing low levels of style="fixed-case">RXFP receptor, we cloned style="fixed-case">CAMYEL into a lentiviral vector and transduced style="fixed-case">THP‐1 cells, which express low levels of style="fixed-case">RXFP1. style="fixed-case">THP‐1 style="fixed-case">CAMYEL cells demonstrated robust style="fixed-case">cAMP activation in response to relaxin. However, the style="fixed-case">CAMYEL assay was unable to detect the Gαi3‐mediated phase of style="fixed-case">RXFP1 style="fixed-case">cAMP activation in style="fixed-case">PTX‐treated style="fixed-case">THP‐1 cells or style="fixed-case">HEK293A cells with knockout of Gαs. Our data demonstrate that cytoplasmically‐expressed style="fixed-case">CAMYEL efficiently detects real‐time style="fixed-case">cAMP activation by Gαs or inhibition by Gαi/o but may not detect style="fixed-case">cAMP generated in specific intracellular compartments such as that generated by Gαi3 upon RXFP1 activation.