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

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 Gα_s, whereas RXFP3 and RXFP4 inhibit cAMP via Gα_i/o. RXFP1 also shows a delayed increase in cAMP downstream of Gα_i3. 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. CAMYEL detected concentration‐dependent changes in cAMP activity at RXFP1‐4 in recombinant cell lines, using a variety of ligands with potencies comparable to those seen in conventional cAMP assays. We used RXFP2 and RXFP3 antagonists to demonstrate that CAMYEL detects dynamic changes in cAMP by reversing cAMP activation or inhibition respectively, with real‐time addition of antagonist after agonist stimulation. To demonstrate the utility of CAMYEL to detect cAMP activation in native cells expressing low levels of RXFP receptor, we cloned CAMYEL into a lentiviral vector and transduced THP‐1 cells, which express low levels of RXFP1. THP‐1 CAMYEL cells demonstrated robust cAMP activation in response to relaxin. However, the CAMYEL assay was unable to detect the Gα_i3‐mediated phase of RXFP1 cAMP activation in PTX‐treated THP‐1 cells or HEK293A cells with knockout of Gα_s. Our data demonstrate that cytoplasmically‐expressed CAMYEL efficiently detects real‐time cAMP activation by Gα_s or inhibition by Gα_i/o but may not detect cAMP generated in specific intracellular compartments such as that generated by Gα_i3 upon RXFP1 activation.

DOI: 10.1002/prp2.432

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