Modulation of adrenergic-mediated signaling by cell swelling: a fluorescence spectroscopy investigation

Introduction: Osmotic swelling is a physiological process, which takes place in several pathological conditions like the ischemia/reperfusion scenario or cellular acidosis. It is known to cause substantial alteration to intracellular architecture, ranging from membrane curvature to actin polymerization, altering the environment of the transmembrane proteins.

Objectives: Our aim is to study if and how the signaling of G protein-coupled receptors, a key family of transmembrane signaling proteins, is altered in the context of cell swelling and to determine how their signaling efficiency can be affected at the molecular level by alterations to the subplasmalemmal biophysical environment.

Materials and methods: Here, we employ a comprehensive set of fluorescence-based methods, ranging from FRET-based biosensors to nanobody recruitment assays, to dissect the individual steps of the signaling cascade, from upstream stimulation of ß-adrenergic receptors (ß-AR) down to cAMP production. Results: Notably, we observe that swollen cells yield increased cAMP production in response to stimulation of Gα_s-coupled receptors. Upon direct adenylyl cyclase stimulation with forskolin, cell swelling increases cAMP production, but fails to elicit comparable effects when using a Gα_s-knockout cell line. Further studying the ß2-AR, we don't observe any changes to ligand binding affinity by osmotic swelling. At the same time, nanobody recruitment experiments show an increased extent of receptor activation along with elevated levels of active G_s. Based on these results, we hypothesize that signal transduction by the G_s-coupled receptors and the Gαs-protein is enhanced by the intracellular reorganization arising upon cell swelling.

Conclusion: Our findings uncover a novel effect of cell swelling on cellular signaling, but also open interesting perspectives on whether what we observe here is a general mechanism common to other GPCRs, with biophysical changes of the local environment of a receptor acting as an allosteric modulator of its function.