G protein‐coupled cell surface receptors (GPCR) trigger complex intracellular signaling cascades upon agonist binding. Classic pharmacological assays provide information about binding affinities, activation or blockade at different stages of the signaling cascade, but real time dynamics and reversibility of these processes remain often disguised. We show that combining photochromic NPY receptor ligands, which can be toggled in their receptor activation ability by irradiation with light of different wavelengths, with whole cell label‐free impedance assays allows observing the cell response to receptor activation and its reversibility over time. The concept demonstrated on NPY receptors may be well applicable to many other GPCRs providing a deeper insight into the time course of intracellular signaling processes.

Due to its high metastatic potential, malignant melanoma is one of the deadliest skin cancers. In melanoma as well as in other cancers, acidification of the tumor microenvironment (=TME, inverse pH‐gradient) is a well‐known driver of tumor progression and metastasis. Membrane‐bound receptors, such as the proton‐sensitive GPCR (pH‐GPCR) GPR4, are considered as potential initiators of the signalling cascades relevant to malignant transformation. In this study, we investigated the pH‐dependent migration of GPR4 wildtype/overexpressing SK‐Mel‐28 cells using an impedance‐based electrical wounding and migration assay and classical Boyden chamber experiments. Migration of GPR4 overexpressing SK‐Mel‐28 cells was enhanced in a range of pH 6.5–7.5 as compared to controls in the impedance‐based electrical wounding and migration assay. In Boyden chamber experiments, GPR4 overexpression only increased migration at pH 7.5 in a Matrigel‐free setup, but not at pH 6.5. Results indicate that GPR4 is involved in the migration of melanoma cells, especially in the tumor periphery, and that this process is affected by pH in the TME.

G protein‐coupled cell surface receptors (GPCR) trigger complex intracellular signaling cascades upon agonist binding. Classic pharmacological assays provide information about binding affinities, activation or blockade at different stages of the signaling cascade, but real time dynamics and reversibility of these processes remain often disguised. We show that combining photochromic NPY receptor ligands, which can be toggled in their receptor activation ability by irradiation with light of different wavelengths, with whole cell label‐free impedance assays allows observing the cell response to receptor activation and its reversibility over time. The concept demonstrated on NPY receptors may be well applicable to many other GPCRs providing a deeper insight into the time course of intracellular signaling processes.