G protein-coupled receptors (GPCRs) form the largest class of cell membrane receptors and belong to the most important drug targets that are exploited in the treatment of the majority of human diseases [1,2]. GPCRs sense a myriad of chemically diverse extracellular ligands and relay their information to the inner cell by activation-related large-scale conformational changes. Canonically, this results in the recruitment and activation of intracellular heterotrimeric G proteins [3,4]. Cells amplify these signals mainly through modulation of second messenger levels such as 3′,5′-cyclic adenosine monophosphate (cAMP) and calcium, eventually eliciting precise cellular functions. In addition to soluble second messengers, GPCR signaling affects the function of various ion channels and intracellular kinases, such as extracellular signal-regulated kinases (ERK). For a long time, GPCRs have been regarded as simple on/off switches that exist in an equilibrium of one inactive and one active state. In this light, essentially all GPCR drugs fall into three groups: orthosteric agonists that stimulate endogenous signaling pathways, orthosteric antagonists that block endogenous GPCR signaling, and allosteric modulators that bind to a topographically distinct site of the receptor protein and that can thus fine-tune orthosteric agonism or antagonism.

The development of novel biophysical techniques has drastically changed our current view on how GPCRs operate. It is now well established that GPCRs are very dynamic proteins that exist in an equilibrium of many different inactive and active states [5,6] and that can form dimers and higher-order oligomers [3,4]. Moreover, GPCRs can activate multiple members of different G-protein families, and recruit β-arrestin and other GPCR-interacting proteins. The conformational equilibrium of GPCRs can be modulated with ligands, and it has been shown that different ligands can stabilize different GPCR states. These different, ligand-specific active states can result in unique GPCR signaling profiles that are different from the profile induced by the respective endogenous ligand. This so-called “biased agonism” may have therapeutic advantages and exploration of this concept is a major focus of current drug development programs [7].

The discovery of spatiotemporal GPCR cell signaling adds further complexity to the diversity of GPCR activation and signaling [8]. It is now widely accepted that some GPCRs can reside at intracellular locations such as early endosomes and the trans-Golgi network (TGN) [9, 10, 11, 12, 13, 14, 15, 16]. GPCR activation at the cell membrane can additionally promote receptor internalization into these subcellular compartments. From those internal sites, GPCRs can resume cell signaling. In contrast to the confinement of G-protein activation to larger cell organelles, recent work on intracellular cAMP compartmentation has revealed that signaling modules downstream of GPCRs are spatially organized in so-called nanodomains [17∗∗, 18∗∗, 19, 20, 21]. Importantly, it has been suggested that the cellular location from which GPCR signaling occurs can define specific cellular functions in response to GPCR activation [22,23]. Along this line, it has further been demonstrated that disruption of such GPCR signaling compartmentation can cause noncommunicable diseases such as heart failure [24] and cancer [25] that are among the leading causes of human death.

Genetically encoded fluorescent biosensors have revolutionized our understanding of spatiotemporal aspects of GPCR signaling. Fluorescent biosensors are genetically encoded tools that allow visualization of cellular processes by singe-cell fluorescence microscopy. They can sense the concentration of specific signaling molecules, conformational changes of signaling proteins or their posttranslational modifications. Most biosensors report on those changes with a change in fluorescence intensity or fluorescence/bioluminescence resonance energy transfer (FRET/BRET). The technical details of biosensing have been discussed extensively in excellent recent reviews [26, 27, 28].

Here, we showcase novel exciting technologies based on the elegant use of genetically encoded fluorescent biosensors that have proven pivotal in the discovery of novel concepts in spatiotemporal signaling. We highlight recent updates in the GPCR field that have provided new conceptual insight into the complexity of spatiotemporal GPCR signaling using the GPCR/cAMP signaling axis as a paradigm. We hypothesize that the concepts discovered in spatiotemporal GPCR/cAMP signaling may be transferable to other GPCR-regulated signaling pathways such as kinase signaling or even to other receptor classes.