When it comes to signal transduction, cellular context matters. The cellular context is crucial for understanding the diverse signaling outcomes mediated by different receptors including G protein coupled receptors (GPCRs). The localization of GPCRs to specific intracellular compartments dictates the spatial and temporal dynamics of signaling, ultimately shaping cellular responses1. For example, the lipid composition of intracellular membranes may influence GPCR dynamics and signaling outcomes, changing the receptor conformation and accessibility to ligands2.
Traditionally, GPCR signaling was believed to primarily occur at the plasma membrane. However, emerging evidence suggests that GPCRs also signal from intracellular membranes including endosomes, the Golgi apparatus, and the nuclear membrane, leading to spatially biased signaling responses. In the endosomes, sustained G protein signaling has been associated with prolonged interactions between GPCRs, G proteins, and arrestins on endosomal membranes3,4. This phenomenon has been demonstrated for receptors such as the parathyroid hormone receptor (PTHR) and the vasopressin receptor 2 (V2R). The formation of stable complexes between GPCRs and signaling effectors on endosomes supports multiple rounds of G protein activation, leading to sustained cellular responses5,6.
The role of β-arrestins in endosomal signaling has been extensively studied, highlighting their ability to scaffold various kinases and modulate downstream signaling pathways. Showing that this spatially biased signaling through arrestins can influence transcriptional responses and contribute to physiological processes such as meiosis in ovarian follicles and pain neurotransmission7,1.
In addition to endosomal signaling, GPCRs are also known to localize to the Golgi apparatus, where they can be activated by ligands and initiate signaling cascades. The Golgi-localized signaling of GPCRs such as the β1-adrenergic receptor (B1AR) and the thyroid-stimulating hormone receptor (TSHR) has been shown to regulate distinct cellular processes, including phospholipase C epsilon (PLCε)-dependent signaling and transcriptional responses mediated by CREB phosphorylation1,8,9.
Furthermore, GPCRs localized to the nuclear membrane have emerged as important regulators of gene transcription and cellular responses. Activation of nuclear GPCRs, such as the metabotropic glutamate receptor 5 (mGluR5), can lead to sustained calcium signaling and activation of transcription factors like CREB and Elk1, influencing synaptic plasticity and neuronal function10.
GPCRs have also been identified within the mitochondria. Initial investigations revealed the positioning of the cannabinoid CB1 receptor on the outer mitochondrial membrane of skeletal and myocardial cells, where stimulation of mitochondrial CB1 receptors by lipophilic agonists in these tissues was associated with the modulation of oxidative activity via enzymes involved in pyruvate metabolism11.
As the mechanisms underlying spatial bias in GPCR signaling involve both temporal and spatial components, sustained signaling responses from intracellular compartments can be regulated by the kinetics of signaling and the spatial organization of signaling effectors, including G proteins, adenylyl cyclases, and kinases. Therefore the presence of membrane microdomains and protein scaffolds, further contributes to the spatial regulation of GPCR signaling and downstream responses1,2.
The clinical relevance of understanding these signaling mechanism is linked with the fact that dysregulation of compartmentalized GPCR signaling has been implicated in various diseases. For instance, aberrant endosomal signaling by GPCRs has been linked to disorders such as cancer, neurodegenerative diseases, and cardiovascular disorders. Therefore, targeting specific intracellular compartments and signaling pathways associated with GPCRs holds promise for the development of novel therapeutic interventions12.
Tagging GPCRs expressed in cellular compartments such as the nucleus or endosomes presents several pharmaceutical challenges for many reasons. These compartments have distinct biochemical environments, which may affect the stability and functionality of the tagging molecules. For example, the nuclear envelope presents a barrier that restricts the passage of large molecules, making it challenging to deliver tagging agents effectively. Additionally, the conditions within endosomes, such as low pH and high protease activity, can degrade tagging molecules, reducing their effectiveness1,8,12.
Furthermore, targeting GPCRs in specific cellular compartments requires precise delivery mechanisms to ensure the tagging molecules reach their intended destination. This may involve the development of specialized delivery vehicles, such as nanoparticles or liposomes, capable of penetrating cellular membranes and delivering the tagging agents to the desired compartment.
Another challenge is the potential interference of tagging molecules with GPCR function. Tagging molecules may alter the conformation or activity of GPCRs, leading to unintended effects on downstream signaling pathways. Therefore, it is essential to design tagging strategies that minimize interference with GPCR function while allowing for accurate detection and localization.
Overall, the pharmaceutical challenges associated with tagging GPCRs expressed in cellular compartments require innovative approaches to overcome barriers related to stability, delivery, interference with GPCR function, and off-target effects.
References
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Gonçalves-Monteiro, S., Ribeiro-Oliveira, R., Vieira-Rocha, M. S., Vojtek, M., Sousa, J. B., & Diniz, C. (2021). Insights into Nuclear G-Protein-Coupled Receptors as Therapeutic Targets in Non-Communicable Diseases. Pharmaceuticals (Basel, Switzerland), 14(5), 439. https://doi.org/10.3390/ph14050439
Chen, K. E., Healy, M. D., & Collins, B. M. (2019). Towards a molecular understanding of endosomal trafficking by Retromer and Retriever. Traffic (Copenhagen, Denmark), 20(7), 465–478. https://doi.org/10.1111/tra.12649
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Lyga, S., Volpe, S., Werthmann, R. C., Götz, K., Sungkaworn, T., Lohse, M. J., & Calebiro, D. (2016). Persistent cAMP Signaling by Internalized LH Receptors in Ovarian Follicles. Endocrinology, 157(4), 1613–1621. https://doi.org/10.1210/en.2015-1945
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Godbole, A., Lyga, S., Lohse, M. J., & Calebiro, D. (2017). Internalized TSH receptors en route to the TGN induce local Gs-protein signaling and gene transcription. Nature communications, 8(1), 443. https://doi.org/10.1038/s41467-017-00357-2
Jong, Y. I., & O'Malley, K. L. (2017). Mechanisms Associated with Activation of Intracellular Metabotropic Glutamate Receptor, mGluR5. Neurochemical research, 42(1), 166–172. https://doi.org/10.1007/s11064-016-2026-6
Mendizabal-Zubiaga, J., Melser, S., Bénard, G., Ramos, A., Reguero, L., Arrabal, S., Elezgarai, I., Gerrikagoitia, I., Suarez, J., Rodríguez De Fonseca, F., Puente, N., Marsicano, G., & Grandes, P. (2016). Cannabinoid CB1 Receptors Are Localized in Striated Muscle Mitochondria and Regulate Mitochondrial Respiration. Frontiers in physiology, 7, 476. https://doi.org/10.3389/fphys.2016.00476
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