G protein-coupled receptors (GPCRs) constitute the largest class of transmembrane receptors and regulate several key physiological processes, such as neurotransmission.
In addition to the plasma membrane (PM), GPCRs localize to various intracellular compartments, including nuclear membranes, endoplasmic reticulum (ER), the Golgi apparatus, endosomes, and mitochondria. Yet, GPCR function has been mainly studied with a focus on cell surface-localized receptors, not capturing the possible influence of subcellular GPCR localization on downstream signaling. As GPCRs are key therapeutic targets, deciphering their diverse signaling mechanisms within cells is crucial for the development of future drugs.
Activation of PM-localized GPCRs by ligands triggers the recruitment and activation of several transducers, including heterotrimeric G-proteins. Activated G-proteins then trigger downstream signaling cascades, that can involve changes in intracellular cyclic adenosine monophosphate (cAMP) concentration. Activated PM GPCRs also recruit members of the GPCR kinase (GRK) family, which phosphorylate the receptor at multiple sites. Phosphorylated GPCRs then bind to β-arrestins, which often induces their internalization. In contrast, the recruitment of transducers upon activation of Golgi-localized GPCRs remains largely uncharacterized.
i) Here, we investigated the dynamic recruitment of transducers to activated PM- and Golgi-localized opioid receptors (ORs) using a combination of fluorescence and luminescence-based assays. We revealed a location-dependent bias in transducer recruitment. More specifically, β-arrestins and a specific miniG probe selectively interacted with activated PM-localized ORs, directly demonstrating location bias in OR signal transduction. Further, through collaborative work using molecular dynamics simulation, we uncovered a likely role of the lipid environment in promoting the location-specific OR coupling. Finally, we observed distinct effects of activated PM- and Golgi-localized ORs on transcription and protein phosphorylation.
ii) Using phosphosite-specific antibodies, we also demonstrate that mu-OR (MOR) and delta-OR (DOR) are differentially regulated via phosphorylation at the Golgi apparatus. While both Golgi- and PM-localized MOR were phosphorylated upon agonist stimulation, DOR phosphorylation was restricted to the PM. We further utilized CRISPR knock-out cell lines lacking specific members of the GRK family and GRK inhibitors to dissect the specificity of MOR and DOR interactions with individual kinases and with β-arrestins.
iii) The subcellular localization of GPCRs in the Golgi apparatus has been extensively studied only for a small fraction of GPCRs. To expand the insights into subcellular GPCR distribution, we cloned 120 GPCRs mainly belonging to the class A family and profiled their cellular localizations via immunostaining, high throughput imaging, and automated image quantification. The approach revealed the intracellular retention and Golgi localization in a large subset of analyzed GPCRs.
Taken together, the thesis uncovers novel aspects of location bias between PM and Golgi-localized GPCRs, with a major focus on the clinically relevant ORs, and reveals that Golgi localization is a common feature of the GPCR family.
