Investigation of the role of US28 subcellular location in the upregulation of oncomodulatory genes in the context of glioblastoma

US28 is a viral GPCR (vGPCR) encoded by the human cytomegalovirus (HCMV) reported to have an oncomodulatory role in GBM. US28 shares the highest homology to human chemokine receptors and can bind to a wide range of CC chemokines and the CX3C chemokine fractalkine. This enables chemokine sequestering believed to aid immunoevasion which is particularly important to HCMV as it is a latent virus.

Furthermore, US28 has a high constitutive activity which is reported to occur principally by Gαq. In addition, US28 constitutively internalises and this results in approximately 80 % of the US28 receptor pool located intracellularly predominantly within endosomes. <br><br>It was originally believed that GPCRs could only signal from the plasma membrane, however studies over the last two decades have demonstrated that receptor signalling can occur from intracellular locations, a term called compartmentalised signalling.

Studies using drugs or nanobodies targeting US28 at the plasma membrane have only achieved partial results, therefore we hypothesised that US28 predominantly signals from early endosomes. Using bioluminescence resonance energy transfer (BRET)-based biosensors, confocal microscopy and nanobodies combined with mini G protein technology, we demonstrate that US28 mainly activates Gαq from endosomes, with only a small proportion of signalling occurring at the plasma membrane.

As the constitutive phosphorylation of serine and threonine residues on the carboxy-terminal (CT) is in part responsible for the constitutive internalisation of US28, we rescued US28 at the plasma membrane by mutating all these residues to alanine and valine respectively. Using BRET, confocal microscopy and mini G proteins, we show that this mutant (termed as US28-PDT) is functional and induces a partial displacement of Gαq signalling from endosomes to plasma membrane.<br>To determine if the subcellular location of a Gαq-coupled receptor signalling impacts its transcriptional activity, we performed RNAseq on glioblastoma U251 cells expressing equivalent amount of Renilla luciferase II (RlucII)-fused US28 and US28-PDT, or RlucII alone.

We hypothesised that a displacement of receptor signalling location from endosomes to the plasma membrane would decrease the transcription of genes involved in the oncomodulatory role of US28 in GBM. This hypothesis was based on Gαs signalling from endosomes, which has a more potent impact on transcription than Gαs signalling from plasma membrane. Surprisingly, our results suggest that increasing expression and Gαq signalling of US28 at the plasma membrane increases US28-mediated upregulation of oncomodulatory genes, notably a range of important inflammatory cytokines linked to cancer, which were also more secreted.<br><br>Overall, our data show that despite a predominant proportion of US28 signalling through Gαq from endosomes, the small population of US28 signalling at plasma membrane rather than from endosomes drives gene transcription.

Understanding how signalling location shapes cellular responses in the context of GBM will help to design better treatments for HCMV-associated GBM. Importantly, our data also raise important questions related to the translation of Gαq signalling from endosomes into a cellular responses, such as gene regulation. Further studies are required to identify the cellular factors at the origin of the relationship between the spatial properties of US28 signalling and downstream responses.<br>