A virally encoded GPCR drives glioblastoma through feed-forward activation of the SK1-S1P1 signaling axis.

The G protein-coupled receptor (GPCR) US28 encoded by the human cytomegalovirus (HCMV) is associated with accelerated progression of glioblastomas, aggressive brain tumors with a generally poor prognosis. Here, we showed that US28 increased the malignancy of U251 glioblastoma cells by enhancing signaling mediated by sphingosine-1-phosphate (S1P), a bioactive lipid that stimulates oncogenic pathways in glioblastoma. US28 expression increased the abundance of the key components of the S1P signaling axis, including an enzyme that generates S1P [sphingosine kinase 1 (SK1)], an S1P receptor [S1P receptor 1 (S1P1)], and S1P itself. Enhanced S1P signaling promoted glioblastoma cell proliferation and survival by activating the kinases AKT and CHK1 and the transcriptional regulators cMYC and STAT3 and by increasing the abundance of cancerous inhibitor of PP2A (CIP2A), driving several feed-forward signaling loops. Inhibition of S1P signaling abrogated the proliferative and anti-apoptotic effects of US28. US28 also activated the S1P signaling axis in HCMV-infected cells. This study uncovers central roles for S1P and CIP2A in feed-forward signaling that contributes to the US28-mediated exacerbation of glioblastoma.

Exosomal release of the virus-encoded chemokine receptor US28 contributes to chemokine scavenging.

The human cytomegalovirus (HCMV)-encoded chemokine receptor US28 contributes to various aspects of the viral life cycle and promotes immune evasion by scavenging chemokines from the microenvironment of HCMV-infected cells. In contrast to the plasma membrane localization of most human chemokine receptors, US28 has a predominant intracellular localization. In this study, we used immunofluorescence and electron microscopy to determine the localization of US28 upon exogenous expression, as well as in HCMV-infected cells. We observed that US28 localizes to late endosomal compartments called multivesicular bodies (MVBs), where it is sorted in intraluminal vesicles. Live-cell total internal reflection fluorescence (TIRF) microscopy revealed that US28-containing MVBs can fuse with the plasma membrane, resulting in the secretion of US28 on exosomes. Exosomal US28 binds the chemokines CX3CL1 and CCL5, and US28-containing exosomes inhibited the CX3CL1-CX3CR1 signaling axis. These findings suggest that exosomal release of US28 contributes to chemokine scavenging and immune evasion by HCMV.

Fusicoccin-A Targets Cancerous Inhibitor of Protein Phosphatase 2A by Stabilizing a C-Terminal Interaction with 14-3-3.

The cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein found overexpressed in many types of cancer. CIP2A has been shown to stabilize oncoproteins such as cMYC by shielding them from PP2A-mediated dephosphorylation. Here we report that the penultimate residue Ser904 in the C-terminus of CIP2A can be phosphorylated to create a binding site for the regulatory protein 14-3-3. We demonstrate that 14-3-3 is a new interaction partner of CIP2A. The 14-3-3/CIP2A C-terminal interaction complex can be targeted by the protein-protein interaction (PPI) stabilizer fusicoccin-A (FC-A), resulting in enhanced levels of phosphorylated Ser904. FC-A treatment of TNBC cells leads to the increased association of CIP2A with 14-3-3. We show that the composite interface between 14 and 3-3 and CIP2A's C-terminus can be targeted by the PPI stabilizer FC-A, providing a new interface that could potentially be exploited to modulate CIP2A's activity.

Use of CRISPR/Cas9-edited HEK293 cells reveals that both conventional and novel protein kinase C isozymes are involved in mGlu5a receptor internalization.

The internalization of G protein-coupled receptors (GPCRs) can be regulated by PKC. However, most tools available to study the contribution of PKC isozymes have considerable limitations, including a lack of selectivity. In this study, we generated and characterized human embryonic kidney 293A (HEK293A) cell lines devoid of conventional or novel PKC isozymes (ΔcPKC and ΔnPKC) and employ these to investigate the contribution of PKC isozymes in the internalization of the metabotropic glutamate receptor 5 (mGlu5). Direct activation of PKC and mutation of rat mGlu5a Ser901, a PKC-dependent phosphorylation site in the receptor C-tail, both showed that PKC isozymes facilitate approximately 40% of the receptor internalization. Nonetheless, we determined that mGlu5a internalization was not altered upon the loss of cPKCs or nPKCs. This indicates that isozymes from both classes are involved, compensate for the absence of the other class, and thus fulfill dispensable functions. Additionally, using the Gαq/11 inhibitor YM-254890, GPCR kinase 2 and 3 (GRK2 and GRK3) KO cells, and a receptor containing a mutated putative adaptor protein complex 2 (AP-2) interaction motif, we demonstrate that internalization of rat mGlu5a is mediated by Gαq/11 proteins (77% of the response), GRK2 (27%), and AP-2 (29%), but not GRK3. Our PKC KO cell lines expand the repertoire of KO HEK293A cell lines available to research GPCR pharmacology. Moreover, since pharmacological tools to study PKC isozymes generally lack specificity and/or potency, we present the PKC KO cell lines as more specific research tools to investigate PKC-mediated aspects of cell biology.

Arrestin-Dependent and -Independent Internalization of G Protein-Coupled Receptors: Methods, Mechanisms, and Implications on Cell Signaling.

Agonist-induced endocytosis is a key regulatory mechanism for controlling the responsiveness of the cell by changing the density of cell surface receptors. In addition to the role of endocytosis in signal termination, endocytosed G protein-coupled receptors (GPCRs) have been found to signal from intracellular compartments of the cell. Arrestins are generally believed to be the master regulators of GPCR endocytosis by binding to both phosphorylated receptors and adaptor protein 2 (AP-2) or clathrin, thus recruiting receptors to clathrin-coated pits to facilitate the internalization process. However, many other functions have been described for arrestins that do not relate to their role in terminating signaling. Additionally, there are now more than 30 examples of GPCRs that internalize independently of arrestins. Here we review the methods, pharmacological tools, and cellular backgrounds used to determine the role of arrestins in receptor internalization, highlighting their advantages and caveats. We also summarize key examples of arrestin-independent GPCR endocytosis in the literature and their suggested alternative endocytosis pathway (e.g., the caveolae-dependent and fast endophilin-mediated endocytosis pathways). Finally, we consider the possible function of arrestins recruited to GPCRs that are endocytosed independently of arrestins, including the catalytic arrestin activation paradigm. Technological improvements in recent years have advanced the field further, and, combined with the important implications of endocytosis on drug responses, this makes endocytosis an obvious parameter to include in molecular pharmacological characterization of ligand-GPCR interactions. SIGNIFICANCE STATEMENT: G protein-coupled receptor (GPCR) endocytosis is an important means to terminate receptor signaling, and arrestins play a central role in the widely accepted classical paradigm of GPCR endocytosis. In contrast to the canonical arrestin-mediated internalization, an increasing number of GPCRs are found to be endocytosed via alternate pathways, and the process appears more diverse than the previously defined "one pathway fits all."

Dissecting the roles of GRK2 and GRK3 in µ-opioid receptor internalization and ß-arrestin2 recruitment using CRISPR/Cas9-edited HEK293 cells.

Most G protein-coupled receptors (GPCRs) recruit ß-arrestins and internalize upon agonist stimulation. For the µ-opioid receptor (µ-OR), this process has been linked to development of opioid tolerance. GPCR kinases (GRKs), particularly GRK2 and GRK3, have been shown to be important for µ-OR recruitment of ß-arrestin and internalization. However, the contribution of GRK2 and GRK3 to ß-arrestin recruitment and receptor internalization, remain to be determined in their complete absence. Using CRISPR/Cas9-mediated genome editing we established HEK293 cells with knockout of GRK2, GRK3 or both to dissect their individual contributions in ß-arrestin2 recruitment and µ-OR internalization upon stimulation with four different agonists. We showed that GRK2/3 removal reduced agonist-induced µ-OR internalization and ß-arrestin2 recruitment substantially and we found GRK2 to be more important for these processes than GRK3. Furthermore, we observed a sustained and GRK2/3 independent component of ß-arrestin2 recruitment to the plasma membrane upon µ-OR activation. Rescue expression experiments restored GRK2/3 functions. Inhibition of GRK2/3 using the small molecule inhibitor CMPD101 showed a high similarity between the genetic and pharmacological approaches, cross-validating the specificity of both. However, off-target effects were observed at high CMPD101 concentrations. These GRK2/3 KO cell lines should prove useful for a wide range of studies on GPCR function.

Human Cytomegalovirus-Encoded G Protein-Coupled Receptor UL33 Facilitates Virus Dissemination via the Extracellular and Cell-to-Cell Route.

Human cytomegalovirus (HCMV) encodes four G protein-coupled receptor (GPCR) homologs. Three of these receptors, UL78, US27 and US28, are known for their roles in HCMV dissemination and latency. Despite importance of its rodent orthologs for viral replication and pathogenesis, such a function is not reported for the HCMV-encoded GPCR UL33. Using the clinical HCMV strain Merlin, we show that UL33 facilitates both cell-associated and cell-free virus transmission. A UL33-deficient virus derivative revealed retarded virus spread, formation of less and smaller plaques, and reduced extracellular progeny during multi-cycle growth analysis in fibroblast cultures compared to parental virus. The growth of UL33-revertant, US28-deficient, and US28-revertant viruses were similar to parental virus under multistep growth conditions. UL33- and US28-deficient Merlin viruses impaired cell-associated virus spread to a similar degree. Thus, the growth defect displayed by the UL33-deficient virus but not the US28-deficient virus reflects UL33's contribution to extracellular transmission. In conclusion, UL33 facilitates cell-associated and cell-free spread of the clinical HCMV strain Merlin in fibroblast cultures.

Viral G protein-coupled receptors as modulators of cancer hallmarks.

Herpesviruses encode transmembrane G protein-coupled receptors (GPCRs), which share structural homology to human chemokine receptors. These viral GPCRs include KSHV-encoded ORF74, EBV-encoded BILF1, and HCMV-encoded US28, UL33, UL78 and US27. Viral GPCRs hijack various signaling pathways and cellular networks, including pathways involved in the so-called cancer hallmarks as defined by Hanahan and Weinberg. These hallmarks describe cellular characteristics crucial for transformation and tumor progression. The cancer hallmarks involve growth factor-independent proliferation, angiogenesis, avoidance of apoptosis, invasion and metastasis, metabolic reprogramming, genetic instability and immune evasion amongst others. The role of beta herpesviruses modulating these cancer hallmarks is clearly highlighted by the proliferative and pro-angiogenic phenotype associated with KSHV infection which is largely ascribed to the ORF74-mediated modulation of signaling networks in host cells. For HCMV and Epstein-Bar encoded GPCRs, oncomodulatory effects have been described which contribute to the cancer hallmarks, thereby enhancing oncogenic development. In this review, we describe the main signaling pathways controlling the hallmarks of cancer which are affected by the betaherpesvirus encoded GPCRs. Most prominent among these involve the JAK-STAT, PI(3)K-AKT, NFkB and MAPK signaling nodes. These insights are important to effectively target these viral GPCRs and their signaling networks in betaherpesvirus-associated malignancies.

The human cytomegalovirus-encoded G protein-coupled receptor UL33 exhibits oncomodulatory properties.

Herpesviruses can rewire cellular signaling in host cells by expressing viral G protein-coupled receptors (GPCRs). These viral receptors exhibit homology to human chemokine receptors, but some display constitutive activity and promiscuous G protein coupling. Human cytomegalovirus (HCMV) has been detected in multiple cancers, including glioblastoma, and its genome encodes four GPCRs. One of these receptors, US28, is expressed in glioblastoma and possesses constitutive activity and oncomodulatory properties. UL33, another HCMV-encoded GPCR, also displays constitutive signaling via Gαq, Gαi, and Gαs proteins. However, little is known about the nature and functional effects of UL33-driven signaling. Here, we assessed UL33's signaling repertoire and oncomodulatory potential. UL33 activated multiple proliferative, angiogenic, and inflammatory signaling pathways in HEK293T and U251 glioblastoma cells. Notably, upon infection, UL33 contributed to HCMV-mediated STAT3 activation. Moreover, UL33 increased spheroid growth in vitro and accelerated tumor growth in different in vivo tumor models, including an orthotopic glioblastoma xenograft model. UL33-mediated signaling was similar to that stimulated by US28; however, UL33-induced tumor growth was delayed. Additionally, the spatiotemporal expression of the two receptors only partially overlapped in HCMV-infected glioblastoma cells. In conclusion, our results unveil that UL33 has broad signaling capacity and provide mechanistic insight into its functional effects. UL33, like US28, exhibits oncomodulatory properties, elicited via constitutive activation of multiple signaling pathways. UL33 and US28 might contribute to HCMV's oncomodulatory effects through complementing and converging cellular signaling, and hence UL33 may represent a promising drug target in HCMV-associated malignancies.

Nanobody-Targeted Photodynamic Therapy Selectively Kills Viral GPCR-Expressing Glioblastoma Cells.

Photodynamic therapy (PDT) eradicates tumors by the local activation of a photosensitizer with near-infrared light. One of the aspects hampering the clinical use of PDT is the poor selectivity of the photosensitizer. To improve this, we have recently introduced a new approach for targeted PDT by conjugating photosensitizers to nanobodies. Diverse G protein-coupled receptors (GPCRs) show aberrant overexpression in tumors and are therefore interesting targets in cancer therapy. Here we show that GPCR-targeting nanobodies can be used in targeted PDT. We have developed a nanobody binding the extracellular side of the viral GPCR US28, which is detected in tumors like glioblastoma. The nanobody was site-directionally conjugated to the water-soluble photosensitizer IRDye700DX. This nanobody-photosensitizer conjugate selectively killed US28-expressing glioblastoma cells both in 2D and 3D cultures upon illumination with near-infrared light. This is the first example employing a GPCR as target for nanobody-directed PDT. With the emerging role of GPCRs in cancer, this data provides a new angle for exploiting this large family of receptors for targeted therapies.

The constitutive activity of the virally encoded chemokine receptor US28 accelerates glioblastoma growth.

Glioblastoma (GBM) is the most aggressive and an incurable type of brain cancer. Human cytomegalovirus (HCMV) DNA and encoded proteins, including the chemokine receptor US28, have been detected in GBM tumors. US28 displays constitutive activity and is able to bind several human chemokines, leading to the activation of various proliferative and inflammatory signaling pathways. Here we show that HCMV, through the expression of US28, significantly enhanced the growth of 3D spheroids of U251- and neurospheres of primary glioblastoma cells. Moreover, US28 expression accelerated the growth of glioblastoma cells in an orthotopic intracranial GBM-model in mice. We developed highly potent and selective US28-targeting nanobodies, which bind to the extracellular domain of US28 and detect US28 in GBM tissue. The nanobodies inhibited chemokine binding and reduced the constitutive US28-mediated signaling with nanomolar potencies and significantly impaired HCMV/US28-mediated tumor growth in vitro and in vivo. This study emphasizes the oncomodulatory role of HCMV-encoded US28 and provides a potential therapeutic approach for HCMV-positive tumors using the nanobody technology.

Human cytomegalovirus encoded chemokine receptor US28 activates the HIF-1α/PKM2 axis in glioblastoma cells.

The human cytomegalovirus (HCMV) encoded chemokine receptor US28 promotes tumorigenesis through activation of various proliferative and angiogenic signaling pathways. Upon infection, US28 displays constitutive activity and signals in a G protein-dependent manner, hijacking the host's cellular machinery. In tumor cells, the hypoxia inducible factor-1α/pyruvate kinase M2 (HIF-1α/PKM2) axis plays an important role by supporting proliferation, angiogenesis and reprogramming of energy metabolism. In this study we show that US28 signaling results in activation of the HIF-1α/PKM2 feedforward loop in fibroblasts and glioblastoma cells. The constitutive activity of US28 increases HIF-1 protein stability through a Gαq-, CaMKII- and Akt/mTOR-dependent mechanism. Furthermore, we found that VEGF and lactate secretion are increased and HIF-1 target genes, glucose transporter type 1 (GLUT1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), involved in glucose metabolism, are upregulated in US28 expressing cells. In addition, PKM2 is phosphorylated and found to be in a tumor-associated dimeric state upon US28 expression. Also in HCMV-infected cells HIF-1 activity is enhanced, which in part is US28-dependent. Finally, increased proliferation of cells expressing US28 is abolished upon inhibition of the HIF-1α/PKM2 cascade. These data highlight the importance of HIF-1α and PKM2 in US28-induced proliferation, angiogenesis and metabolic reprogramming.

Pharmacological characterization of [3H]VUF11211, a novel radiolabeled small-molecule inverse agonist for the chemokine receptor CXCR3.

Chemokine receptor CXCR3 has attracted much attention, as it is thought to be associated with a wide range of immune-related diseases. As such, several small molecules with different chemical structures targeting CXCR3 have been discovered. Despite limited clinical success so far, these compounds serve as interesting tools for investigating receptor activation and antagonism. Accumulating evidence suggests that many of these compounds are allosteric modulators for CXCR3. One feature of allosteric ligands is that the magnitude of the mediated allosteric effect is dependent on the orthosteric probe that is used. Consequently, there is a risk for incorrect assessment of affinity for allosteric modulators with orthosteric radioligands, which has so far been the most applied approach for chemokine receptors. Therefore, we aimed to use a small-molecule allosteric ligand from the piperazinyl-piperidine class, also known as VUF11211 [(S)-5-chloro-6-(4-(1-(4-chlorobenzyl)piperidin-4-yl)-3-ethylpiperazin-1-yl)-N-ethylnicotinamide]. VUF11211 acts as an inverse agonist at a constitutively active mutant of CXCR3. Radiolabeling of VUF11211 gave [(3)H]VUF11211, which in radioligand binding studies shows high affinity for CXCR3 (Kd = 0.65 nM) and reasonably fast association (kon= 0.03 minute(-1)nM(-1)) and dissociation kinetics (koff = 0.02 minute(-1)). The application of the [(3)H]VUF11211 to assess CXCR3 pharmacology was validated with diverse classes of CXCR3 compounds, including both antagonists and agonists, as well as VUF11211 analogs. Interestingly, VUF11211 seems to bind to a different population of CXCR3 conformations compared with the CXCR3 agonists CXC chemokine ligand 11 (CXCL11), VUF11418 [1-((1R,5S)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)-N-((2'-iodobiphenyl-4-yl)methyl)-N,N-dimethylmethanaminium Iodide], and VUF10661 [N-(6-amino-1-(2,2-diphenylethylamino)-1-oxohexan-2-yl)-2-(4-oxo-4-phenylbutanoyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide]. These findings, taken together, indicate that this allosteric inverse agonist radioligand for CXCR3 may facilitate the discovery, characterization, and optimization of allosteric modulators for the chemokine receptor CXCR3.