Search

Glucagon is secreted from pancreatic α cells, and hypersecretion (hyperglucagonemia) contributes to diabetic hyperglycemia. Molecular heterogeneity in hyperglucagonemia is poorly investigated. By screening human plasma using high-resolution-proteomics, we identified several glucagon variants, among which proglucagon 1-61 (PG 1-61) appears to be the most abundant form. PG 1-61 is secreted in subjects with obesity, both before and after gastric bypass surgery, with protein and fat as the main drivers for secretion before surgery, but glucose after. Studies in hepatocytes and in β cells demonstrated that PG 1-61 dose-dependently increases levels of cAMP, through the glucagon receptor, and increases insulin secretion and protein levels of enzymes regulating glycogenolysis and gluconeogenesis. In rats, PG 1-61 increases blood glucose and plasma insulin and decreases plasma levels of amino acids in vivo. We conclude that glucagon variants, such as PG 1-61, may contribute to glucose regulation by stimulating hepatic glucose production and insulin secretion.

Glucagon is secreted from pancreatic α cells, and hypersecretion (hyperglucagonemia) contributes to diabetic hyperglycemia. Molecular heterogeneity in hyperglucagonemia is poorly investigated. By screening human plasma using high-resolution-proteomics, we identified several glucagon variants, among which proglucagon 1-61 (PG 1-61) appears to be the most abundant form. PG 1-61 is secreted in subjects with obesity, both before and after gastric bypass surgery, with protein and fat as the main drivers for secretion before surgery, but glucose after. Studies in hepatocytes and in β cells demonstrated that PG 1-61 dose-dependently increases levels of cAMP, through the glucagon receptor, and increases insulin secretion and protein levels of enzymes regulating glycogenolysis and gluconeogenesis. In rats, PG 1-61 increases blood glucose and plasma insulin and decreases plasma levels of amino acids in vivo. We conclude that glucagon variants, such as PG 1-61, may contribute to glucose regulation by stimulating hepatic glucose production and insulin secretion.

Varicella zoster virus (VZV) is a highly prevalent human pathogen that establishes latency in neurons of the peripheral nervous system. Primary infection causes varicella whereas reactivation results in zoster, which is often followed by chronic pain in adults. Following infection of epithelial cells in the respiratory tract, VZV spreads within the host by hijacking leukocytes, including T cells, in the tonsils and other regional lymph nodes, and modifying their activity. In spite of its importance in pathogenesis, the mechanism of dissemination remains poorly understood. Here we addressed the influence of VZV on leukocyte migration and found that the purified recombinant soluble ectodomain of VZV glycoprotein C (rSgC) binds chemokines with high affinity. Functional experiments show that VZV rSgC potentiates chemokine activity, enhancing the migration of monocyte and T cell lines and, most importantly, human tonsillar leukocytes at low chemokine concentrations. Binding and potentiation of chemokine activity occurs through the C-terminal part of gC ectodomain, containing predicted immunoglobulin-like domains. The mechanism of action of VZV rSgC requires interaction with the chemokine and signalling through the chemokine receptor. Finally, we show that VZV viral particles enhance chemokine-dependent T cell migration and that gC is partially required for this activity. We propose that VZV gC activity facilitates the recruitment and subsequent infection of leukocytes and thereby enhances VZV systemic dissemination in humans.

Immunotoxins as antiviral therapeutics are largely unexplored but have promising prospective due to their high selectivity potential and their unparalleled efficiency. One recent example targeted the virus-encoded G protein-coupled receptor US28 as a strategy for specific and efficient treatment of human cytomegalovirus (HCMV) infections. US28 is expressed on virus-infected cells and scavenge chemokines by rapid internalization. The chemokine-based fusion-toxin protein (FTP) consisted of a variant (F49A) of CX3CL1 specifically targeting US28 linked to the catalytic domain of Pseudomonas exotoxin A (PE). Here, we systematically seek to improve F49A-FTP by modifications in its three structural domains; we generated variants with (1) altered chemokine sequence (K14A, F49L, and F49E), (2) shortened and elongated linker region, and (3) modified toxin domain. Only F49L-FTP displayed higher selectivity in its binding to US28 versus CX3CR1, the endogenous receptor for CX3CL1, but this was not matched by a more selective killing of US28-expressing cells. A longer linker and different toxin variants decreased US28 affinity and selective killing. Thereby, F49A-FTP represents the best candidate for HCMV treatment. Many viruses encode internalizing receptors suggesting that not only HCMV but also, for instance, Epstein-Barr virus and Kaposi’s sarcoma-associated herpesvirus may be targeted by FTPs.

Human and mouse chronic lymphocytic leukemia (CLL) develop from CD5+ B cells that in mice and macaques are known to define the distinct B1a B cell lineage. B1a cells are characterized by lack of germinal center development and the B1a cell population is increased in mice with reduced germinal center formation. As a major mediator of follicular B cell migration, the G protein-coupled receptor (GPCR) Epstein Barr virus-induced gene 2 (EBI2 or GPR183) directs B cell migration in the lymphoid follicles in response to its endogenous ligands, oxysterols. Thus, upregulation of EBI2 drives the B cells towards the extrafollicular area, whereas downregulation is essential for germinal center formation. We therefore speculated whether increased expression of EBI2 would lead to an expanded B1 cell subset and, ultimately, progression to chronic lymphocytic leukemia. Here we demonstrate that B cell-targeted expression of human EBI2 in mice reduces germinal center-dependent immune responses, reduces total IgM and IgG levels, and leads to increased proliferation and upregulation of cellular oncogenes. Furthermore, hEBI2 overexpression leads to an abnormally expanded CD5+ B1a B cell subset present as early as 4 days after birth, late-onset lymphoid cancer development and premature death. These findings are highly similar to those observed in CLL patients and identify EBI2 as a promoter of B cell malignancies.

The human cytomegalovirus-encoded G protein-coupled receptor US28 is a constitutively active receptor, which can recognize various chemokines. Despite the recent determination of its 2.9 angstrom crystal structure, potent and US28-specific tool compounds are still scarce. Here, we used structural information from a refined US28: VUF2274 complex for virtual screening of >12 million commercially available small molecule compounds. Using a combined receptor-and ligand-based approach, we tested 98 of the top 0.1% ranked compounds, revealing novel chemotypes as compared to the similar to 1.45 million known ligands in the ChEMBL database. Two compounds were confirmed as agonist and inverse agonist, respectively, in both IP accumulation and Ca2+ mobilization assays. The screening setup presented in this work is computationally inexpensive and therefore particularly useful in an academic setting as it enables-simultaneous testing in binding as well as in different functional assays and/or-species without actual chemical synthesis.

The chemokine CXCL12/stromal cell-derived factor-1 is important for leukocyte migration to lymphoid organs and inflamed tissues and stimulates tumor development. In vitro, CXCL12 activity through CXCR4 is abolished by proteolytic processing. However, limited information is available on in vivo effects of posttranslationally modified CXCL12. Natural CXCL12 was purified from the coculture supernatant of stromal cells stimulated with leukocytes and inflammatory agents. In this conditioned medium, CXCL12 with a nitration on Tyr7, designated [3-NT7]CXCL12, was discovered via Edman degradation. CXCL12 and [3-NT7]CXCL12 were chemically synthesized to evaluate the biological effects of this modification. [3-NT7]CXCL12 recruited β-arrestin 2 and phosphorylated the Akt kinase similar to CXCL12 in receptor-transfected cells. Also the affinity of CXCL12 and [3-NT7]CXCL12 for glycosaminoglycans, the G protein-coupled chemokine receptor CXCR4 and the atypical chemokine receptor ACKR3 were comparable. However, [3-NT7]CXCL12 showed a reduced ability to enhance intracellular calcium concentrations, to generate inositol triphosphate, to phosphorylate ERK1/2 and to induce monocyte and lymphocyte chemotaxis in vitro. Moreover, nitrated CXCL12 failed to induce in vivo extravasation of lymphocytes to the joint. In summary, nitration on Tyr7 under inflammatory conditions is a novel natural posttranslational regulatory mechanism of CXCL12 which may downregulate the CXCR4-mediated inflammatory and tumor-promoting activities of CXCL12.

The CCR7 ligands CCL19 and CCL21 are increasingly recognized as functionally different (biased). Using mature human dendritic cells (DCs), we show that CCL19 is more potent than CCL21 in inducing 3D chemotaxis. Intriguingly, CCL21 induces prolonged and more efficient ERK1/2 activation compared with CCL19 and a C-terminal truncated (tailless) CCL21 in DCs. In contrast, tailless-CCL21 displays increased potency in DC chemotaxis compared with native CCL21. Using a CCL21-specific antibody, we show that CCL21, but not tailless-CCL21, accumulates at the cell surface. In addition, removal of sialic acid from the cell surface by neuraminidase treatment impairs ERK1/2 activation by CCL21, but not by CCL19 or tailless-CCL21. Using standard laboratory cell lines, we observe low potency of both CCL21 and tailless-CCL21 in G protein activation and β-arrestin recruitment compared with CCL19, indicating that the tail itself does not improve receptor interaction. Chemokines interact with their receptors in a stepwise manner with ultimate docking of their N-terminus into the main binding pocket. Employing site-directed mutagenesis we identify residues in this pocket of selective CCL21 importance. We also identify a molecular switch in the top of TM7 important for keeping CCR7 in an inactive conformation (Tyr312), as introduction of the chemokine receptor-conserved Glu (or Ala) induces high constitutive activity. Summarized, we show that the interaction of the tail of CCL21 with polysialic acid is needed for strong ERK signaling, whereas it impairs CCL21-mediated chemotaxis and has no impact on receptor docking consistent with the current model of chemokine:receptor interaction. This indicates that future selective pharmacological targeting of CCL19 versus CCL21 should focus on a differential targeting of the main receptor pocket, while selective targeting of tailless-CCL21 versus CCL21 and CCL19 requires targeting of the glycosaminoglycan (GAG) interaction.

GPR119 is a Gαs-coupled lipid-sensor in the gut, where it mediates release of incretin hormones from the enteroendocrine cells and in pancreatic α-cells, where it releases insulin. Naturally occurring lipids such as monoacylglycerols (MAGs) and N-acylethanolamines (NAEs), like oleoylethanolamide (OEA), activate GPR119, and multiple synthetic ligands have been described. Here, we extend the GPR119 signaling profile to Gαq and Gαi in addition to β-arrestin recruitment and the downstream transcription factors CRE (cAMP response element), SRE (serum response element) and NFAT (nuclear factor of activated T cells). The endogenous OEA and the synthetic AR231453 were full agonists in all pathways except for NFAT, where no ligand-modulation was observed. The potency of AR231453 varied <16-fold (EC50 from 6 to 95nM) across the different signaling pathways, whereas that of OEA varied >175-fold (from 85nM to 15μM) indicating a biased signaling for OEA. The degree of constitutive activity was 1-10%, 10-30% and 30-70% of OEA-induced Emax in Gαi, Gαq and Gαs-driven pathways, respectively. This coincided with the lowest and highest OEA potency observed in Gαi and Gαs-driven pathways, respectively. Incubation for 2h with the 2-MAG-lipase inhibitor JZL84 doubled the constitutive activity, indicating that endogenous lipids contribute to the apparent constitutive activity. Finally, besides being an agonist, AR231453 acted as a positive allosteric modulator of OEA and increased its potency by 54-fold at 100nM AR231453. Our studies uncovering broad and biased signaling, masked constitutive activity by endogenous MAGs, and ago-allosteric properties of synthetic ligands may explain why many GPR119 drug-discovery programs have failed so far.

Background Some herpesviruses like human cytomegalovirus (HCMV) encode viral G protein-coupled receptors that cause reprogramming of cell signaling to facilitate dissemination of the virus, prevent immune surveillance and establish life-long latency. Human GPCRs are known to function in complex signaling networks involving direct physical interactions as well as indirect crosstalk of orthogonal signaling networks. The human chemokine receptor CXCR4 is expressed on hematopoietic stem cells, leukocytes, endothelial and epithelial cells, which are infected by HCMV or display reservoirs of latency. Results We investigated the potential heteromerization of US28 with CXCR4 as well as the influence of US28 on CXCR4 signaling. Using Bioluminescence Resonance Energy Transfer and luciferase-complementation based methods we show that US28 expression exhibits negative effects on CXCR4 signaling and constitutive surface expression in HEK293T cells. Furthermore, we demonstrate that this effect is not mediated by receptor heteromerization but via signaling crosstalk. Additionally, we show that in HCMV, strain TB40E, infected HUVEC the surface expression of CXCR4 is strongly downregulated, whereas in TB40E-delUS28 infected cells, CXCR4 surface expression is not altered in particular at late time points of infection. Conclusions We show that the vGPCR US28 is leading to severely disturbed signaling and surface expression of the chemokine receptor CXCR4 thereby representing an effective mechanism used by vGPCRs to reprogram host cell signaling. In contrast to other studies, we demonstrate that these effects are not mediated via heteromerization.

Since the discovery of HIV's use of CCR5 as the primary coreceptor in fusion, the focus on developing small-molecule receptor antagonists for inhibition hereof has only resulted in one single drug, Maraviroc. We therefore investigated the possibility of using small-molecule CCR5 agonists as HIV-1 fusion inhibitors. A virus-free cell-based fusion reporter assay, based on mixing "effector cells" (expressing HIV Env and luciferase activator) with "target cells" (expressing CD4, CCR5 wild type or a selection of well-described mutations, and luciferase reporter), was used as fusion readout. Receptor expression was evaluated by ELISA and fluorescence microscopy. On CCR5 WT, Maraviroc and Aplaviroc inhibited fusion with high potencies (EC 50 values of 91 and 501 nM, respectively), whereas removal of key residues for both antagonists (Glu283Ala) or Maraviroc alone (Tyr251Ala) prevented fusion inhibition, establishing this assay as suitable for screening of HIV entry inhibitors. Both ligands inhibited HIV fusion on signaling-deficient CCR5 mutations (Tyr244Ala and Trp248Ala). Moreover, the steric hindrance CCR5 mutation (Gly286Phe) impaired fusion, presumably by a direct hindrance of gp120 interaction. Finally, the efficacy switch mutation (Leu203Phe) - converting small-molecule antagonists/inverse agonists to full agonists biased toward G-protein activation - uncovered that also small-molecule agonists can function as direct HIV-1 cell entry inhibitors. Importantly, no agonist-induced receptor internalization was observed for this mutation. Our studies of the pharmacodynamic requirements for HIV-1 fusion inhibitors highlight the possibility of future development of biased ligands with selective targeting of the HIV-CCR5 interaction without interfering with the normal functionality of CCR5.

There is a constant need for new therapeutic interventions in a wide range of infectious diseases. Over the past few years, the immunotoxins have entered the stage as promising antiviral treatments. Immunotoxins have been extensively explored in cancer treatment and have achieved FDA approval in several cases. Indeed, the design of new anticancer immunotoxins is a rapidly developing field. However, at present, several immunotoxins have been developed targeting a variety of different viruses with high specificity and efficacy. Rather than blocking a viral or cellular pathway needed for virus replication and dissemination, immunotoxins exert their effect by killing and eradicating the pool of infected cells. By targeting a virus-encoded target molecule, it is possible to obtain superior selectivity and drastically limit the side effects, which is an immunotoxin-related challenge that has hindered the success of immunotoxins in cancer treatment. Therefore, it seems beneficial to use immunotoxins for the treatment of virus infections. One recent example showed that targeting of virus-encoded 7 transmembrane (7TM) receptors by immunotoxins could be a future strategy for designing ultraspecific antiviral treatment, ensuring efficient internalization and hence efficient eradication of the pool of infected cells, both in vitro and in vivo. In this review, we provide an overview of the mechanisms of action of immunotoxins and highlight the advantages of immunotoxins as future anti-viral therapies.

Chemokine receptors play important roles in the immune system and are linked to several human diseases. The initial contact of chemokines with their receptors depends on highly specified extracellular receptor features. Here we investigate the importance of conserved extracellular disulfide bridges and aromatic residues in extracellular loop 2 (ECL2) for ligand binding and activation in the chemokine receptor CCR8. We used IP3 accumulation and radioligand binding experiments to determine the impact of receptor mutagenesis on both chemokine and small molecule agonist and antagonist binding and action in CCR8. We find that the 7 transmembrane (7TM) receptor conserved disulfide bridge (7TM bridge) linking transmembrane helix (TM)III and ECL2 is crucial for chemokine and small molecule action, whereas the chemokine receptor conserved disulfide bridge between the N terminus and TMVII is needed only for chemokines. Furthermore, we find that two distinct aromatic residues in ECL2, Y184 (Cys+1) and Y187 (Cys+4), are crucial for binding of the CC chemokines CCL1 (agonist) and MC148 (antagonist), respectively, but not for small molecule binding. Finally, using in silico modeling, we predict an aromatic cluster of interaction partners for Y187 in TMIV (F171) and TMV (W194). We show in vitro that these residues are crucial for the binding and action of MC148, thus supporting their participation in an aromatic cluster with Y187. This aromatic cluster appears to be present in a large number of CC chemokine receptors and thereby could play a more general role to be exploited in future drug development targeting these receptors.

There is a constant need for new therapeutic interventions in a wide range of infectious diseases. Over the past few years, the immunotoxins have entered the stage as promising antiviral treatments. Immunotoxins have been extensively explored in cancer treatment and have achieved FDA approval in several cases. Indeed, the design of new anticancer immunotoxins is a rapidly developing field. However, at present, several immunotoxins have been developed targeting a variety of different viruses with high specificity and efficacy. Rather than blocking a viral or cellular pathway needed for virus replication and dissemination, immunotoxins exert their effect by killing and eradicating the pool of infected cells. By targeting a virus-encoded target molecule, it is possible to obtain superior selectivity and drastically limit the side effects, which is an immunotoxin-related challenge that has hindered the success of immunotoxins in cancer treatment. Therefore, it seems beneficial to use immunotoxins for the treatment of virus infections. One recent example showed that targeting of virus-encoded 7 transmembrane (7TM) receptors by immunotoxins could be a future strategy for designing ultraspecific antiviral treatment, ensuring efficient internalization and hence efficient eradication of the pool of infected cells, both in vitro and in vivo. In this review, we provide an overview of the mechanisms of action of immunotoxins and highlight the advantages of immunotoxins as future anti-viral therapies.

Herein, we investigated whether G protein-coupled signaling via the vasopressin receptors of the V1a and V2 subtypes (V1aR and V2R) could be obtained as a direct response to hyperosmolar challenges and/or whether hyperosmolar challenges could augment classical vasopressin-dependent V1aR signaling. The V1aR-dependent response was monitored indirectly via its effects on aquaporin 4 (AQP4) when heterologously expressed in Xenopus oocytes and V1aR and V2R function was directly monitored following heterologous expression in COS-7 cells. A tendency toward an osmotically induced, V1aR-mediated reduction in AQP4-dependent water permeability was observed, although osmotic challenges failed to mimic vasopressin-dependent V1aR-mediated internalization of AQP4. Direct monitoring of inositol phosphate (IP) production of V1aR-expressing COS-7 cells demonstrated an efficient vasopressin-dependent response that was, however, independent of hyperosmotic challenges. Similarly, the cAMP production by the V2R was unaffected by hyperosmotic challenges although, in contrast to the V1aR, the V2R displayed an ability to support alternative signaling (IP production) at higher concentration of vasopressin. V1aR and V2R respond directly to vasopressin exposure, but they do not have an ability to act as osmo- or volume sensors when exposed to an osmotic gradient in the absence or presence of vasopressin.

Glucagon-like peptide 2 (GLP-2) is a potent intestinotrophic growth factor with therapeutic potential in the treatment of intestinal deficiencies. It has recently been approved for the treatment of short bowel syndrome. The effects of GLP-2 are mediated by specific binding of the hormone to the GLP-2 receptor (GLP-2R) which was cloned in 1999. However, consensus about the exact receptor localization in the intestine has never been established. By physical, chemical and enzymatic tissue fragmentation, we were able to divide rat jejunum into different compartments consisting of: (1) epithelium alone, (2) mucosa with lamina propria and epithelium, (3) the external muscle coat including myenteric plexus, (4) a compartment enriched for the myenteric plexus and (5) intestine without epithelium. Expression of Glp2r; chromogranin A; tubulin, beta 3; actin, gamma 2, smooth muscle, enteric and glial fibrillary acidic protein in these isolated tissue fractions was quantified with qRT-PCR. Expression of the Glp2r was confined to compartments containing enteric neurons and receptor expression was absent in the epithelium. Our findings provide evidence for the expression of the GLP-2R in intestinal compartments rich in enteric neurons and, importantly they exclude significant expression in the epithelium of rat jejunal mucosa.

Glucose-dependent insulinotropic polypeptide (GIP) is glucagonotropic, and glucagon-like peptide-1 (GLP-1) is glucagonostatic. We studied the effects of GIP and GLP-1 on glucagon responses to insulin-induced hypoglycemia in patients with type 1 diabetes mellitus (T1DM). Ten male subjects with T1DM (C-peptide negative, age [mean ± SEM] 26 ± 1 years, BMI 24 ± 0.5 kg/m(2), HbA1c 7.3 ± 0.2%) were studied in a randomized, double-blinded, crossover study, with 2-h intravenous administration of saline, GIP, or GLP-1. The first hour, plasma glucose was lowered by insulin infusion, and the second hour constituted a "recovery phase." During the recovery phase, GIP infusions elicited larger glucagon responses (164 ± 50 [GIP] vs. 23 ± 25 [GLP-1] vs. 17 ± 46 [saline] min ⋅ pmol/L, P < 0.03) and endogenous glucose production was higher with GIP and lower with GLP-1 compared with saline (P < 0.02). On the GIP days, significantly less exogenous glucose was needed to keep plasma glucose above 2 mmol/L (155 ± 36 [GIP] vs. 232 ± 40 [GLP-1] vs. 212 ± 56 [saline] mg ⋅ kg(-1), P < 0.05). Levels of insulin, cortisol, growth hormone, and noradrenaline, as well as hypoglycemic symptoms and cognitive function, were similar on all days. Our results suggest that during hypoglycemia in patients with T1DM, exogenous GIP increases glucagon responses during the recovery phase after hypoglycemia and reduces the need for glucose administration.