Identifying Functional Hotspot Residues for Biased Ligand Design in G-Protein-Coupled Receptors.

G-protein-coupled receptors (GPCRs) mediate multiple signaling pathways in the cell, depending on the agonist that activates the receptor and multiple cellular factors. Agonists that show higher potency to specific signaling pathways over others are known as "biased agonists" and have been shown to have better therapeutic index. Although biased agonists are desirable, their design poses several challenges to date. The number of assays to identify biased agonists seems expensive and tedious. Therefore, computational methods that can reliably calculate the possible bias of various ligands ahead of experiments and provide guidance, will be both cost and time effective. In this work, using the mechanism of allosteric communication from the extracellular region to the intracellular transducer protein coupling region in GPCRs, we have developed a computational method to calculate ligand bias ahead of experiments. We have validated the method for several ß-arrestin-biased agonists in ß2-adrenergic receptor (ß2AR), serotonin receptors 5-HT1B and 5-HT2B and for G-protein-biased agonists in the κ-opioid receptor. Using this computational method, we also performed a blind prediction followed by experimental testing and showed that the agonist carmoterol is ß-arrestin-biased in ß2AR. Additionally, we have identified amino acid residues in the biased agonist binding site in both ß2AR and κ-opioid receptors that are involved in potentiating the ligand bias. We call these residues functional hotspots, and they can be used to derive pharmacophores to design biased agonists in GPCRs.

Structure of a nanobody-stabilized active state of the ß(2) adrenoceptor.

G protein coupled receptors (GPCRs) exhibit a spectrum of functional behaviours in response to natural and synthetic ligands. Recent crystal structures provide insights into inactive states of several GPCRs. Efforts to obtain an agonist-bound active-state GPCR structure have proven difficult due to the inherent instability of this state in the absence of a G protein. We generated a camelid antibody fragment (nanobody) to the human ß(2) adrenergic receptor (ß(2)AR) that exhibits G protein-like behaviour, and obtained an agonist-bound, active-state crystal structure of the receptor-nanobody complex. Comparison with the inactive ß(2)AR structure reveals subtle changes in the binding pocket; however, these small changes are associated with an 11 Å outward movement of the cytoplasmic end of transmembrane segment 6, and rearrangements of transmembrane segments 5 and 7 that are remarkably similar to those observed in opsin, an active form of rhodopsin. This structure provides insights into the process of agonist binding and activation.

ß2 long-acting and anticholinergic drugs control TGF-ß1-mediated neutrophilic inflammation in COPD.

We quantified TGF-ß1 and acetylcholine (ACh) concentrations in induced sputum supernatants (ISSs) from 18 healthy controls (HC), 22 healthy smokers (HS) and 21 COPDs. ISSs from HC, HS and COPD as well as rhTGF-ß1 were also tested in neutrophil adhesion and in mAChR2, mAChR3 and ChAT expression experiments in human bronchial epithelial cells (16-HBE). Finally, we evaluated the effects of Olodaterol (a novel inhaled ß(2)-adrenoceptor agonist) and Tiotropium Spiriva®, alone or in combination, on neutrophil adhesion and mAChRs and ChAT expression in stimulated 16-HBE. The results showed that 1) TGF-ß1 and ACh concentrations are increased in ISSs from COPD in comparison to HC and HS, and TGF-ß1 in HS is higher than in HC; 2) ISSs from COPD and HS caused increased neutrophil adhesion to 16-HBE when compared to ISSs from HC. The effect of ISSs from COPD was significantly reduced by TGF-ß1 depletion or by the pretreatment with Olodaterol or Tiotropium alone or in combination, while the effect of ISSs from HS was significantly reduced by the pretreatment with Olodaterol alone; 3) mAChR2, mAChR3 and ChAT expression was increased in 16-HBE stimulated with ISSs from COPD and TGF-ß1 depletion significantly reduced this effect on mAChR3 and ChAT expression; 4) rhTGF-ß1 increased mAChR2, mAChR3 and ChAT expression in 16-HBE; 5) Olodaterol did not affect the expression of mAChRs and ChAT in 16-HBE. Our findings support the use of ß2 long-acting and anticholinergic drugs to control the bronchoconstriction and TGF-ß1-mediated neutrophilic inflammation in COPD.

Functional and biochemical rationales for the 24-hour-long duration of action of olodaterol.

ß(2)-Adrenoceptor (ß(2)-AR) agonists are powerful bronchodilators and play a pivotal role in the management of pulmonary obstructive diseases, such as asthma and chronic obstructive pulmonary disease. Although these agents first were used many years ago, progress in drug development has resulted in better tolerated, long-acting ß(2)-AR agonists (LABAs), such as formoterol and salmeterol. Although LABAs have been on the market for several years, relatively little is known on the rationale(s) behind their long duration of action. In this study, we focused on olodaterol (previously known as BI1744CL), a novel inhaled LABA, which provides a bronchodilating effect lasting 24 h and is currently in Phase III clinical trials. To understand the rationale behind its long duration of action, different aspects of olodaterol were analyzed (i.e., its lipophilicity and propensity to accumulate in the lipid bilayer as well as its tight binding to the ß(2)-AR). In line with its physicochemical properties, olodaterol associated moderately with lipid bilayers. Instead, kinetic as well as equilibrium binding studies indicated the presence of a stable [(3)H]olodaterol/ß(2)-AR complex with a dissociation half-life of 17.8 h due to ternary complex formation. The tight binding of olodaterol to the human ß(2)-AR and stabilization of the ternary complex were confirmed in functional experiments monitoring adenylyl cyclase activity after extensive washout. Taken together, binding, kinetic, and functional data support the existence of a stable complex with the ß(2)-AR that, with a dissociation half-life >17 h, might indeed be a rationale for the 24-h duration of action of olodaterol.

Significance of N-terminal proteolysis of CCL14a to activity on the chemokine receptors CCR1 and CCR5 and the human cytomegalovirus-encoded chemokine receptor US28.

The CC chemokine CCL14a is constitutively expressed in a large variety of tissues and its inactive proform CCL14a(1-74) circulates in high concentrations in plasma. CCL14a(1-74) is converted into CCL14a(9-74) by the proteases urokinase-type plasminogen activator and plasmin and is a highly active agonist for the chemokine receptors CCR1 and CCR5. In this study, a new CCL14a analog, CCL14a(12-74), was isolated from blood filtrate. To elucidate the functional role of the N terminus, a panel of N-terminally truncated CCL14a analogs were tested on the receptors CCR1 to CCR5 and on the human cytomegalovirus (HCMV)-encoded chemokine receptor US28. The rank order of binding affinity to these receptors and of the activation of CCR1 and CCR5-mediated intracellular Ca(2+) concentration mobilization is CCL14a(6-74)<(7-74)<(8-74)<<(9-74) = (10-74)>>(11-74)>>(12-74). The almost identical affinities of CCL14a(7-74), CCL14a(9-74), and CCL14a(10-74) for the US28 receptor and the inhibition of US28-mediated HIV infection of 293T cells by all of the N-terminally truncated CCL14a analogs support the promiscuous nature of the viral chemokine receptor US28. In high concentrations, CCL14a(12-74) did reveal antagonistic activity on intracellular Ca(2+) concentration mobilization in CCR1- and CCR5-transfected cells, which suggests that truncation of Tyr(11) might be of significance for an efficient inactivation of CCL14a. A putative inactivation pathway of CCL14a(9-74) to CCL14a(12-74) may involve the dipeptidase CD26/dipeptidyl peptidase IV (DPPIV), which generates CCL14a(11-74), and the metalloprotease aminopeptidase N (CD13), which displays the capacity to generate CCL14a(12-74) from CCL14a(11-74). Our results suggest that the activity of CCL14a might be regulated by stringent proteolytic activation and inactivation steps.

The constitutive activity of the human muscarinic M3 receptor unmasks differences in the pharmacology of anticholinergics.

An activator protein 1-driven luciferase reporter assay was developed to monitor the activation of the human muscarinic M3 receptor (hM3-R) and evaluate functional potencies of different anticholinergics in Chinese hamster ovary cells. This assay proved to be superior to previously used functional assays [i.e., inositol phosphate accumulation (J Pharmacol Exp Ther 330:660-668, 2009)], thanks to the longer incubation times that allow reaching of pseudoequilibrium for ligands with slower dissociation kinetics, the long-acting muscarinic antagonists. Interestingly, within this system the hM3-R efficiently signaled in an agonist-independent manner. All the antagonists tested were able to inhibit the hM3-R constitutive activity in a concentration-dependent fashion, behaving as full inverse agonists. Curiously, significant differences in potency as antagonists (against carbachol) and inverse agonists were seen for some compounds (N-methyl scopolamine and tiotropium). Given the potential for inverse agonists to cause receptor up-regulation, the effect of chronic exposure to anticholinergics on the expression levels of hM3-R was also tested. Again, significant differences were seen, with some ligands (e.g., tiotropium) producing less than half of the receptor up-regulation caused by other anticholinergics. This study shows that anticholinergics can exhibit differential behaviors, which depend on the pathway investigated, and therefore provides evidence that the molecular mechanism of inverse agonism is likely to be more complex than the stabilization of a single inactive receptor conformation. In addition, differences in the potential of anticholinergics to induce hM3-R up-regulation might have clinical relevance, because many are on the market or in clinical trials as chronic treatment for chronic obstructive pulmonary disease, for example.

Preclinical evaluation of long-acting muscarinic antagonists: comparison of tiotropium and investigational drugs.

Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow limitation caused by persistent inflammatory processes in the airways. An increased cholinergic tone mediates different pathophysiological features of COPD, such as bronchoconstriction and mucus hypersecretion, mostly through activation of the human muscarinic M(3) receptor (hM(3)) subtype. Tiotropium bromide (Spiriva) is a well established muscarinic antagonist in the pharmacological management of COPD with a once-daily posology. The rationale behind the sustained bronchodilation obtained with tiotropium consists in its slow dissociation from hM(3) receptors. In this study, we performed a comprehensive preclinical comparison of tiotropium with other long-acting muscarinic antagonists (LAMAs) currently in clinical development, namely aclidinium bromide and glycopyrrolate. The different muscarinic antagonists were characterized for their 1) affinity toward the different human muscarinic receptor subtypes expressed in Chinese hamster ovary cells and kinetics of receptor dissociation, 2) potency in inhibiting the agonist-induced activation of muscarinic receptors through measurement of second messengers, and 3) efficacy and duration of bronchoprotection, as tested in a model of acetylcholine-induced bronchoconstriction in anesthetized dogs over a period of 24 h. All of the tested LAMAs showed high affinity and potency toward the hM(3) receptor (tiotropium, pA(2) = 10.4; aclidinium, pA(2) = 9.6; and glycopyrrolate, pA(2) = 9.7). However, dissociation half-lives of the LAMAs from the hM(3) receptor differed significantly (tiotropium, t((1/2)) = 27 h; aclidinium, t((1/2)) = 10.7 h; and glycopyrrolate, t((1/2)) = 6.1 h). In line with their kinetic properties at the hM(3), the tested LAMAs provided different levels of bronchoprotection in the in vivo setting 24 h after administration (tiotropium = 35%, aclidinium = 21%, and glycopyrrolate = 0% at 24 h) when applied at equieffective doses.

Smoke, choline acetyltransferase, muscarinic receptors, and fibroblast proliferation in chronic obstructive pulmonary disease.

Acetylcholine (ACh), synthesized by choline acetyltransferase (ChAT), and muscarinic M(1), M(2), and M(3) receptors (MRs) are involved in fibroblast proliferation. We evaluated ChAT, MRs, and extracellular signal-regulated kinase (ERK) 1/2 and nuclear factor (NF) kappaB activation in lung fibroblasts from patients with chronic obstructive pulmonary disease (COPD), control smokers, and controls. Human fetal lung fibroblasts (HFL-1) stimulated with interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, and cigarette smoke extracts (CSEs) were evaluated for ChAT and MR expression. We tested the effects of ACh on fibroblast proliferation and its ability to bind fibroblasts from patients with COPD, control smokers, controls, and HFL-1 stimulated with IL-1beta, TNF-alpha, and CSE. ChAT, M(1), and M(3) expression and ERK1/2 and NFkappaB activation were increased, whereas M(2) was reduced, in COPD and smoker subjects compared with controls. IL-1beta increased the ChAT and M(3), TNF-alpha down-regulated M(2), and CSE increased ChAT and M(3) expression while down-regulating the expression of M(2) in HFL-1 cells. ACh stimulation increased fibroblast proliferation in patients with COPD, control smokers, and controls, with higher effect in control smokers and patients with COPD and increased HFL-1 proliferation only in CSE-treated cells. The binding of ACh was higher in patients with COPD and in control smokers than in controls and in CSE-treated than in IL-1beta- and TNF-alpha-stimulated HFL-1 cells. Tiotropium (Spiriva; [1alpha,2beta,4beta,5alpha,7beta-7-hydroxydi-2-thienylacetyl)oxy]-9,9-dimethyl-3-oxa-9-azoniatrcyclo[3.3.1.0(24)], C(19)H(22) NO(4)S(2)Br.H(2)O), gallamine triethiodide (C(19)H(22)N(4)O(2)S.2HCl.H(2)O), telenzepine [4,9-d-dihydro-3-methyl-4-[(4-methyl-1piperazinyl) acetyl]-10H-thieno [3,4-b][1,5]benzodiazepine-10-one dihydrobromide, C(30)H(60)I(3)N(3)O(3)], 4-diphenylacetoxy-N-methylpiperidine, PD098059 [2-(2-amino-3methoxyphenyl)-4H-1benzopyran-4-one, C(16)H(13)NO(3)], and BAY 11-7082 [(E)-3-(4-methylphenylsulfonyl)-2-propenetrile, C(10)H(9)NO(2)C], down-regulated the ACh-induced fibroblast proliferation, promoting the MRs and ERK1/2 and NFkappaB pathways involvement in this phenomenon. These results suggest that cigarette smoke might alter the expression of ChAT and MRs, promoting airway remodeling in COPD and that anticholinergic drugs, including tiotropium, might prevent these events.

The human cytomegalovirus-encoded chemokine receptor US28 induces caspase-dependent apoptosis.

Viral subversion of apoptosis regulation plays an important role in the outcome of host/virus interactions. Although human cytomegalovirus (HCMV) encodes several immediate early (IE) antiapoptotic proteins (IE1, IE2, vMIA and vICA), no proapoptotic HCMV protein has yet been identified. Here we show that US28, a functional IE HCMV-encoded chemokine receptor, which may be involved in both viral dissemination and immune evasion, constitutively induces apoptosis in several cell types. In contrast, none of nine human cellular chemokine receptors, belonging to three different subfamilies, induced any significant level of apoptosis. US28-induced cell death involves caspase 10 and caspase 8 activation, but does not depend on the engagement of cell-surface death receptors of the tumour necrosis factor receptor/CD95 family. US28 cell-death induction is prevented by coexpression of C-FLIP, a protein that inhibits Fas-associated death domain protein (FADD)-mediated activation of caspase 10 and caspase 8, and by coexpression of the HCMV antiapoptotic protein IE1. The use of US28 mutants indicated that the DRY sequence of its third transmenbrane domain, required for constitutive G-protein signalling, and the US28 intracellular terminal domain required for constitutive US28 endocytosis, are each partially required for cell-death induction. Thus, in HCMV-infected cells, US28 may function either as a chemokine receptor, a phospholipase C activator, or a proapoptotic factor, depending on expression levels of HCMV and/or cellular antiapoptotic proteins.

Synthesis and structure-activity relationship of the first nonpeptidergic inverse agonists for the human cytomegalovirus encoded chemokine receptor US28.

US28 is a human cytomegalovirus (HCMV) encoded G-protein-coupled receptor that signals in a constitutively active manner. Recently, we identified 1 [5-(4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl)-2,2-diphenylpentanenitrile] as the first reported nonpeptidergic inverse agonist for a viral-encoded chemokine receptor. Interestingly, this compound is able to partially inhibit the viral entry of HIV-1. In this study we describe the synthesis of 1 and several of its analogues and unique structure-activity relationships for this first class of small-molecule ligands for the chemokine receptor US28. Moreover, the compounds have been pharmacologically characterized as inverse agonists on US28. By modification of lead structure 1, it is shown that a 4-phenylpiperidine moiety is essential for affinity and activity. Other structural features of 1 are shown to be of less importance. These compounds define the first SAR of ligands on a viral GPCR (US28) and may therefore serve as important tools to investigate the significance of US28-mediated constitutive activity during viral infection.

Molecular basis for the long duration of action and kinetic selectivity of tiotropium for the muscarinic M3 receptor.

Antagonizing the human M3 muscarinic receptor (hM3R) over a long time is a key feature of modern bronchodilating COPD drugs aiming at symptom relief. The long duration of action of the antimuscarinic drug tiotropium and its kinetic subtype selectivity over hM2R are investigated by kinetic mapping of the binding site and the exit channel of hM3R. Hence, dissociation experiments have been performed with a set of molecular matched pairs of tiotropium on a large variety of mutated variants of hM3R. The exceedingly long half-life of tiotropium (of more than 24 h) is attributed to interactions in the binding site; particularly a highly directed interaction of the ligands' hydroxy group with an asparagine (N508(6.52)) prevents rapid dissociation via a snap-lock mechanism. The kinetic selectivity over hM2R, however, is caused by differences in the electrostatics and in the flexibility of the extracellular vestibule. Extensive molecular dynamics simulations (several microseconds) support experimental results.

In vitro anticholinergic drugs affect CD8+ peripheral blood T-cells apoptosis in COPD.

Novel pharmacological strategies are aimed at the resolution of systemic inflammation in COPD potentiating peripheral blood T-cell (PBT-cell) apoptosis. Although muscarinic acetylcholine receptors (mAChRs) M(3) and choline-acetyltransferase (ChAT) participate in the airway inflammation of COPD, their role in PBT-cell apoptosis remains unexplained. We evaluated in PBT-cells from COPD patients, smoker (S) and control (C) subjects: (1) apoptosis (by annexin V binding), (2) mAChR M(3) and ChAT expression, acetylcholine (ACh)-binding; (3) choline levels in serum and PBT-cells extracts. We tested the effects of Tiotropium (Spiriva(®)) and hemicholinium-3 (HCh-3) on apoptosis, NFκB pathway, caspases 3 and 8 activity and choline levels, in PBT-cells from COPD patients. We showed that: (1) apoptosis, mAChR M(3) and ChAT expression and the CD3+ and CD8+ ACh-binding are increased in PBT-cells from COPD patients when compared to C subjects, while CD4+/CD8+ ratio of ACh-binding to PBT cells was reduced in COPD; (2) choline levels are higher in serum and PBT-cells extracts from COPD patients than in S and C; (3) Tiotropium and HCh-3 reduced CD4+ and increased CD8+ apoptosis via caspases 3 and 8 activities and via IκB mediated mechanisms in COPD patients. This study suggests the involvement of non-neuronal components of cholinergic system in the regulation of PBT-cell apoptosis in COPD and demonstrates that Tiotropium regulates CD4+ and CD8+ PBT-cell apoptosis. It provides novel putative pharmacological targets for the resolution of systemic inflammation in COPD.

Cigarette smoke extract activates human bronchial epithelial cells affecting non-neuronal cholinergic system signalling in vitro.

AIMS: Acetylcholine (ACh) is synthesized by Choline Acetyl-Transferase (ChAT) that exerts its physiological effects in airway epithelial cells via muscarinic receptor (MR) activation. We evaluate the effect of ACh stimulation on human bronchial epithelial cells (16-HBE) and test whether cigarette smoke extract (CSE) can modify the basal cellular response to ACh affecting the non-neuronal cholinergic system signalling. MAIN METHODS: ACh stimulated 16-HBE were tested for ACh-binding, Leukotriene B(4) (LTB(4)) release and ERK1/2 and NFkB pathway activation. Additionally, we investigated all the aforementioned parameters as well as ChAT and MR proteins and mRNA expression and endogenous ACh production in CSE-treated 16-HBE. KEY FINDINGS: We showed that ACh induced in 16-HBE, in a concentration-dependent manner, LTB(4) release via the activation of ERK1/2 and NFkB pathways. The addition of Tiotropium (Spiriva®), Gallamine, Telenzepine and 4-DAMP (muscarinic receptor antagonists), as well as of PD 098059 (MAPKK inhibitor) and BAY117082 (inhibitor of IkBα phosphorilation), down-regulated the ACh-induced effects. Additionally, CSE treatment of 16-HBE increased the binding of ACh, and shifted the LTB4 release from the concentration ACh 1µM to 10nM. Finally, we observed that the treatment of 16-HBE with CSE increased the expression of ChAT, M(2) and M(3) and of endogenous ACh production in 16-HBE. Tiotropium regulated the LTB4 release and ACh production in CSE treated 16-HBE. SIGNIFICANCE: CSE increases the pro-inflammatory activity of human bronchial epithelial cells, and promotes the cellular response to lower concentrations of ACh, by affecting the expression of ChAT and MRs. Tiotropium might prevent pro-inflammatory events generated by ACh together with CSE.

Differential inverse agonism at the human muscarinic M3 receptor.

Human muscarinic M3 receptors (hM3Rs) induce smooth muscle contraction and mucus gland secretion in response to parasympathetic stimulation. As a consequence of hM3R function, muscarinic antagonists have wide therapeutic use to treat overactive bladder, abdominal pain (irritable bowel syndrome), and chronic obstructive pulmonary disease (COPD). In this chapter, we describe the set up and results obtained with different in vitro assays to monitor hM3R activation (agonist-dependent and constitutive) and evaluate functional potencies of different anticholinergics in CHO cells. Given the G(q) coupling of hM3R, assays measuring the second messengers inositol phosphates (InsP) and an AP-1-driven reporter luciferase were developed. In our hands, the reporter gene assay shows advantages: firstly, thanks to the longer incubation times, it allows reaching of pseudo-equilibrium also for ligands with slower receptor dissociation kinetics (e.g., tiotropium). Secondly, the AP-1-driven luciferase detects significant constitutive activity of the hM3R, which allows characterizing the different anticholinergics for their inverse agonist properties. Given the potential for inverse agonists to cause changes in receptor expression, monitoring hM3R upregulation is another important pharmacological parameter. Here, we describe how to measure the effect of chronic exposure to anticholinergics on the expression levels of hM3R, with particular attention to ensure full antagonist removal from receptor pool before hM3R quantification. Taken together, our results indicate that anticholinergics exhibit differential pharmacological behaviors, which are dependent on the pathway investigated, and therefore provide evidence that the molecular mechanism of inverse agonism is likely to be more complex than the stabilization of a single inactive receptor conformation.

CC and CX3C chemokines differentially interact with the N terminus of the human cytomegalovirus-encoded US28 receptor.

Human cytomegalovirus (HCMV) is the causative agent of life-threatening systemic diseases in immunocompromised patients as well as a risk factor for vascular pathologies, like atherosclerosis, in immunocompetent individuals. HCMV encodes a G-protein-coupled receptor (GPCR), referred to as US28, that displays homology to the human chemokine receptor CCR1 and binds several chemokines of the CC family as well as the CX3C chemokine fractalkine with high affinity. Most importantly, following HCMV infection, US28 activates several intracellular pathways, either constitutively or in a chemokine-dependent manner. In this study, our goal was to understand the molecular interactions between chemokines and the HCMV-encoded US28 receptor. To achieve this goal, a double approach has been used, consisting in the analysis of both receptor and ligand mutants. This approach has led us to identify several amino acids located in the N terminus of US28 that differentially contribute to the high affinity binding of CC versus CX3C chemokines. Additionally, our results highlight the importance of secondary modifications occurring at US28, such as sulfation, for ligand recognition. Finally, the effects of chemokine dimerization and interaction with glycosaminoglycans (GAGs) on chemokine binding and activation of US28 were investigated as well using CCL4 as model ligand. In line with the two-state model describing chemokine/receptor interaction, we show that an aromatic residue in the N-loop region of CCL4 promotes tight binding to US28, whereas receptor activation depends on the presence of the N terminus of CCL4, as shown previously for CCR5.

Kaposi's sarcoma-associated herpesvirus-encoded G protein-coupled receptor ORF74 constitutively activates p44/p42 MAPK and Akt via G(i) and phospholipase C-dependent signaling pathways.

The G protein-coupled receptor encoded by Kaposi's sarcoma-associated herpesvirus, also referred to as ORF74, has been shown to stimulate oncogenic and angiogenic signaling pathways in a constitutively active manner. The biochemical routes linking ORF74 to these signaling pathways are poorly defined. In this study, we show that ORF74 constitutively activates p44/p42 mitogen-activated protein kinase (MAPK) and Akt via G(i)- and phospholipase C (PLC)-mediated signaling pathways. Activation of Akt by ORF74 appears to be phosphatidylinositol 3-kinase (PI3-K) dependent but, interestingly, is also mediated by activation of protein kinase C (PKC) and p44/p42 MAPK. ORF74 may signal to Akt via p44/p42 MAPK, which can be activated by G(i), through activation of PI3-K or through PKC via the PLC pathway. Signaling of ORF74 to these proliferative and antiapoptotic signaling pathways can be further modulated positively by growth-related oncogene (GROalpha/CXCL1) and negatively by human gamma interferon-inducible protein 10 (IP-10/CXCL10), thus acting as an agonist and an inverse agonist, respectively. Despite the ability of the cytomegalovirus-encoded chemokine receptor US28 to constitutively activate PLC, this receptor does not increase phosphorylation of p44/p42 MAPK or Akt in COS-7 cells. Hence, ORF74 appears to signal through a larger diversity of G proteins than US28, allowing it to couple to proliferative and antiapoptotic signaling pathways. ORF74 can therefore be envisioned as an attractive target for novel treatment of Kaposi's sarcoma.

Constitutively active Gq/11-coupled receptors enable signaling by co-expressed G(i/o)-coupled receptors.

Co-expression of guanine nucleotide-binding regulatory (G) protein-coupled receptors (GPCRs), such as the G(i/o)-coupled human 5-hydroxytryptamine receptor 1B (5-HT(1B)R), with the G(q/11)-coupled human histamine 1 receptor (H1R) results in an overall increase in agonist-independent signaling, which can be augmented by 5-HT(1B)R agonists and inhibited by a selective inverse 5-HT(1B)R agonist. Interestingly, inverse H1R agonists inhibit constitutively H1R-mediated as well as 5-HT(1B)R agonist-induced signaling in cells co-expressing both receptors. This phenomenon is not solely characteristic of 5-HT(1B)R; it is also evident with muscarinic M2 and adenosine A1 receptors and is mimicked by mastoparan-7, an activator of G(i/o) proteins, or by over-expression of Gbetagamma subunits. Likewise, expression of the G(q/11)-coupled human cytomegalovirus (HCMV)-encoded chemokine receptor US28 unmasks a functional coupling of G(i/o)-coupled CCR1 receptors that is mediated via the constitutive activity of receptor US28. Consequently, constitutively active G(q/11)-coupled receptors, such as the H1R and HCMV-encoded chemokine receptor US28, constitute a regulatory switch for signal transduction by G(i/o)-coupled receptors, which may have profound implications in understanding the role of both constitutive GPCR activity and GPCR cross-talk in physiology as well as in the observed pathophysiology upon HCMV infection.

The carboxyl terminus of human cytomegalovirus-encoded 7 transmembrane receptor US28 camouflages agonism by mediating constitutive endocytosis.

US28 is one of four 7 transmembrane (7TM) chemokine receptors encoded by human cytomegalovirus and has been shown to both signal and endocytose in a ligand-independent, constitutively active manner. Here we show that the constitutive activity and constitutive endocytosis properties of US28 are separable entities in this viral chemokine receptor. We generated chimeric and mutant US28 proteins that were altered in either their constitutive endocytic (US28 Delta 300, US28 Delta 317, US28-NK1-ctail, and US28-ORF74-ctail) or signaling properties (US28R129A). By using this series of mutants, we show that the cytoplasmic tail domain of US28 per se regulates receptor endocytosis, independent of the signaling ability of the core domain of US28. The constitutive endocytic property of the US28 c-tail was transposable to other 7TM receptors, the herpes virus 8-encoded ORF74 and the tachykinin NK1 receptor (ORF74-US28-ctail and NK1-US28-ctail). Deletion of the US28 C terminus resulted in reduced constitutive endocytosis and consequently enhanced signaling capacity of all receptors tested as assessed by inositol phosphate turnover, NF-kappa B, and cAMP-responsive element-binding protein transcription assays. We further show that the constitutive endocytic property of US28 affects the action of its chemokine ligand fractalkine/CX3CL1 and show that in the absence of the US28 C terminus, fractalkine/CX3CL1 acts as an agonist on US28. This demonstrates for the first time that the endocytic properties of a 7TM receptor can camouflage the agonist properties of a ligand.

Human cytomegalovirus chemokine receptor US28-induced smooth muscle cell migration is mediated by focal adhesion kinase and Src.

The human cytomegalovirus-encoded chemokine receptor US28 induces arterial smooth muscle cell (SMC) migration; however, the underlying mechanisms involved in this process are unclear. We have previously shown that US28-mediated SMC migration occurs by a ligand-dependent process that is sensitive to protein-tyrosine kinase inhibitors. We demonstrate here that US28 signals through the non-receptor protein-tyrosine kinases Src and focal adhesion kinase (FAK) and that this activity is necessary for US28-mediated SMC migration. In the presence of RANTES (regulated on activation normal T cell expressed and secreted), US28 stimulates the production of a FAK.Src kinase complex. Interestingly, Src co-immunoprecipitates with US28 in a ligand-dependent manner. This association occurs earlier than the formation of the FAK.Src kinase complex, suggesting that US28 activates Src before FAK. US28 binding to RANTES also promotes the formation of a Grb2.FAK complex, which is sensitive to treatment with the Src inhibitor PP2, further highlighting the critical role of Src in US28 activation of FAK. Human cytomegalovirus US28-mediated SMC migration is inhibited by treatment with PP2 and through the expression of either of two dominant negative inhibitors of FAK (F397Y and NH2-terminal amino acids 1-401). These findings demonstrate that activation of FAK and Src plays a critical role in US28-mediated signaling and SMC migration.