G protein coupled receptors (GPCRs) exhibit a spectrum of functional behaviors 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 β2 adrenergic receptor (β2AR) that exhibits G protein-like behavior, and obtained an agonist-bound, active-state crystal structure of the receptor-nanobody complex. Comparison with the inactive β2AR structure reveals subtle changes in the binding pocket; however, these small changes are associated with an 11Å 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.

Previously it was shown that the HHV-8-encoded chemokine receptor ORF74 shows considerable agonist-independent, constitutive activity giving rise to oncogenic transformation (Arvanitakis, L., Geras-Raaka, E., Varma, A., Gershengorn, M. C., and Cesarman, E. (1997) Nature 385, 347-350). In this study we report that a second viral-encoded chemokine receptor, the human cytomegalovirus-encoded US28, also efficiently signals in an agonist-independent manner. Transient expression of US28 in COS-7 cells leads to the constitutive activation of phospholipase C and NF-B signaling via G q/11 proteindependent pathways. Whereas phospholipase C activation is mediated via G␣ q/11 subunits, the activation of NF-B strongly depends on ␤␥ subunits with a preference for the ␤ 2 ␥ 1 dimer. The CC chemokines RANTES (regulated on activation, normal T cell expressed and secreted) and MCP-1 (monocyte chemotactic protein-1) act as neutral antagonists at US28, whereas the CX 3 C chemokine fractalkine acts as a partial inverse agonist with IC 50 values of 1-5 nM. Our data suggest that a high level of constitutive activity might be a more general characteristic of viral G protein-coupled receptors and that human cytomegalovirus might exploit this G protein-coupled receptor property to modulate the homeostasis of infected cells via the early gene product US28.

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⌬300, US28⌬317, US28-NK 1 -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 NK 1 receptor (ORF74-US28-ctail and NK 1 -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-B, and cAMPresponsive element-binding protein transcription assays. We further show that the constitutive endocytic property of US28 affects the action of its chemokine ligand fractalkine/CX 3 CL1 and show that in the absence of the US28 C terminus, fractalkine/CX 3 CL1 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. Endocytosis of 7 transmembrane (7TM)1 G protein-coupled receptors is a mechanism that allows control of an appropriate cell surface receptor density in a given physiological setting. Typically, a receptor is activated by an agonist and initiates a signaling cascade before it undergoes endocytosis. However, there are receptors that can signal in a ligand-independent manner, the so-called constitutively active receptors. For some of these, constitutive internalization is an accompanying effect, e.g. for mutant parathyroid hormone receptors (PTH1Rc) (1), the constitutively active mutants of the angiotensin II (1A) receptor (2), or the human complement factor C5a receptor (3). Two examples of constitutively endocytosing 7TM receptors have been described in the chemokine system, one of which is the CXCR4 receptor. However, its constitutive internalization rate with ϳ2.5% of total surface receptor/min in the absence of ligand is rather slow (4). The other receptor is US28, a 7TM receptor encoded by human cytomegalovirus (HCMV) that has been shown to both signal (5, 6) and endocytose in a ligandindependent manner at the rate of ϳ7% of the cell surface pool/min (7).Of the four chemokine receptors encoded by this large DNA virus, UL33, UL78, US27, and US28 (8), US28 is so far the most extensively characterized receptor. It binds several CCchemokines such as MIP-1␣/CCL3, MCP-1/CCL2, MIP-1␤/ CCL4, and RANTES/CCL5 (9) as well as the CX 3 C-chemokine fractalkine/CX 3 CL1 with high affinit...

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 ϭ 10.4; aclidinium, pA 2 ϭ 9.6; and glycopyrrolate, pA 2 ϭ 9.7). However, dissociation half-lives of the LAMAs from the hM 3 receptor differed significantly (tiotropium, t 1 ⁄2 ϭ 27 h; aclidinium, t 1 ⁄2 ϭ 10.7 h; and glycopyrrolate, t 1 ⁄2 ϭ 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 ϭ 35%, aclidinium ϭ 21%, and glycopyrrolate ϭ 0% at 24 h) when applied at equieffective doses.

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.

Human cytomegalovirus (HCMV) encodes a G proteincoupled receptor (GPCR), named US28, which shows homology to chemokine receptors and binds several chemokines with high affinity. US28 induces migration of smooth muscle cells, a feature essential for the development of atherosclerosis, and may serve as a co-receptor for human immunodeficiency virus-type 1 entry into cells. Previously, we have shown that HCMV-encoded US28 displays constitutive activity, whereas its mammalian homologs do not. In this study we have identified a small nonpeptidergic molecule (VUF2274) that inhibits US28-mediated phospholipase C activation in transiently transfected COS-7 cells and in HCMV-infected fibroblasts. Moreover, VUF2274 inhibits US28-mediated HIV entry into cells. In addition, VUF2274 fully displaces radiolabeled RANTES (regulated on activation normal T cell expressed and secreted) binding at US28, apparently with a noncompetitive behavior. Different analogues of VUF2274 have been synthesized and pharmacologically characterized, to understand which features are important for its inverse agonistic activity. Finally, by means of mutational analysis of US28, we have identified a glutamic acid in transmembrane 7 (TM 7), which is highly conserved among chemokine receptors, as a critical residue for VUF2274 binding to US28. The identification of a full inverse agonist provides an important tool to investigate the relevance of US28 constitutive activity in viral pathogenesis.

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 proteintyrosine 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 FAK⅐Src kinase complex. Interestingly, Src co-immunoprecipitates with US28 in a ligand-dependent manner. This association occurs earlier than the formation of the FAK⅐Src kinase complex, suggesting that US28 activates Src before FAK. US28 binding to RANTES also promotes the formation of a Grb2⅐FAK 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 NH 2 -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. Human cytomegalovirus (HCMV)1 is a ubiquitous herpesvirus that establishes a life-long latent infection after the primary infection has been cleared. Although anti-viral therapy has appreciably reduced disease in transplant and AIDS patients, HCMV is still a significant problem in congenital disease and bone marrow transplant patients. In addition, HCMV has also been associated with long term diseases such as atherosclerosis, restenosis after angioplasty, chronic rejection after solid organ transplantation, and malignancies (1-4). The development of vascular disease involves a chronic inflammatory process with many contributing factors, and of these, chemokines and their receptors have been identified as key mediators. Interestingly, HCMV encodes a CXC chemokine (UL146), a potential CC chemokine (UL128), and four potential chemokine receptors (US27, US28, UL33, and UL78) with the most characterized being US28 (5-8). We have previously reported that US28 mediates arterial smooth muscle cell (SMC) migration and that this activity may contribute to viral dissemination and/or acceleration of vascular disease development (9).US28 contains homology to the CC-chemokine receptors (10) and binds to a broad spectrum of chemokines including the CC chemokines RANTES, MCP-1, MCP-3, and MIP-1␤ and the CX 3 C chemokine Fractalkine/CX3CL1 (11, 12). That CC chemokines fail to compete out Fractalkine binding suggests that Fractalkine binds to additional unique regions of US28 compared with the CC chemokines (11). In 293 cells, RANTES binding to US28 activates ERK1/2 pathways through the Gproteins G␣ i1 and G␣ 16 (13). We have...

The preclinical pharmacological profile of 6-hydroxy-8-[(1R)-1-hydroxy-2-[[2-(4-methoxyphenyl)-1,1-dimethylethyl]amino]ethyl]-2H-1,4-benzoxazin-3(4H)-one monohydrochloride (olodaterol, previously known as BI 1744 CL), a novel, enantiomeric pure, inhaled human ␤ 2 -adrenoceptor (h␤ 2 -AR) agonist, was compared with marketed drugs, such as salmeterol and formoterol. In vitro, olodaterol showed a potent, nearly full agonistic response at the h␤ 2 -AR (EC 50 ϭ 0.1 nM; intrinsic activity ϭ 88% compared with isoprenaline) and a significant selectivity profile (219-and 1622-fold against the h␤ 1 -and h␤ 3 -ARs, respectively). Likewise, olodaterol was able to potently reverse contraction induced by different stimuli in isolated human bronchi. In vivo, antagonistic effects of single doses of olodaterol and formoterol were measured against acetylcholine challenges in anesthetized guinea pigs and dogs for up to 24 h by using the Respimat Soft Mist inhaler. Heart rate and metabolic parameters (serum potassium, lactate, and glucose) were monitored to evaluate systemic pharmacodynamic effects in the dog model. In both models, olodaterol provided bronchoprotection over 24 h. Formoterol applied at an equally effective dose did not retain efficacy over 24 h. In both models olodaterol showed a rapid onset of action comparable with formoterol. Taken together, the preclinical behavior of olodaterol suggests that this novel ␤ 2 -AR agonist has the profile for once-daily dosing in humans concomitant with a fast onset of action and a favorable systemic pharmacodynamic profile.Asthma and chronic obstructive pulmonary disease (COPD) are conditions characterized by airway obstruction, which is variable and reversible in asthma but is progressive in COPD (Guerra, 2009). Both diseases are very common, and their incidence is increasing globally, placing a growing burden on patients and health services in industrialized and developing countries (Pauwels and Rabe, 2004;Braman, 2006). ␤ 2 -Adrenoceptor (␤ 2 -AR) agonists are among the most potent and rapidly acting bronchodilators currently available for clinical use. In asthma, rapid-acting inhaled ␤ 2 -AR agonists are the therapy of choice as a reliever therapy for episodes of dyspnea and the pretreatment of exercise-induced bronchoconstriction (Bateman et al., 2008). In asthma patients with persistent symptoms long-acting ␤-agonists (LABAs), such as salmeterol and formoterol, are administered as an add-on controller therapy when the first-line treatment of medium dose-inhaled corticosteroids alone fails to achieve control of asthma (Bateman et al., 2008). Recently, formoterol has gained some recognition as an as needed controller therapy because of its fast onset of action. In addition, inhaled ␤ 2 -AR agonists provide major therapeutic benefits in the treatment of COPD, such as reduction in symptoms and exacerbations, increases in exercise capacity, and improvements of health-related quality of life (Gold, 2009).␤ 2 -AR agonists exert a bronchodilatory effect through activation of ...