Structural basis for HCMV Pentamer receptor recognition and antibody neutralization

Human cytomegalovirus (HCMV) represents the viral leading cause of congenital birth defects and uses the gH/ gL/UL128-130-131A complex (Pentamer) to enter different cell types, including epithelial and endothelial cells. Upon infection, Pentamer elicits the most potent neutralizing response against HCMV, representing a key vaccine candidate. Despite its relevance, the structural basis for Pentamer receptor recognition and antibody neutralization is largely unknown. Here, we determine the structures of Pentamer bound to neuropilin 2 (NRP2) and a set of potent neutralizing antibodies against HCMV. Moreover, we identify thrombomodulin (THBD) as a functional HCMV receptor and determine the structures of the Pentamer-THBD complex. Unexpectedly, both NRP2 and THBD also promote dimerization of Pentamer. Our results provide a framework for understanding HCMV receptor engagement, cell entry, antibody neutralization, and outline strategies for antiviral therapies against HCMV

Isoproterenol Acts as a Biased Agonist of the Alpha-1A-Adrenoceptor that Selectively Activates the MAPK/ERK Pathway

<div><p>The α_1A-AR is thought to couple predominantly to the Gα_q/PLC pathway and lead to phosphoinositide hydrolysis and calcium mobilization, although certain agonists acting at this receptor have been reported to trigger activation of arachidonic acid formation and MAPK pathways. For several G protein-coupled receptors (GPCRs) agonists can manifest a bias for activation of particular effector signaling output, <i>i.e.</i> not all agonists of a given GPCR generate responses through utilization of the same signaling cascade(s). Previous work with Gα_q coupling-defective variants of α_1A-AR, as well as a combination of Ca²⁺ channel blockers, uncovered cross-talk between α_1A-AR and β₂-AR that leads to potentiation of a Gα_q-independent signaling cascade in response to α_1A-AR activation. We hypothesized that molecules exist that act as biased agonists to selectively activate this pathway. In this report, isoproterenol (Iso), typically viewed as β-AR-selective agonist, was examined with respect to activation of α_1A-AR. α_1A-AR selective antagonists were used to specifically block Iso evoked signaling in different cellular backgrounds and confirm its action at α_1A-AR. Iso induced signaling at α_1A-AR was further interrogated by probing steps along the Gα_q /PLC, Gα_s and MAPK/ERK pathways. In HEK-293/EBNA cells transiently transduced with α_1A-AR, and CHO_α_1A-AR stable cells, Iso evoked low potency ERK activity as well as Ca²⁺ mobilization that could be blocked by α_1A-AR selective antagonists. The kinetics of Iso induced Ca²⁺ transients differed from typical Gα_q- mediated Ca²⁺ mobilization, lacking both the fast IP₃R mediated response and the sustained phase of Ca²⁺ re-entry. Moreover, no inositol phosphate (IP) accumulation could be detected in either cell line after stimulation with Iso, but activation was accompanied by receptor internalization. Data are presented that indicate that Iso represents a novel type of α_1A-AR partial agonist with signaling bias toward MAPK/ERK signaling cascade that is likely independent of coupling to Gα_q.</p></div

Treatment of α_1A-AR transduced HEK-293/EBNA with A-61603 and Iso does not trigger intracellular redistribution of arrestins.

<p>HEK293 cells were co-transfected with FLAG-tagged α_1A – AR and GFP- tagged β-arrestin-1 (left panels) or β-arrestin-2 (right panels). Following serum-deprivation for 24h, cells were left untreated (top panels), or stimulated with 1μM A-61603 (middle panels) or 1mM Iso (bottom panels) for the indicated amount of time. Cells were then fixed, permeabilized, stained with Alexa Fluor-568 conjugated anti-FLAG antibodies, and analyzed employing confocal microscopy.</p

Structural basis of antibody inhibition and chemokine activation of the human CC chemokine receptor 8

Abstract The C-C motif chemokine receptor 8 (CCR8) is a class A G-protein coupled receptor that has emerged as a promising therapeutic target in cancer. Targeting CCR8 with an antibody has appeared to be an attractive therapeutic approach, but the molecular basis for chemokine-mediated activation and antibody-mediated inhibition of CCR8 are not fully elucidated. Here, we obtain an antagonist antibody against human CCR8 and determine structures of CCR8 in complex with either the antibody or the endogenous agonist ligand CCL1. Our studies reveal characteristic antibody features allowing recognition of the CCR8 extracellular loops and CCL1-CCR8 interaction modes that are distinct from other chemokine receptor - ligand pairs. Informed by these structural insights, we demonstrate that CCL1 follows a two-step, two-site binding sequence to CCR8 and that antibody-mediated inhibition of CCL1 signaling can occur by preventing the second binding event. Together, our results provide a detailed structural and mechanistic framework of CCR8 activation and inhibition that expands our molecular understanding of chemokine - receptor interactions and offers insight into the development of therapeutic antibodies targeting chemokine GPCRs

The Iso-induced Ca²⁺ mobilization response in α_1A-AR transduced HEK-293/EBNA cells is slower in onset and shorter in duration than the response to NE.

<p>Ca²⁺ transient response kinetics (expressed as ΔF_t/F₀) are shown for fluo3-loaded cells monitored fluorometrically following addition of Iso or NE. HEK-293/EBNA cells were first exposed to recombinant baculoviral strains (3–4 h) encoding either α_1A-AR or an irrelevant negative control protein (aldehyde oxidase), then cultured in fresh culture medium containing 4 mM NaBu for 18 h prior to use in experiments as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115701#sec002" target="_blank">Materials and Methods</a>. <b>Panel A</b>: Slow onset of the Iso agonist response which returns to baseline within a 2 min interval is evident in representative traces from α_1A-AR transduced HEK-293/EBNA cells (black lines) and negative control cells (gray lines) during responses elicited by addition of Iso (↓) at 100 nM (dashed lines) or 100 μM (solid lines). <b>Panel B</b>: Representative Ca²⁺ transients in α_1A-AR transduced HEK-293/EBNA cells showing rapid onset and sustained NE response following application of NE (↓) at 100 nM (dashed lines) or 100 μM (solid lines) to α_1A-AR transduced (black lines) or negative control-transduced cells (gray lines). <b>Panel C</b>: α_1A-AR transduced HEK-293/EBNA cells exhibit distinct kinetics for Iso-mediated responses via occupancy of β-AR vs α_1A-AR transduced, as revealed by monitoring of responses following pre-treatment of cells for 20 min with vehicle (solid black line), 10 nM RS-100329 (dashed gray line) or 10 nM ICI118551 (dashed black line). Representative traces are shown for responses to 100 μM Iso (↓).</p

Pertussis toxin pretreatment does not impair Ca²⁺ responses to Iso.

<p>HEK-293/EBNA cells were exposed for 3 h to a baculoviral strain carrying α_1A-AR or to viral medium. Cells were treated with 100 ng/mL of pertussis toxin (PTX) or vehicle for 18 h prior to experiments. <b>Panel A</b>: untransduced HEK-293/EBNA cells exposed to PTX (filled symbols) or vehicle (open symbols) were treated with Iso (■,□) during fluorometric imaging of the Ca²⁺-tracking dye. <b>Panel B:</b> untreated (open symbols) or PTX-pretreated (filled symbols) α_1A-AR HEK-293/EBNA cells were stimulated with Iso (■,□) during fluorometric imaging of the Ca²⁺-tracking dye. Each experiment was performed in duplicate two independent times.</p

Iso-induced Ca²⁺ mobilization in α_1A-AR transduced HEK-293/EBNA cells is partially dependent on the presence of extracellular Ca²⁺.

<p>Ca²⁺ transient responses (expressed as ΔF/F₀) were measured as a function of Iso concentration in untransduced control cells (<b>Panel A</b>) or α_1A-AR transduced HEK-293/EBNA cells (<b>Panel B</b>), either in the presence (filled symbols) or absence (open symbols) of 2 mM Ca²⁺ in the assay buffer. Responses in untransduced cells were virtually abolished in the absence of extracellular Ca²⁺ (<b>Panel A</b>). In α_1A-AR transduced cells, responses to low concentrations of Iso in the absence of extracellular Ca²⁺ were also essentially abolished whereas the low potency phase of response was diminished by approximately 50% (<b>Panel B</b>). Each data point is an average of duplicate determinations; this experiment was repeated twice.</p

Inositol phosphates production in α_1A-AR transduced HEK-293/EBNA cells occurs in response to A-61603 and NE, but not in response to Iso.

<p>HEK-293/EBNA cells were exposed to baculovirus encoding α_1A-AR for 3–4 h, then cultured in fresh medium containing 4 mM NaBu for 18 h prior to use in experiments as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115701#sec002" target="_blank">Materials and Methods</a>. IP₁ (top), IP₂ (middle) and IP₃ (bottom) formation was measured in α_1A-AR transduced HEK-293/EBNA cells stimulated with increasing concentrations of A-61603 (▲), NE (■) or Iso(●). IP₁, IP₂, and IP₃ levels were determined via LC-MS. Plots are representative of three independent experiments with each data point being the average of triplicates.</p