Endocannabinoids (eCBs) modulate the activity of proteins expressed at the plasma and intracellular membranes. Nothing is known about the dynamic changes in eCB levels in these subcellular compartments. We leveraged the eCB sensor, GRABeCB2.0, to establish the stimulus-induced increases in the eCB, 2-arachidonoyl glycerol (2-AG), at the plasma and intracellular membranes of undifferentiated Neuro2a cells in culture. Activating G protein-coupled B 2 receptors with bradykinin increased 2-AG levels at both the plasma and intracellular membranes within approximate to 5 and approximate to 15 s, respectively. By contrast, the activation of G proteins by the small peptide mastoparan and the ensuing opening of plasma membrane calcium channels increased 2-AG levels in plasma membrane within approximate to 1-2 s and in intracellular membranes after approximate to 30 s. While both these stimuli-induced increases in 2-AG production involved canonical lipases, they required distinct sources of calcium. Thus, distinct stimuli differentially increase 2-AG levels at plasma and intracellular membranes via distinct molecular mechanisms.

Cellular retinoic acid binding proteins (CRABP1 and CRABP2) bind all-trans-retinoic acid (atRA), the active metabolite of vitamin A, with high affinity. CRABP1 and CRABP2 have been shown to interact with the atRA-clearing cytochrome P450 enzymes CYP26B1 and CYP26C1 and with nuclear retinoic acid receptors (RARs). We hypothesized that CRABP1 and CRABP2 also alter atRA metabolism and clearance by CYP26A1, the third key atRA-metabolizing enzyme in the CYP26 family. Based on stopped-flow experiments, atRA bound CRABP1 and CRABP2 with K-d values of 4.7 nM and 7.6 nM, respectively. The unbound atRA K-m values for 4-OH-atRA formation by CYP26A1 were 4.7 +/- 0.8 nM with atRA, 6.8 +/- 1.7 nM with holo-CRABP1 and 6.1 +/- 2.7 nM with holo-CRABP2 as a substrate. In comparison, the apparent k(cat) value was about 30% lower (0.71 +/- 0.07 min(-1) for holo-CRABP1 and 0.75 +/- 0.09 min(-1) for holo-CRABP2) in the presence of CRABPs than with free atRA (1.07 +/- 0.08 min(-1)). In addition, increasing concentrations in apo-CRABPs decreased the 4-OH-atRA formation rates by CYP26A1. Kinetic analyses suggest that apo-CRABP1 and apo-CRABP2 inhibit CYP26A1 (K-i = 0.39 nM and 0.53 nM, respectively) and holo-CRABPs channel atRA for metabolism by CYP26A1. These data suggest that CRABPs play a critical role in modulating atRA metabolism and cellular atRA concentrations.

Voltage-gated sodium channels initiate electrical signals and are frequently targeted by deadly gating-modifier neurotoxins, including tarantula toxins, which trap the voltage sensor in its resting state. The structural basis for tarantula-toxin action remains elusive because of the difficulty of capturing the functionally relevant form of the toxin-channel complex. Here, we engineered the model sodium channel Na(V)Ab with voltage-shifting mutations and the toxin-binding site of human Na(V)1.7, an attractive pain target. This mutant chimera enabled us to determine the cryoelectronmicroscopy (cryo-EM) structure of the channel functionally arrested by tarantula toxin. Our structure reveals a high-affinity resting-state-specific toxin-channel interaction between a key lysine residue that serves as a "stinger'' and penetrates a triad of carboxyl groups in the S3-S4 linker of the voltage sensor. By unveiling this high-affinity binding mode, our studies establish a high-resolution channel-docking and resting-state locking mechanism for huwentoxin-IV and provide guidance for developing future resting-state-targeted analgesic drugs.

Monoclonal antibodies (mAbs) have become an important class of therapeutics, particularly in the realm of anticancer immunotherapy. While the two antigen-binding fragments (Fabs) of an mAb allow for high-avidity binding to molecular targets, the crystallizable fragment (Fc) engages immune effector elements. mAbs of the IgG class are used for the treatment of autoimmune diseases and can elicit antitumor immune functions not only by several mechanisms including direct antigen engagement via their Fab arms but also by Fab binding to tumors combined with Fc engagement of complement component C1q and Fc gamma receptors. Additionally, IgG binding to the neonatal Fc receptor (FcRn) allows for endosomal recycling and prolonged serum half-life. To augment the effector functions or half-life of an IgG1 mAb, we constructed a novel "2Fc" mAb containing two Fc domains in addition to the normal two Fab domains. Structural and functional characterization of this 2Fc mAb demonstrated that it exists in a tetrahedral-like geometry and retains binding capacity via the Fab domains. Furthermore, duplication of the Fc region significantly enhanced avidity for Fc receptors Fc gamma RI, Fc gamma RIIIa, and FcRn, which manifested as a decrease in complex dissociation rate that was more pronounced at higher densities of receptor. At intermediate receptor density, the dissociation rate for Fc receptors was decreased 6- to 130-fold, resulting in apparent affinity increases of 7- to 42-fold. Stoichiometric analysis confirmed that each 2Fc mAb may simultaneously bind two molecules of Fc gamma RI or four molecules of FcRn, which is double the stoichiometry of a wild-type mAb. In summary, duplication of the IgG Fc region allows for increased avidity to Fc receptors that could translate into clinically relevant enhancement of effector functions or pharmacokinetics.

Oxylipins derived from the oxidation of polyunsaturated fatty acids (PUFAs) act as important paracrine and autocrine signaling molecules. A subclass of oxylipins, the eicosanoids, have a broad range of physiological outcomes in inflammation, the immune response, cardiovascular homeostasis, and cell growth regulation. Consequently, eicosanoids are implicated in the pathophysiology of various diseases, most notably cancer, where eicosanoid mediated signaling is involved in tumor development, progression, and angiogenesis. Cytochrome P450s (CYPs) are a superfamily of heme monooxygenases generally involved in the clearance of xenobiotics while a subset of isozymes oxidize PUFAs to eicosanoids. Several eicosanoid forming CYPs are overexpressed in tumors, elevating eicosanoid levels and suggesting a key function in tumorigenesis and progression of tumors in the lung, breast, prostate, and kidney. This review summarizes the current understanding of CYPs' involvement in solid tumor etiology and progression providing supporting public data for gene expression from The Cancer Genome Atlas.

G protein-coupled receptor (GPCR) biogenesis, trafficking, and function are regulated by post-translational modifications, including N-glycosylation of asparagine residues. alpha (1D)-adrenergic receptors (alpha (1D)-ARs) - key regulators of central and autonomic nervous system function - contain two putative N-glycosylation sites within the large N-terminal domain at N65 and N82. However, determining the glycosylation state of this receptor has proven challenging. Towards understanding the role of these putative glycosylation sites, site-directed mutagenesis and lectin affinity purification identified N65 and N82 as bona fide acceptors for N-glycans. Surprisingly, we also report that simultaneously mutating N65 and N82 causes early termination of alpha (1D)-AR between transmembrane domain 2 and 3. Label-free dynamic mass redistribution and cell surface trafficking assays revealed that single and double glycosylation deficient mutants display limited function with impaired plasma membrane expression. Confocal microscopy imaging analysis and SNAP-tag sucrose density fractionation assays revealed the dual glycosylation mutant alpha (1D)-AR is widely distributed throughout the cytosol and nucleus. Based on these novel findings, we propose alpha (1D-)AR transmembrane domain 2 acts as an ER localization signal during active protein biogenesis, and that alpha (1D)-AR N-terminal glycosylation is required for complete translation of nascent, functional receptor.

Many G protein-coupled receptors (GPCRs) are organized as dynamic macromolecular complexes in human cells. Unraveling the structural determinants of unique GPCR complexes may identify unique protein: protein interfaces to be exploited for drug development. We previously reported alpha(1D)-adrenergic receptors (alpha(1D)-ARs) - key regulators of cardiovascular and central nervous system function - form homodimeric, modular PDZ protein complexes with cell-type specificity. Towards mapping alpha(1D)-AR complex architecture, biolayer interferometry (BLI) revealed the alpha(1D)-AR C-terminal PDZ ligand selectively binds the PDZ protein scribble (SCRIB) with >8x higher affinity than known interactors syntrophin, CASK and DLG1. Complementary in situ and in vitro assays revealed SCRIB PDZ domains 1 and 4 to be high affinity alpha(1D)-AR PDZ ligand interaction sites. SNAP-GST pull-down assays demonstrate SCRIB binds multiple alpha(1D)-AR PDZ ligands via a co-operative mechanism. Structure-function analyses pinpoint R1110(PDZ4) as a unique, critical residue dictating SCRIB: alpha(1D)-AR binding specificity. The crystal structure of SCRIB PDZ4 R1110G predicts spatial shifts in the SCRIB PDZ4 carboxylate binding loop dictate alpha(1D)-AR binding specificity. Thus, the findings herein identify SCRIB PDZ domains 1 and 4 as high affinity alpha(1D)-AR interaction sites, and potential drug targets to treat diseases associated with aberrant alpha(1D)-AR signaling.

Efficient stem cell differentiation into pancreatic islet cells is of critical importance for the development of cell replacement therapies for diabetes. Here, we identify the expression pattern of connexin 43 (Cx43), a gap junction (GJ) channel protein, in human embryonic stem cell (hESC)-derived definitive endoderm (DE) and primitive gut tube cells, representing early lineages for posterior foregut (PF), pancreatic progenitors (PP), pancreatic endocrine progenitors (PE), and islet cells. As the function of GJ channels is dependent on their gating status, we tested the impact of supplementing hESC-derived PP cell cultures with AAP10, a peptide that promotes Cx43 GJ channel opening. We found that this treatment promotes the expression of DE markers FoxA2 and Sox17, leads to a more efficient derivation of DE, and improves the yield of PF, PP, and PE cells. These results demonstrate a functional involvement of GJ channels in the differentiation of embryonic stem cells into pancreatic cell lineages.

All-trans-retinoic acid (atRA) downregulates cytochrome P450 (CYP)2D6 in several model systems. The aim of this study was to determine whether all active retinoids downregulate CYP2D6 and whether in vitro downregulation translates to in vivo drug-drug interactions (DDIs). The retinoids atRA, 13cisRA, and 4-oxo-13cisRA all decreased CYP2D6 mRNA in human hepatocytes in a concentration-dependent manner. The in vitro data predicted similar to 50% decrease in CYP2D6 activity in humans after dosing with 13cisRA. However, the geometric mean area under plasma concentration-time curve (AUC) ratio for dextromethorphan between treatment and control was 0.822, indicating a weak induction of dextromethorphan clearance following 13cisRA treatment. Similarly, in mice treatment with 4-oxo-13cisRA-induced mRNA expression of multiple mouse Cyp2d genes. In comparison, a weak induction of CYP3A4 in human hepatocytes translated to a weak in vivo induction of CYP3A4. These data suggest that in vitro CYP downregulation may not translate to in vivo DDIs, and better understanding of the mechanisms of CYP downregulation is needed.