Differences In Ligand Specificity Research Articles

Regulation of PXR Function by Coactivator and Corepressor Proteins: Ligand Binding Is Just the Beginning.

The pregnane X receptor (PXR, NR1I2) is a nuclear receptor which exerts its regulatory function by heterodimerization with the retinoid-X-receptor α (RXRα, NR2B1) and binding to the promoter and enhancer regions of diverse target genes. PXR is involved in the regulation of drug metabolism and excretion, metabolic and immunological functions and cancer pathogenesis. PXR activity is strongly regulated by the association with coactivator and corepressor proteins. Coactivator proteins exhibit histone acetyltransferase or histone methyltransferase activity or associate with proteins having one of these activities, thus promoting chromatin decondensation and activation of the gene expression. On the contrary, corepressor proteins promote histone deacetylation and therefore favor chromatin condensation and repression of the gene expression. Several studies pointed to clear cell- and ligand-specific differences in the activation of PXR. In this article, we will review the critical role of coactivator and corepressor proteins as molecular determinants of the specificity of PXR-mediated effects. As already known for other nuclear receptors, understanding the complex mechanism of PXR activation in each cell type and under particular physiological and pathophysiological conditions may lead to the development of selective modulators with therapeutic potential.

Ligand-Specific Factors Influencing GLP-1 Receptor Post-Endocytic Trafficking and Degradation in Pancreatic Beta Cells.

The glucagon-like peptide-1 receptor (GLP-1R) is an important regulator of blood glucose homeostasis. Ligand-specific differences in membrane trafficking of the GLP-1R influence its signalling properties and therapeutic potential in type 2 diabetes. Here, we have evaluated how different factors combine to control the post-endocytic trafficking of GLP-1R to recycling versus degradative pathways. Experiments were performed in primary islet cells, INS-1 832/3 clonal beta cells and HEK293 cells, using biorthogonal labelling of GLP-1R to determine its localisation and degradation after treatment with GLP-1, exendin-4 and several further GLP-1R agonist peptides. We also characterised the effect of a rare GLP1R coding variant, T149M, and the role of endosomal peptidase endothelin-converting enzyme-1 (ECE-1), in GLP1R trafficking. Our data reveal how treatment with GLP-1 versus exendin-4 is associated with preferential GLP-1R targeting towards a recycling pathway. GLP-1, but not exendin-4, is a substrate for ECE-1, and the resultant propensity to intra-endosomal degradation, in conjunction with differences in binding affinity, contributes to alterations in GLP-1R trafficking behaviours and degradation. The T149M GLP-1R variant shows reduced signalling and internalisation responses, which is likely to be due to disruption of the cytoplasmic region that couples to intracellular effectors. These observations provide insights into how ligand- and genotype-specific factors can influence GLP-1R trafficking.

Arrestin Recruitment to C-C Chemokine Receptor 5: Potent C-C Chemokine Ligand 5 Analogs Reveal Differences in Dependence on Receptor Phosphorylation and Isoform-Specific Recruitment Bias.

C-C chemokine receptor 5 (CCR5) is a chemokine receptor belonging to the G protein-coupled receptor (GPCR) superfamily. An established anti-human immunodeficiency virus drug target, CCR5 is attracting significant additional interest in both cancer and neuroinflammation. Several N-terminally engineered analogs of C-C chemokine ligand 5 (CCL5), a natural ligand of CCR5, are highly potent CCR5 inhibitors. The inhibitory mechanisms of certain analogs relate to modulation of receptor desensitization, but the cellular and molecular mechanisms have not been fully elucidated. Here we made use of a collection of CCR5 phosphorylation mutants and arrestin variants to investigate how CCL5 analogs differ from CCL5 in their capacity to elicit both CCR5 phosphorylation and arrestin recruitment, with reference to the current "core" and "tail" interaction model for arrestin-GPCR interaction. We showed that CCL5 recruits both arrestin 2 and arrestin 3 to CCR5 with recruitment, particularly of arrestin 2, strongly dependent on the arrestin tail interaction. 5P12-RANTES does not elicit receptor phosphorylation or arrestin recruitment. In contrast, PSC-RANTES induces CCR5 hyperphosphorylation, driving enhanced arrestin recruitment with lower dependence on the arrestin tail interaction. 5P14-RANTES induces comparable levels of receptor phosphorylation to CCL5, but arrestin recruitment is absolutely dependent on the arrestin tail interaction, and in one of the cellular backgrounds used, recruitment showed isoform bias toward arrestin 3 versus arrestin 2. No evidence for ligand-specific differences in receptor phosphorylation patterns across the four implicated serine residues was observed. Our results improve understanding of the molecular pharmacology of CCR5 and help further elucidate the inhibitory mechanisms of a group of potent inhibitors. SIGNIFICANCE STATEMENT: C-C chemokine receptor 5 (CCR5) is a key drug target for human immunodeficiency virus, cancer, and inflammation. Highly potent chemokine analog inhibitors act via the modulation of receptor desensitization, a process initiated by the recruitment of arrestin proteins. This study shows that potent C-C chemokine ligand 5 analogs differ from each other and from the parent chemokine in the extent and quality of CCR5-arrestin association that they elicit, providing valuable insights into CCR5 pharmacology and cell biology that will facilitate the development of new medicines targeting this important receptor.

A Machine Learning Approach for the Discovery of Ligand-Specific Functional Mechanisms of GPCRs.

G protein-coupled receptors (GPCRs) play a key role in many cellular signaling mechanisms, and must select among multiple coupling possibilities in a ligand-specific manner in order to carry out a myriad of functions in diverse cellular contexts. Much has been learned about the molecular mechanisms of ligand-GPCR complexes from Molecular Dynamics (MD) simulations. However, to explore ligand-specific differences in the response of a GPCR to diverse ligands, as is required to understand ligand bias and functional selectivity, necessitates creating very large amounts of data from the needed large-scale simulations. This becomes a Big Data problem for the high dimensionality analysis of the accumulated trajectories. Here we describe a new machine learning (ML) approach to the problem that is based on transforming the analysis of GPCR function-related, ligand-specific differences encoded in the MD simulation trajectories into a representation recognizable by state-of-the-art deep learning object recognition technology. We illustrate this method by applying it to recognize the pharmacological classification of ligands bound to the 5-HT2A and D2 subtypes of class-A GPCRs from the serotonin and dopamine families. The ML-based approach is shown to perform the classification task with high accuracy, and we identify the molecular determinants of the classifications in the context of GPCR structure and function. This study builds a framework for the efficient computational analysis of MD Big Data collected for the purpose of understanding ligand-specific GPCR activity.

Ultrasensitivity dynamics of diverse aryl hydrocarbon receptor modulators in a hepatoma cell line

The aryl hydrocarbon receptor (AhR) is a nuclear receptor that facilitates a wide transcriptional response and causes a variety of adaptive and maladaptive physiological functions. Such functions are entirely dependent on the type of ligand activating it, and therefore, the nuances in the activation of this receptor at the single-cell level have become a research interest for different pharmacological and toxicological applications. Here, we investigate the activation of the AhR by diverse classes of compounds in a Hepa1c1c7-based murine hepatoma cell line. The exogenous compounds analyzed produced different levels of ultrasensitivity in AhR activation as measured by XRE-coupled EGFP production and analyzed by both flow cytometric and computational simulation techniques. Interestingly, simulation experiments reported herein were able to reproduce and quantitate the natural single-cell stochasticity inherent to mammalian cell lines as well as the ligand-specific differences in ultrasensitivity. Classical AhR modulators 2,3,7,8-tetrachlorodibenzodioxin (10− 1–105 pM), PCB-126 (10− 1–107 pM), and benzo[a]pyrene (10− 1–107 pM) produced the greatest levels of single-cell ultrasensitivity and most maximal responses, while consumption-based ligands indole-3-carbinol (103–109 pM), 3,3′-diindolylmethane (103–108 pM), and cannabidiol (103–108 pM) caused low-level AhR activation in more purely graded single-cell fashions. All compounds were tested and analyzed over a 24 h period for consistency. The comparative quantitative results for each compound are presented within. This study aids in defining the disparity between different types of AhR modulators that produce distinctly different physiological outcomes. In addition, the simulation tool developed for this study can be used in future studies to predict the quantitative effects of diverse types of AhR ligands in the context of pharmacological therapies or toxicological concerns.

Comparative estrogenicity of endogenous, environmental and dietary estrogens in pregnant women II: Total estrogenicity calculations accounting for competitive protein and receptor binding and potency

Evaluating the biological significance of human-relevant exposures to environmental estrogens involves assessing the individual and total estrogenicity of endogenous and exogenous estrogens found in serum, for example from biomonitoring studies. We developed a method for this assessment by integrating approaches for (i) measuring total hormone concentrations by mass spectrometry (Fleck et al., 2018), (ii) calculating hormone bioavailable concentrations in serum and, (iii) solving multiple equilibria between estrogenic ligands and receptors, and (iv) quantitatively describing key elements of estrogen potency. The approach was applied to endogenous (E1, E2, E3, E4), environmental (BPA), and dietary Genistein (GEN), Daidzein (DDZ) estrogens measured in the serum of thirty pregnant women. Fractional receptor occupancy (FRO) based estrogenicity was dominated by E1, E2 and E3 (ER-α, 94.4–99.2% (median: 97.3%), ER-β, 82.7–97.7% (median: 92.8%), as was the total response (TR), which included ligand specific differences in recruitment of co-activator proteins (RCA). The median FRO for BPA was at least five orders of magnitude lower than E1, E2 and E3, and three orders of magnitude lower than the fetal derived E4 and GEN and DDZ. BPA contributed less than 1/1000th of the normal daily variability in total serum estrogenicity in this cohort of pregnant women.

Structures of two aptamers with differing ligand specificity reveal ruggedness in the functional landscape of RNA.

Two classes of riboswitches related to the ykkC guanidine-I riboswitch bind phosphoribosyl pyrophosphate (PRPP) and guanosine tetraphosphate (ppGpp). Here we report the co-crystal structure of the PRPP aptamer and its ligand. We also report the structure of the G96A point mutant that prefers ppGpp over PRPP with a dramatic 40,000-fold switch in specificity. The ends of the aptamer form a helix that is not present in the guanidine aptamer and is involved in the expression platform. In the mutant, the base of ppGpp replaces G96 in three-dimensional space. This disrupts the S-turn, which is a primary structural feature of the ykkC RNA motif. These dramatic differences in ligand specificity are achieved with minimal mutations. ykkC aptamers are therefore a prime example of an RNA fold with a rugged fitness landscape. The ease with which the ykkC aptamer acquires new specificity represents a striking case of evolvability in RNA.

GDF-5 can act as a context-dependent BMP-2 antagonist.

BackgroundBone morphogenetic protein (BMP)-2 and growth and differentiation factor (GDF)-5 are two related transforming growth factor (TGF)-β family members with important functions in embryonic development and tissue homeostasis. BMP-2 is best known for its osteoinductive properties whereas GDF-5—as evident from its alternative name, cartilage derived morphogenetic protein 1—plays an important role in the formation of cartilage. In spite of these differences both factors signal by binding to the same subset of BMP receptors, raising the question how these different functionalities are generated. The largest difference in receptor binding is observed in the interaction with the type I receptor BMPR-IA. GDF-5, in contrast to BMP-2, shows preferential binding to the isoform BMPR-IB, which is abrogated by a single amino acid (A57R) substitution. The resulting variant, GDF-5 R57A, represents a “BMP-2 mimic” with respect to BMP receptor binding. In this study we thus wanted to analyze whether the two growth factors can induce distinct signals via an identically composed receptor. ResultsUnexpectedly and dependent on the cellular context, GDF-5 R57A showed clear differences in its activity compared to BMP-2. In ATDC-5 cells, both ligands induced alkaline phosphatase (ALP) expression with similar potency. But in C2C12 cells, the BMP-2 mimic GDF-5 R57A (and also wild-type GDF-5) clearly antagonized BMP-2-mediated ALP expression, despite signaling in both cell lines occurring solely via BMPR-IA. The BMP-2- antagonizing properties of GDF-5 and GDF-5 R57A could also be observed in vivo when implanting BMP-2 and either one of the two GDF-5 ligands simultaneously at heterotopic sites.ConclusionsAlthough comparison of the crystal structures of the GDF-5 R57A:BMPR-IAEC- and BMP-2:BMPR-IAEC complex revealed small ligand-specific differences, these cannot account for the different signaling characteristics because the complexes seem identical in both differently reacting cell lines. We thus predict an additional component, most likely a not yet identified GDF-5-specific co-receptor, which alters the output of the signaling complexes. Hence the presence or absence of this component then switches GDF-5′s signaling capabilities to act either similar to BMP-2 or as a BMP-2 antagonist. These findings might shed new light on the role of GDF-5, e.g., in cartilage maintenance and/or limb development in that it might act as an inhibitor of signaling events initiated by other BMPs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0183-8) contains supplementary material, which is available to authorized users.

Ligand Control of G Protein-Coupled Receptor Activity: New Insights

In this issue of Chemistry & Biology, Srinivasan and colleagues describe their study of ligand-protein interactions in visual pigments. Comparing the more stable isomeric ligand 9-cis retinal to the physiologically occurring 11-cis retinal, they report differences in ligand specificity and opsin conformational stability not previously described for bleached rhodopsin.

Two Distinct Determinants of Ligand Specificity in T1R1/T1R3 (the Umami Taste Receptor)

Umami taste perception in mammals is mediated by a heteromeric complex of two G-protein-coupled receptors, T1R1 and T1R3. T1R1/T1R3 exhibits species-dependent differences in ligand specificity; human T1R1/T1R3 specifically responds to L-Glu, whereas mouse T1R1/T1R3 responds more strongly to other L-amino acids than to L-Glu. The mechanism underlying this species difference remains unknown. In this study we analyzed chimeric human-mouse receptors and point mutants of T1R1/T1R3 and identified 12 key residues that modulate amino acid recognition in the human- and mouse-type responses in the extracellular Venus flytrap domain of T1R1. Molecular modeling revealed that the residues critical for human-type acidic amino acid recognition were located at the orthosteric ligand binding site. In contrast, all of the key residues for the mouse-type broad response were located at regions outside of both the orthosteric ligand binding site and the allosteric binding site for inosine-5'-monophosphate (IMP), a known natural umami taste enhancer. Site-directed mutagenesis demonstrated that the newly identified key residues for the mouse-type responses modulated receptor activity in a manner distinct from that of the allosteric modulation via IMP. Analyses of multiple point mutants suggested that the combination of two distinct determinants, amino acid selectivity at the orthosteric site and receptor activity modulation at the non-orthosteric sites, may mediate the ligand specificity of T1R1/T1R3. This hypothesis was supported by the results of studies using nonhuman primate T1R1 receptors. A complex molecular mechanism involving changes in the properties of both the orthosteric and non-orthosteric sites of T1R1 underlies the determination of ligand specificity in mammalian T1R1/T1R3.

Role of pregnane X receptor (PXR) in diet‐induced obesity

Clinical obesity is a complex metabolic disorder that affects 1 in 3 adults. While a high‐fat diet (HFD) causes obesity and insulin resistance, the specific genes that determine sensitivity to obesity are not known. Recent reports suggest that the nuclear receptor pregnane X receptor (PXR), a xenobiotic receptor that defends against toxic agents, may also play a role in obesity: PXR ligands alter patient plasma lipid levels and affect lipid homeostatic and energy metabolic gene expression. Because of differences in ligand specificity between human and mouse PXRs, we examined the role of PXR in diet‐induced obesity using male PXR‐humanized (hPXR) transgenic and PXR‐knockout (PXR‐KO) vs. wild type (WT) mice. After 16 weeks of high (45%) or normal (12%) fat diets, WT mice had a higher susceptibility to obesity compared to hPXR and PXR‐KO mice. PXR‐KO mice showed the smallest gain in white adipose tissue mass. In contrast, only HFD‐fed hPXR mice had elevated serum glucose and triglycerides and reduced hepatic adiponectin, even though all three genotypes on a HFD displayed insulin resistance, hyperleptinemia and hepatotoxicity. Taken together, our data indicate that while the mouse PXR promoted HFD‐induced obesity, the hPXR mouse carries a genetic predisposition for type 2 diabetes and can be used to explore the role of human PXR in metabolic syndrome. [Supported by NIH grants RO1 HL064761, R25HL059868, P20 MD000175, and SC1 HL099139]

The adenosine deaminases of Plasmodium vivax and Plasmodium falciparum exhibit surprising differences in ligand specificity

Plasmodium vivax and Plasmodium falciparum cause malaria, so proteins essential for their survival in vivo are potential anti-malarial drug targets. Adenosine deaminases (ADA) catalyze the irreversible conversion of adenosine into inosine, and play a critical role in the purine salvage pathways of Plasmodia and their mammalian hosts. Currently, the number of selective inhibitors of Plasmodium ADAs is limited. One potent and widely used inhibitor of the human ADA (hADA), erythro-9-(2-hydroxy-3-nonly)adenine (EHNA), is a very weak inhibitor (Ki=120μM) of P. falciparum ADA (pfADA). EHNA-like compounds are thus excluded from consideration as potential inhibitors of Plasmodium ADA in general. However, EHNA activity in P. vivax ADA (pvADA) has not been reported. Here we applied computational molecular modeling to identify ligand recognition mechanisms unique to P. vivax and P. falciparum ADA. Our biochemical experiments show that EHNA is at least 60-fold more potent against pvADA (Ki=1.9μM) than against pfADA. The D172A pvADA mutant is bound even more tightly (Ki=0.9μM). These results improve our understanding of the mechanisms of ADA ligand recognition and species-selectivity, and facilitate the rational design of novel EHNA-based ADA inhibitors as anti-malarial drugs. To demonstrate a practical application of our findings we have computationally predicted a novel potential inhibitor of pvADA that will not interact with the human ADA.

Agonist-dependent effects of mutations in the sphingosine-1-phosphate type 1 receptor

The sphingosine-1-phosphate type 1 (S1P 1) receptor is a new target in the treatment of auto-immune diseases as evidenced by the recent approval of FTY720 (Fingolimod). The ligand-binding pocket of the S1P 1 receptor has been generally characterised but detailed insight into ligand-specific differences is still lacking. The aim of the current study is to determine differences in ligand-induced S1P 1 receptor activation using an in silico guided site-directed mutagenesis strategy. S1P 1 mutant receptors (modifications of residues Y98 2.57, R120 3.28, F125 3.33) were probed with a chemically diverse set of S1P 1 agonists (S1P, dihydro-S1P (dhS1P), R-, S- and racemic FTY720-P, VPC24191, SEW2871). Mutation of the R 3.28 residue generally results in a reduction of the potency of all ligands although the synthetic ligands including FTY720-P are less sensitive to these mutations. The Y 2.57F mutation does not affect the potency of any of the ligands tested, but for all ligands except FTY720-P a significant decrease in potency is observed at the Y 2.57A mutant. The F 3.33A mutation significantly decreased the potency of FTY720-P and is detrimental for SEW2871 and VPC24191. The non-aromatic endogenous ligands S1P and dhS1P are less affected by this mutation. Our in silico guided mutagenesis studies identified new molecular determinants of ligand-induced S1P 1 receptor activation: 1) the flexibility of the polar head of the agonist to maintain a tight H-bond network with R 3.28 and 2) the ability of the agonist to make aromatic π-stacking interactions with F 3.33. Interestingly, FTY720-P has both chemical properties and is the only ligand that can efficiently activate the Y 2.57A mutant.

Differential nuclear localisation and promoter occupancy play a role in glucocorticoid receptor ligand-specific transcriptional responses

The glucocorticoid receptor (GR) is a ligand-activated transcription factor for which a number of endogenous and synthetic ligands exist. A key question in steroid receptor biology is how different ligands elicit different maximal transcriptional responses via the same receptor and on the same promoter. This question was addressed quantitatively for the GR, using a panel of agonists, partial agonists and antagonists, on the endogenous GILZ gene in two different human cell lines. It was found that the extent of GR nuclear localization correlated with the efficacy for GILZ transactivation by the GR in U2OS cells. However, in A549 cells there was no significant correlation, with all ligands resulting in similar levels of GR nuclear localization, despite different levels of transcriptional activation of the GILZ gene. Chromatin immunoprecipitation analysis on the other hand, revealed ligand-specific differences in GILZ promoter occupancy in the A549 cells, which correlated with the transcriptional efficacy of the subset of ligands investigated. This suggests that ligand-specific differences in promoter occupancy by activated GR play a major role in discrimination between agonist, partial agonist and antagonist responses on the endogenous GILZ gene in A549 cells, while differences in nuclear localisation of liganded GR play a role in determining the transcriptional outcome in U2OS cells. These cell line-specific differences were not dependent on the amount of GR present, since transient overexpression of GR in U2OS did not alter the relative ligand-selective nuclear localisation. Our results show that there is a relationship between ligand-specific transactivation efficacy, extent of nuclear translocation and recruitment of GR to the promoter. However, the relative contribution of nuclear translocation and GR promoter recruitment to ligand-specific transactivation efficacy is cell-specific.

Molecular cloning of two gonadotropin receptors and their distinct mRNA expression profiles in daily oogenesis of the wrasse Pseudolabrus sieboldi

In fish, asynchronous development of ovarian follicles, the simultaneous advance of vitellogenesis and oocyte maturation in one ovary, is a rational reproductive strategy to spawn consecutively in one spawning season. In this study, to clarify the mode of action of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in asynchronous ovarian follicle development in daily egg production, we cloned cDNAs of the follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) in the bambooleaf wrasse ( Pseudolabrus sieboldi), which exhibits clear diurnal spawning rhythms over 1 month. In addition, different developmental stages of ovarian follicles were isolated from whole ovaries at various daily time points on 1 day in the spawning season, and mRNA expression levels of FSHR and LHR were analyzed. Sequence analysis showed distinct differences in the number of putative leucine-rich repeats at the extracellular domain between FSHR and LHR, suggesting a difference in ligand-specificity. Real-time PCR analyses revealed that FSHR mRNA was highly expressed in early yolk-stage follicles but decreased at the end of vitellogenesis. In contrast, the expression of LHR mRNA was maintained at low levels in vitellogenic stage follicles but markedly elevated at the end of the vitellogenic and early migratory nucleus stages, thereafter markedly dropping in the late migratory nucleus stage. The present results suggest that co-regulation of vitellogenesis and oocyte maturation in one ovary is controlled by the stage-distinctive expression levels of FSHR and LHR mRNA in ovarian follicles, and daily switching of sensitivity from FSH to LH is required for daily egg production.

Imaging ensemble activity in arthropod olfactory receptor neurons in situ

We show that lobster olfactory receptor neurons (ORNs), much like their vertebrate counterparts, generate a transient elevation of intracellular calcium (Ca i) in response to odorant activation that can be used to monitor ensemble ORN activity. This is done in antennal slice preparation in situ maintaining the polarity of the cells and the normal micro-environment of the olfactory cilia. The Ca i signal is ligand-specific and increases in a dose-dependent manner in response to odorant stimulation. Saturating stimulation elicits a robust increase of up to 1 μM free Ca i within 1–2 s of stimulation. The odor-induced Ca i response closely follows the discharge pattern of extracellular spikes elicited by odorant application, with the maximal rise in Ca i matching the peak of the spike generation. The Ca i signal can be used to track neuronal activity in a functional subpopulation of rhythmically active ORNs and discriminate it from that of neighboring tonically active ORNs. Being able to record from many ORNs simultaneously over an extended period of time not only allows more accurate estimates of neuronal population activity but also dramatically improves the ability to identify potential new functional subpopulations of ORNs, especially those with more subtle differences in responsiveness, ligand specificity, and/or transduction mechanisms.

Down-regulation of c-Cbl by Morphine Accounts for Persistent ERK1/2 Signaling in δ-Opioid Receptor-expressing HEK293 Cells

Opioids display ligand-specific differences in the time course of ERK1/2 signaling. Whereas full agonists, like etorphine, induce only transient activation of ERK1/2, the partial agonist morphine mediates persistent stimulation of mitogenic signaling. Here we report that in stably delta-opioid receptor (DOR)-expressing HEK293 (HEK/DOR) cells, the transient nature of etorphine-induced ERK1/2 signaling is due to desensitization of epidermal growth factor (EGF) receptor-mediated activation of the Ras/Raf-1/ERK1/2 cascade. Desensitization of ERK1/2 activity by etorphine is associated with down-regulation of EGF receptors, an effect mediated by the ubiquitin ligase c-Cbl. In contrast, chronic morphine treatment failed to desensitize EGF receptors, resulting in unimpeded ERK1/2 signaling. The failure of morphine to desensitize ERK1/2 signaling is mediated by persistent activation of c-Src, which induces degradation of c-Cbl. The role of c-Src in opioid-specific ERK1/2 signaling is further demonstrated by pretreatment of the cells with PP2 and SKI-I as well as overexpression of a dominant negative c-Src mutant (c-Src(dn)) or a c-Src-resistant c-Cbl mutant (CblY3F), both of which facilitate desensitization of ERK1/2 signaling by morphine. Conversely, overexpression of c-Src as well as down-regulation of c-Cbl by small interfering RNA results in persistent etorphine-induced stimulation of ERK1/2 activity. Subcellular fractionation experiments finally attributed the ability of morphine to persistently activate c-Src to its redistribution from Triton X-100-insensitive membrane rafts to DOR and EGF receptor containing high density membrane compartments implicated in ERK1/2 signaling. These results demonstrate that agonist-specific differences in the temporal and spatial pattern of c-Src activation determine the kinetics of DOR-mediated regulation of ERK1/2 signaling.

Crystal structure of the variable domain of the Streptococcus gordonii surface protein SspB

The Antigen I/II (AgI/II) family of proteins are cell wall anchored adhesins expressed on the surface of oral streptococci. The AgI/II proteins interact with molecules on other bacteria, on the surface of host cells, and with salivary proteins. Streptococcus gordonii is a commensal bacterium, and one of the primary colonizers that initiate the formation of the oral biofilm. S. gordonii expresses two AgI/II proteins, SspA and SspB that are closely related. One of the domains of SspB, called the variable (V-) domain, is significantly different from corresponding domains in SspA and all other AgI/II proteins. As a first step to elucidate the differences among these proteins, we have determined the crystal structure of the V-domain from S. gordonii SspB at 2.3 A resolution. The domain comprises a beta-supersandwich with a putative binding cleft stabilized by a metal ion. The overall structure of the SspB V-domain is similar to the previously reported V-domain of the Streptococcus mutans protein SpaP, despite their low sequence similarity. In spite of the conserved architecture of the binding cleft, the cavity is significantly smaller in SspB, which may provide clues about the difference in ligand specificity. We also verified that the metal in the binding cleft is a calcium ion, in concurrence with previous biological data. It was previously suggested that AgI/II V-domains are carbohydrate binding. However, we tested that hypothesis by screening the SspB V-domain for binding to over 400 glycoconjucates and found that the domain does not interact with any of the carbohydrates.

Ligand Specificity of Constitutive Androstane Receptor as Probed by Induced-Fit Docking and Mutagenesis

Constitutive androstane receptor (CAR, NR1I3) belongs to the nuclear receptor family of transcription factors and acts as a chemical sensor of drugs and endogenous compounds. The ligand-binding preferences of CAR are diverse, and more importantly, there are significant species differences in ligand specificity. Here, we show that while certain residues are critical for the basal activity of mouse CAR (mCAR) and/or affect the binding of all tested ligands, mutation of some ligand-binding pocket (LBP) residues (e.g., F171 and Y336) paradoxically decreased the activity of a specific ligand while increasing that of others. Comparisons to previously reported human CAR (hCAR) residues indicated that the function of key CAR residues (e.g., N175, L253) is dramatically different between species. The docking results provide some mechanistic rationale for the ability of 17alpha-ethinyl-3,17beta-estradiol (EE2) to both activate mCAR and repress hCAR.

Evolution of pharmacologic specificity in the pregnane X receptor

BackgroundThe pregnane X receptor (PXR) shows the highest degree of cross-species sequence diversity of any of the vertebrate nuclear hormone receptors. In this study, we determined the pharmacophores for activation of human, mouse, rat, rabbit, chicken, and zebrafish PXRs, using a common set of sixteen ligands. In addition, we compared in detail the selectivity of human and zebrafish PXRs for steroidal compounds and xenobiotics. The ligand activation properties of the Western clawed frog (Xenopus tropicalis) PXR and that of a putative vitamin D receptor (VDR)/PXR cloned in this study from the chordate invertebrate sea squirt (Ciona intestinalis) were also investigated.ResultsUsing a common set of ligands, human, mouse, and rat PXRs share structurally similar pharmacophores consisting of hydrophobic features and widely spaced excluded volumes indicative of large binding pockets. Zebrafish PXR has the most sterically constrained pharmacophore of the PXRs analyzed, suggesting a smaller ligand-binding pocket than the other PXRs. Chicken PXR possesses a symmetrical pharmacophore with four hydrophobes, a hydrogen bond acceptor, as well as excluded volumes. Comparison of human and zebrafish PXRs for a wide range of possible activators revealed that zebrafish PXR is activated by a subset of human PXR agonists. The Ciona VDR/PXR showed low sequence identity to vertebrate VDRs and PXRs in the ligand-binding domain and was preferentially activated by planar xenobiotics including 6-formylindolo-[3,2-b]carbazole. Lastly, the Western clawed frog (Xenopus tropicalis) PXR was insensitive to vitamins and steroidal compounds and was activated only by benzoates.ConclusionIn contrast to other nuclear hormone receptors, PXRs show significant differences in ligand specificity across species. By pharmacophore analysis, certain PXRs share similar features such as human, mouse, and rat PXRs, suggesting overlap of function and perhaps common evolutionary forces. The Western clawed frog PXR, like that described for African clawed frog PXRs, has diverged considerably in ligand selectivity from fish, bird, and mammalian PXRs.