Direct Interaction Of Ligand Research Articles

Surface CD6 regulates T cell adhesion and migration through direct ligand interaction and stabilization of VLA4

Abstract The CD6-ALCAM pathway is an important regulator of T cell activity and trafficking. However, its precise role in modulating T-cell adhesion and migration during inflammation is unclear. To investigate its role in adhesion and transendothelial migration, we used itolizumab, an anti-CD6 mAb that can induce cleavage of CD6 from the T cell surface when in the presence of monocytes. PBMCs were stimulated overnight or left unstimulated prior to treatment with itolizumab to generate CD6low T cells. Cells were then tested in standard adhesion and migration assays. The CD6 low T cells had ~ 50% decreased adherence to HUVEC cells compared to the control T cells (CD6high); similar results were observed by blocking ALCAM on HUVEC. Modulating CD6 on T cells or blocking ALCAM on the HUVEC also translated to a ~3-fold decrease in migration of Teff cells. Loss of CD6 was associated with reduced protein levels of VLA4, but not of other integrins, under both stimulated (~70%) and non-stimulated conditions (~40%). Antibody blockade of CD6 without cleavage did not affect VLA4 levels, but still decreased migration of unstimulated T cells though less so than in stimulated T cells, where VLA4 reduction is required to modulate migration. Our data demonstrate that CD6 contributes to cell adhesion/migration both through direct ligand interactions with ALCAM, and through the stabilization of cell membrane VLA4 and that these functions can be abrogated by modulation of cell surface levels of CD6.

Structures of the Plasmodium falciparum heat-shock protein 70-x ATPase domain in complex with chemical fragments identify conserved and unique binding sites.

Plasmodium falciparum invades erythrocytes and extensively modifies them in a manner that increases the virulence of this malaria parasite. A single heat-shock 70 kDa-type chaperone, PfHsp70-x, is among the parasite proteins exported to the host cell. PfHsp70-x assists in the formation of a key protein complex that underpins parasite virulence and supports parasite growth during febrile episodes. Previous work resolved the crystallographic structures of the PfHsp70-x ATPase and substrate-binding domains, and showed them to be highly similar to those of their human counterparts. Here, 233 chemical fragments were screened for binding to the PfHsp70-x ATPase domain, resulting in three crystallographic structures of this domain in complex with ligands. Two binding sites were identified, with most ligands binding proximal to the ATPase nucleotide-binding pocket. Although amino acids participating in direct ligand interactions are conserved between the parasite and human erythrocytic chaperones, one nonconserved residue is also present near the ligand. This work suggests that PfHsp70-x features binding sites that may be exploitable by small-molecule ligands towards the specific inhibition of the parasite chaperone.

Discovery of GS-9688 (Selgantolimod) as a Potent and Selective Oral Toll-Like Receptor 8 Agonist for the Treatment of Chronic Hepatitis B.

Toll-like receptor 8 (TLR8) recognizes pathogen-derived single-stranded RNA fragments to trigger innate and adaptive immune responses. Chronic hepatitis B (CHB) is associated with a dysfunctional immune response, and therefore a selective TLR8 agonist may be an effective treatment option. Structure-based optimization of a dual TLR7/8 agonist led to the identification of the selective TLR8 clinical candidate (R)-2-((2-amino-7-fluoropyrido[3,2-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (GS-9688, (R)-7). Potent TLR8 agonism (IL-12p40 EC50 = 220 nM) and >100-fold TLR7 selectivity (IFN-α EC50 > 50 μM) was observed in human peripheral blood mononuclear cells (PBMCs). The TLR8-ectodomain:(R)-7 complex confirmed TLR8 binding and a direct ligand interaction with TLR8 residue Asp545. Oral (R)-7 had good absorption and high first pass clearance in preclinical species. A reduction in viral markers was observed in HBV-infected primary human hepatocytes treated with media from PBMCs stimulated with (R)-7, supporting the clinical development of (R)-7 for the treatment of CHB.

Activation of the GLP-1 receptor by a non-peptidic agonist.

Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, including diabetes and obesity1. Structures of active receptors reveal peptide agonists engage deep within the receptor core, leading to an outward movement of extracellular loop 3 and the tops of transmembrane helices 6 and 7, an inward movement of transmembrane helix 1, reorganization of extracellular loop 2 and outward movement of the intracellular side of transmembrane helix 6, resulting in G-protein interaction and activation2-6. Here we solved the structure of a non-peptide agonist, TT-OAD2, bound to the glucagon-like peptide-1 (GLP-1) receptor. Our structure identified an unpredicted non-peptide agonist-binding pocket in which reorganization of extracellular loop 3 and transmembrane helices 6 and 7 manifests independently of direct ligand interaction within the deep transmembrane domain pocket. TT-OAD2 exhibits biased agonism, and kinetics of G-protein activation and signalling that are distinct from peptide agonists. Within the structure, TT-OAD2 protrudes beyond the receptor core to interact with the lipid or detergent, providing an explanation for the distinct activation kinetics that may contribute to the clinical efficacy of this compound series. This work alters our understanding of the events that drive the activation of class B receptors.

Abstract PR11: Hyperspectral imaging tools capture the spatial organization of cell subsets within the tumor microenvironment

Abstract Immune cells are a major component of the tumor microenvironment (TME). The spatial organization of immune cell subpopulations within the TME is recognized to have biologic significance and clinical relevance. For example, spatial organization of immune cell subsets within the TME is critical for the inhibition of cytotoxic T-cell activity through direct interaction of ligand (PD-L1) with receptor (PD-1)). However, precise spatial deconvolution is limited by the lack of imaging algorithms for in situ multiplex single cell analyses as flow cytometry does not preserve data in the spatial dimension. To this end, we have developed a hyperspectral imaging platform designed for analyzing multichannel immunohistochemical-stained tissue sections for generating cell density data and reconstructing spatial architecture for tumor biology as well as clinical association studies. Whole-tissue sections from 20 lung adenocarcinomas with at least 5 years’ follow-up were stained for CD3 (pan-T cell), CD8 (cytotoxic T cell), and CD79a (B cell and plasma cell) and counterstained with hematoxylin. Multispectral images were acquired for five fields of view and analyzed to quantify cell types. Regions of Interest (ROIs) were then identified and analyzed in order to quantify cell-cell spatial relationships. Nonrandom patterns of immune cell distributions were identified using the Monte Carlo resampling method (500 iterations). Cell counts, densities, spatial relationships, and significant immune cell distributions were associated with clinical features (Kruskal-Wallis p<0.001). Our analysis generated 234 image files for analysis, with an average of 16,400 cells per image. The densities of intratumoral CD8+ cytotoxic T cells were significantly higher in nonrecurrent cases, agreeing with literature reports. Similarly, cell sociology deductions identified relationships associated with metastasis: tumor cells in nonmetastatic cases had increased numbers of CD8+ cytotoxic T-cell neighbors. Following Monte Carlo analysis, nonrandom cell~cell spatial proximities emerged that were not identified at a cell density level. We have developed a hyperspectral imaging platform capable of quantifying cell-cell spatial relationships within tissue sections. This technology can be applied to larger clinical cohorts for the study of therapeutically targetable immune cell subsets with the goal of identifying patterns that correlate with clinical response and patient outcome. This abstract is also being presented as Poster B16. Citation Format: Katey S.S. Enfield, Spencer D. Martin, Sonia H.Y. Kung, Paul Gallagher, Calum E. MacAulay, Martial Guillaud, Wan L. Lam. Hyperspectral imaging tools capture the spatial organization of cell subsets within the tumor microenvironment [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr PR11.

New 4-Amino-1,2,3-Triazole Inhibitors of Indoleamine 2,3-Dioxygenase Form a Long-Lived Complex with the Enzyme and Display Exquisite Cellular Potency.

Indoleamine-2,3 dioxygenase 1 (IDO1) has emerged as a central regulator of immune responses in both normal and disease biology. Due to its established role in promoting tumour immune escape, IDO1 has become an attractive target for cancer treatment. A novel series of highly cell potent IDO1 inhibitors based on a 4-amino-1,2,3-triazole core have been identified. Comprehensive kinetic, biochemical and structural studies demonstrate that compounds from this series have a noncompetitive kinetic mechanism of action with respect to the tryptophan substrate. In co-complex crystal structures, the compounds bind in the tryptophan pocket and make a direct ligand interaction with the haem iron of the porphyrin cofactor. It is proposed that these data can be rationalised by an ordered-binding mechanism, in which the inhibitor binds an apo form of the enzyme that is not competent to bind tryptophan. These inhibitors also form a very tight, long-lived complex with the enzyme, which partially explains their exquisite cellular potency. This novel series represents an attractive starting point for the future development of potent IDO1-targeted drugs.

Abstract 2373: Analysis of the distinct activities of the C1a and C1b domains of protein kinase C theta

Abstract PKC isozymes, the major receptors for tumor-promoting phorbol esters, play a central role in cellular signal transduction downstream of the second messenger diacylglycerol. The PKCθ isozyme has been extensively studied particularly in the context of hematopoiesis and immunity, where is required for the development and maturation of specific T cell subsets. PKCθ also plays a role in Notch-driven T cell leukemia and in a subset of gastrointestinal stromal tumors, melanoma and breast cancers. The C1 domains represent the regulatory motif in the PKCs responsible for their recognition of diacylglycerol / phorbol esters. PKCθ is unique among the conventional and novel PKC isoforms in that one of the two C1 domains of PKCθ, the C1a domain, has been reported to show little or no affinity for diacylglycerol or phorbol ester, unlike the C1b domain. Likewise, the PKCθ C1b domain has been reported to play the predominant role in the membrane translocation and activation process of PKCθ. We observed that the C1a domain in fact has appreciable binding activity for [3H]phorbol 12,13-dibutyrate (PDBu), with a Kd of 254 nM, albeit much less than that of the C1b domain, 1.58 nM. Replacing individually in the C1a domain ten of the amino acid residues that differed between the C1a and C1b domains, using the corresponding residue from the C1b domain, we found that only the P9K replacement restored [3H]phorbol 12,13-dibutyrate binding activity to the C1a domain, yielding a Kd of 3.60 nM, close to that of the C1b domain. Strikingly, the reciprocal replacement in the C1b domain of K9P still preserved appreciable binding affinity, 11.7 nM. We conclude that other features in the C1a domain contribute to the extent to which the P9 residue negatively influences binding. We further explored the role of the C1 domains of PKCθ on translocation using confocal microscopy with a variety of full length constructs in which the C1 domains were manipulated. Consistent with the binding studies, we showed that the C1b domain was the main contributor to translocation, that the full length PKCθ with the C1b K9P retained response, albeit more weakly, and that deletion of the C1a domain or its replacement with a second C1b domain enhanced the translocation to the plasma membrane. Exploitation of the structural differences between the C1 domains in PKCθ and those of other PKC isoforms, both at the level of the direct ligand interactions and the more complicated interplay between C1 domain, ligand, and phospholipid bilayer, may help to afford specific ligands for this member of the PKC family. Citation Format: Agnes Czikora, Satyabrata Pany, Youngki You, Amandeep Saini, Noemi Kedei, Nancy E. Lewin, Adelle Abramovitz, Joydip Das, Peter M. Blumberg. Analysis of the distinct activities of the C1a and C1b domains of protein kinase C theta [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2373. doi:10.1158/1538-7445.AM2017-2373

Reevaluation of the CLV3-receptor interaction in the shoot apical meristem: dissection of the CLV3 signaling pathway from a direct ligand-binding point of view.

The CLAVATA signaling pathway is a key component of the network that controls stem cell renewal and differentiation in Arabidopsis thaliana. CLAVATA3 (CLV3) is a post-translationally arabinosylated secreted peptide signal that regulates WUSHEL (WUS) transcription to affect the balance of stem cell differentiation and proliferation in the shoot apical meristem (SAM). Known membrane-localized receptors involved in the perception of CLV3 signaling include CLV1, the CLV2/CORYNE (CRN) complex and RPK2. The CLV3 peptide can directly bind to CLV1; however, it is unclear whether the CLV3 peptide directly binds to CLV2 or RPK2. In this study, we re-evaluated the direct interaction between CLV3 and its receptors by photoaffinity labeling with photoactivatable arabinosylated CLV3. We showed that CLV2 and RPK2 exhibited no direct binding to the CLV3 peptide. Further analysis showed that the receptor kinase BAM1 directly binds the CLV3 peptide. A loss-of-function clv1 bam1 double mutant exhibited a large number of stem cells that accumulated in the SAM and was insensitive to exogenous treatment with the arabinosylated CLV3 peptide. WUS gene transcripts were up-regulated, and the region of WUS expression was enlarged at the SAM in the clv1 bam1 double mutant. These results indicate that CLV1 and BAM1 are direct receptors that are sufficient to affect the regulatory network controlling stem cell number in the SAM. In contrast, the CLV2/CRN complex and RPK2 are not involved in direct ligand interactions but may act as co-receptors.

Structural Insight into AMPK Regulation: ADP Comes into Play

The AMP-activated protein kinase (AMPK), a sensor of cellular energy status found in all eukaryotes, responds to changes in intracellular adenosine nucleotide levels resulting from metabolic stresses. Here we describe crystal structures of a heterotrimeric regulatory core fragment from Schizosaccharomyces pombe AMPK in complex with ADP, ADP/AMP, ADP/ATP, and 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranotide (AICAR phosphate, or ZMP), a well-characterized AMPK activator. Prior crystallographic studies had revealed a single site in the gamma subunit that binds either ATP or AMP within Bateman domain B. Here we show that ZMP binds at this site, mimicking the binding of AMP. An analogous site in Bateman domain A selectively accommodates ADP, which binds in a distinct manner that also involves direct ligation to elements from the beta subunit. These observations suggest a possible role for ADP in regulating AMPK response to changes in cellular energy status.

Crystal Structure of the T877A Human Androgen Receptor Ligand-binding Domain Complexed to Cyproterone Acetate Provides Insight for Ligand-induced Conformational Changes and Structure-based Drug Design

Cyproterone acetate (CPA) is a steroidal antiandrogen used clinically in the treatment of prostate cancer. Compared with steroidal agonists for the androgen receptor (AR) (e.g. dihydrotestosterone, R1881), CPA is bulkier in structure and therefore seemingly incompatible with the binding pockets observed in currently available x-ray crystal structures of the AR ligand-binding domain (LBD). We solved the x-ray crystal structure of the human AR LBD bound to CPA at 1.8A in the T877A variant, a mutation known to increase the agonist activity of CPA and therefore facilitate purification and crystal formation of the receptor.drug complex. The structure demonstrates that bulk from the 17alpha-acetate group of CPA induces movement of the Leu-701 side chain, which results in partial unfolding of the C-terminal end of helix 11 and displacement of the loop between helices 11 and 12 in comparison to all other AR LBD crystal structures published to date. This structural alteration leads to an expansion of the AR binding cavity to include an additional pocket bordered by Leu-701, Leu-704, Ser-778, Met-780, Phe-876, and Leu-880. Further, we found that CPA invokes transcriptional activation in the L701A AR at low nanomolar concentrations similar to the T877A mutant. Analogous mutations in the glucocorticoid receptor (GR) and progesterone receptor were constructed, and we found that CPA was also converted into a potent agonist in the M560A GR. Altogether, these data offer information for structure-based drug design, elucidate flexible regions of the AR LBD, and provide insight as to how CPA antagonizes the AR and GR.

Structural Basis for Binding of Porphyrin to Human Telomeres,

Maintenance of telomere integrity is a hallmark of human cancer, and the single-stranded 3' ends of telomeric DNA are targets for small-molecule anticancer therapies. We report here the crystal structure of a bimolecular human telomeric quadruplex, of the sequence d(TAGGGTTAGGG), in a complex with the quadruplex-binding ligand 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4) to a resolution of 2.09 A. The DNA quadruplex topology is parallel-stranded with external double-chain-reversal propeller loops, consistent with previous structural determinations. The porphyrin molecules bind by stacking onto the TTA nucleotides, either as part of the external loop structure or at the 5' region of the stacked quadruplex. This involves stacked on hydrogen-bonded base pairs, formed from those nucleotides not involved in the formation of G-tetrads, and there are thus no direct ligand interactions with G-tetrads. This is in accord with the relative nonselectivity by TMPyP4 for quadruplex DNAs compared to duplex DNA. Porphyrin binding is achieved by remodeling of loops compared to the ligand-free structures. Implications for the design of quadruplex-binding ligands are discussed, together with a model for the formation of anaphase bridges, which are observed following cellular treatment with TMPyP4.

Altered CYP Expression and Function in Response to Dietary Factors: Potential Roles in Disease Pathogenesis

Increasing evidence implicates dietary factors in the progression of diseases, including certain cancers, diabetes and obesity. Diet also regulates the expression and function of CYP genes, which impacts on drug elimination and may also significantly affect disease pathogenesis. Upregulation of CYPs 2E1 and 4A occurs after feeding of experimental diets that are high in fats or carbohydrates; these diets also promote hepatic lipid infiltration, which is a component of the metabolic syndrome that characterises obesity. Increased availability of lipid substrates for CYPs can enhance free radical production and exacerbate tissue injury. Similar processes may also occur in other models of experimental disease states that exhibit a component of altered nutrient utilization. Food-derived chemicals, including constituents of cruciferous vegetables and fruits, modulate CYP expression and the expression of genes that encode cytoprotective phase II enzymes. Certain dietary indoles and flavonoids activate CYP1A expression either by direct ligand interaction with the aryl hydrocarbon receptor (AhR) or by augmenting the interaction of the AhR with xenobiotic response elements in CYP1A1 and other target genes. Other dietary chemicals, including methylenedioxyphenyl (MDP) compounds and isothiocyanates also modulate CYP gene expression. Apart from altered CYP regulation, a number of dietary agents also inhibit CYP enzyme activity, leading to pharmacokinetic interactions with coadministered drugs. A well described example is that of grapefruit juice, which contains psoralens and possibly other chemicals, that inactivate intestinal CYP3A4. Decreased presystemic oxidation by this CYP increases the systemic bioavailability of drug substrates and the likelihood of drug toxicity. Dietary interactions may complicate drug therapy but inhibition of certain CYP reactions may also protect the individual against toxic metabolites and free radicals generated by CYPs. Chemicals in teas and cruciferous vegetables may also inhibit human CYP enzymes that have been implicated in the bioactivation of chemical carcinogens. Thus, food constituents modulate CYP expression and function by a range of mechanisms, with the potential for both deleterious and beneficial outcomes.

Glycosylation of the receptor guanylate cyclase C: role in ligand binding and catalytic activity.

GC-C (guanylate cyclase C) is the receptor for heat-stable enterotoxins, guanylin and uroguanylin peptides. Ligand binding to the extracellular domain of GC-C activates the guanylate cyclase domain leading to accumulation of cGMP. GC-C is expressed as differentially glycosylated forms in HEK-293 cells (human embryonic kidney-293 cells). In the present study, we show that the 145 kDa form of GC-C contains sialic acid and galactose residues and is present on the PM (plasma membrane) of cells, whereas the 130 kDa form is a high mannose form that is resident in the endoplasmic reticulum and serves as the precursor for the PM-associated form. Ligand-binding affinities of the differentially glycosylated forms are similar, indicating that glycosylation of GC-C does not play a role in direct ligand interaction. However, ligand-stimulated guanylate cyclase activity was observed only for the fully mature form of the receptor present on the PM, suggesting that glycosylation had a role to play in imparting a conformation to the receptor that allows ligand stimulation. Treatment of cells at 20 degrees C led to intracellular accumulation of a mature glycosylated form of GC-C that now showed ligand-stimulated guanylate cyclase activity, indicating that localization of GC-C was not critical for its catalytic activity. To determine if complex glycosylation was required for ligand-stimulated activation of GC-C, the receptor was expressed in HEK-293 cells that were deficient in N -acetylglucosaminyltransferase 1. This minimally glycosylated form of the receptor was expressed on the cell surface and could bind a ligand with an affinity comparable with the 145 kDa form of the receptor. However, this form of the receptor was poorly activated by the ligand. Therefore our studies indicate a novel role for glycosidic modification of GC-C during its biosynthesis, in imparting subtle conformational changes in the receptor that allow for ligand-mediated activation and perhaps regulation of basal activity.

NMR chemical shift perturbation study of the N-terminal domain of Hsp90 upon binding of ADP, AMP-PNP, geldanamycin, and radicicol.

Hsp90 is one of the most abundant chaperone proteins in the cytosol. In an ATP-dependent manner it plays an essential role in the folding and activation of a range of client proteins involved in signal transduction and cell cycle regulation. We used NMR shift perturbation experiments to obtain information on the structural implications of the binding of AMP-PNP (adenylyl-imidodiphosphate-a non-hydrolysable ATP analogue), ADP and the inhibitors radicicol and geldanamycin. Analysis of (1)H,(15)N correlation spectra showed a specific pattern of chemical shift perturbations at N210 (ATP binding domain of Hsp90, residues 1-210) upon ligand binding. This can be interpreted qualitatively either as a consequence of direct ligand interactions or of ligand-induced conformational changes within the protein. All ligands show specific interactions in the binding site, which is known from the crystal structure of the N-terminal domain of Hsp90. For AMP-PNP and ADP, additional shift perturbations of residues outside the binding pocket were observed and can be regarded as a result of conformational rearrangement upon binding. According to the crystal structures, these regions are the first alpha-helix and the "ATP-lid" ranging from amino acids 85 to 110. The N-terminal domain is therefore not a passive nucleotide-binding site, as suggested by X-ray crystallography, but responds to the binding of ATP in a dynamic way with specific structural changes required for the progression of the ATPase cycle.

Molecular modelling and site-directed mutagenesis of human GALR1 galanin receptor defines determinants of receptor subtype specificity.

Human galanin is a 30 amino acid neuropeptide that elicits a range of biological activities by interaction with G protein-coupled receptors. We have generated a model of the human GALR1 galanin receptor subtype (hGALR1) based on the alpha carbon maps of frog rhodopsin and investigated the significance of potential contact residues suggested by the model using site-directed mutagenesis. Mutation of Phe186 within the second extracellular loop to Ala resulted in a 6-fold decrease in affinity for galanin, representing a change in free energy consistent with hydrophobic interaction. Our model suggests interaction between Phe186 of hGALR1 and Ala7 or Leu11 of galanin. Receptor subtype specificity was investigated by replacement of residues in hGALR1 with the corresponding residues in hGALR2 and use of the hGALR2-specific ligands hGalanin(2-30) and [D-Trp2]hGalanin(1-30). The His267Ile mutant receptor exhibited a pharmacological profile corresponding to that of hGALR1, suggesting that His267 is not involved in a receptor-ligand interaction. The mutation Phe115Ala resulted in a decreased binding affinity for hGalanin and for hGALR2-specific analogues, indicating Phe115 to be of structural importance to the ligand binding pocket of hGALR1 but not involved in direct ligand interaction. Analysis of Glu271Trp suggested that Glu271 of hGALR1 interacts with the N-terminus of galanin and that the Trp residue in the corresponding position in hGALR2 is involved in receptor subtype specificity of binding. Our model supports previous reports of Phe282 of hGALR1 interacting with Trp2 of galanin and His264 of hGALR1 interacting with Tyr9 of galanin.

Bacterial CpG-DNA licenses TLR9.

The family of Toll-like receptors (TLRs) plays an important role in the innate immune response to pathogens. TLRs sense pathogen associated molecular patterns (PAMP) and lead to the stimulation of immune cells. In man, so far ten members (TLR1-10) have been reported. This review focuses on TLR9 which is an essential component for the recognition of bacterial CpG-DNA. Expression of TLR9 and structural consideration as well as direct ligand interaction of TLR9 and CpG-DNA are discussed.

Expression of the amino-terminal domain of platelet glycoprotein Ib alpha: exploitation of a calmodulin tag for determination of its functional activity.

Platelet glycoprotein (GP) Ibalpha is a component of the GPIb-IX receptor complex, which is involved in multiple physiological and pathological processes, including platelet adhesion at sites of vascular injury, thrombin binding, Bernard-Soulier syndrome, platelet-type von Willebrand disease, and immune-mediated thrombocytopenias. The amino-terminal domain of approximately 300 residues of GPIbalpha mediates both normal biological function (by providing the sites for direct ligand interaction) and aberrant function (through amino acid substitutions). To investigate the molecular interactions mediated by this region of GPIbalpha, we have developed a recombinant baculovirus to facilitate its expression as a calmodulin fusion protein from insect cells. By employing the calmodulin tag, the fusion protein could be obtained at >90% purity after a single isolation step at yields of 8 mg/L of insect cell medium (purified fusion protein). The recombinant GPIbalpha fragment was shown to be posttranslationally sulfated and glycosylated, although its glycosylation differed from that of the equivalent GPIbalpha fragment isolated from human platelets. The differential glycosylation, however, did not affect the function of the recombinant GPIbalpha fragment in either von Willebrand factor (vWf) or thrombin binding as these were both found to be identical to those of the same-length GPIbalpha fragment derived from human platelets. The calmodulin tag was also exploited in the development of assays to measure directly vWf and thrombin binding, since it did not interfere with either, demonstrating the feasibility for the use of this soluble receptor fusion protein in detailed biophysical assays to investigate the molecular mode of binding of platelet glycoprotein Ibalpha to these ligands.

9-cis retinoic acid stereoisomer binds and activates the nuclear receptor RXR alpha.

Vitamin A (retinol) and its natural derivatives are required for many physiological processes. The activity of retinoids is thought to be mediated by interactions with two subfamilies of nuclear retinoic acid receptors, RAR and RXR. The RARs bind all-trans retinoic acid (t-RA) with high affinity and alter gene expression as a consequence of this direct ligand interaction. RXR alpha is activated by t-RA, yet has little binding affinity for this ligand. t-RA may be converted to a more proximate ligand that directly binds and activates RXR alpha, and we have developed a method of nuclear receptor-dependent ligand trapping to test this hypothesis. Here we report the identification of a stereoisomer of retinoic acid, 9-cis retinoic acid, which directly binds and activates RXR alpha. These results suggest a new role for isomerization in the physiology of natural retinoids.

Ligand-induced inactivation of type I iodothyronine 5'-deiodinase: protection by propylthiouracil in vivo and reversibility in vitro.

The inactivation of the phenolic ring deiodinases by natural substrates and iodinated contrast agents such as iopanoic acid (IOP) is a principal mechanism regulating the peripheral metabolism of thyroid hormones. The present studies were designed to investigate the cellular and biochemical mechanisms mediating this effect on type I 5'-deiodinase (5'DI) in rat liver and kidney. We reasoned that if inactivation requires the direct interaction of ligand (e.g. IOP) with 5'DI, then this effect should be blocked by a prior injection of 6n-propyl-2-thiouracil (PTU), which binds to an essential sulfhydryl group at the enzyme's active site and inhibits catalytic cycling. Experiments were performed using liver and kidney microsomes prepared from male rats injected 2.5 or 6 h earlier with various combinations of PTU, IOP, and/or vehicle. In initial studies, conditions were defined whereby active 5'DI could be regenerated in vitro from PTU-injected animals. This involved the treatment of microsomes with 50 mM dithiothreitol, pH 7.0, before assay. After this reductive treatment, 5'DI activity in both liver and kidney microsomes from rats previously injected with PTU, or the combination of PTU followed 30 min later by IOP, was equivalent to that in vehicle-injected control animals, whereas activity was decreased 60% in microsomes from animals injected with only IOP. This demonstrates that PTU protects 5'DI against ligand-induced inactivation, suggesting that this process is initiated by substrate binding to the enzyme's active site. In other experiments, liver microsomes from IOP-injected animals were pretreated with 100 mM dithiothreitol, pH 9.0, then washed, and 5'DI activity was determined. Under these stringent reducing conditions, 5'DI activity was completely recovered in microsomes from rats injected with IOP 2.5 h earlier, but only partially recovered 6 h after the IOP injection. Thus, ligand-induced inactivation of 5'DI may involve initially a reversible oxidation of the enzyme, followed by its irreversible inactivation.