CD44 Receptor Unfolding Enhances Binding by Freeing Basic Amino Acids to Contact Carbohydrate Ligand

The extracellular carbohydrate-binding domain of the Type I transmembrane receptor CD44 is known to undergo affinity switching, where change in conformation leads to enhanced binding of its carbohydrate ligand hyaluronan. Separate x-ray crystallographic and NMR experiments have led to competing explanations, with the former supporting minor conformational changes at the binding site and the latter a major order-to-disorder unfolding transition distant from the binding site. Here, all-atom explicit-solvent molecular dynamics studies employing adaptive biasing force sampling revealed a substantial favorable free-energy change associated with contact formation between the Arg⁴¹ side chain and hyaluronan at the binding site, independent of whether the distant site was ordered or disordered. Analogous computational experiments on Arg⁴¹Ala mutants showed loss of this favorable free-energy change, consistent with existing experimental data. More provocatively, the simulation data revealed the molecular mechanism by which the order-to-disorder transition enhances hyaluronan binding: in the disordered state, a number of basic residues gain sufficient conformational freedom—lacking in the ordered state—to spontaneously form side-chain contacts with hyaluronan. Mutation of these residues to Ala had been known to decrease binding affinity, but there had previously been no structural explanation, given their lack of proximity to the carbohydrate-binding site in existing structures of the complex.     

Identification of Amino Acids in the N-Terminal Domain of Corticotropin-Releasing Factor Receptor 1 that Are Important Determinants of High-Affinity Ligand Binding

Abstract : The aim of the present study was to identify the N-terminal regions of human corticotropin-releasing factor (CRF) receptor type 1 (hCRF-R1) that are crucial for ligand binding. Mutant receptors were constructed by replacing specific residues in hCRF-R1 with amino acids from the corresponding position in the N-terminal region of the human vasoactive intestinal peptide receptor type 2 (hVIP-R2). In cyclic AMP stimulation and CRF binding assays, it was established that two regions within the N-terminal domain were crucial for the binding of CRF receptor agonists and antagonists : one region mapping to amino acids 43-50 and a second amino acid sequence extending from position 76 to 84 of hCRF-R1. Recently, it was found that the latter sequence plays a very important role in determining the high ligand selectivity of the Xenopus CRF-R1 (xCRF-R1). Replacement of amino acids 76-84 of hCRF-R1 with residues from the same segment of the hVIP-R2 N terminus markedly reduced the binding affinity of CRF ligands. Mutation of Arg⁷⁶ or Asn⁸¹ but not Gly⁸³ of hCRF-R1 to the corresponding amino acids of xCRF-R1 or hVIP-R2 resulted in 100-1,000-fold lower affinities for human/rat CRF, rat urocortin, and astressin. These data underline the importance of the N-terminal domain of CRF-R1 in high-affinity ligand binding.     

Ligand binding to anti-cancer target CD44 investigated by molecular simulations

CD44 is a cell-surface glycoprotein and receptor for hyaluronan, one of the major components of the tumor extracellular matrix. There is evidence that the interaction between CD44 and hyaluronan promotes breast cancer metastasis. Recently, the molecule F-19848A was shown to inhibit hyaluronan binding to receptor CD44 in a cell-based assay. In this study, we investigated the mechanism and energetics of F-19848A binding to CD44 using molecular simulation. Using the molecular mechanics/Poisson Boltzmann surface area (MM-PBSA) method, we obtained the binding free energy and inhibition constant of the complex. The van der Waals (vdW) interaction and the extended portion of F-19848A play key roles in the binding affinity. We screened natural products from a traditional Chinese medicine database to search for CD44 inhibitors. From combining pharmaceutical requirements with docking and molecular dynamics simulations, we found ten compounds that are potentially better or equal to the F-19848A ligand at binding to CD44 receptor. Therefore, we have identified new candidates of CD44 inhibitors, based on molecular simulation, which may be effective small molecules for the therapy of breast cancer.     

Evidence That Calmodulin Binding to the Dopamine D2 Receptor Enhances Receptor Signaling

The Ca²⁺ sensor calmodulin (CaM) regulates numerous proteins involved in G protein-coupled receptor (GPCR) signaling. CaM binds directly to some GPCRs, including the dopamine D₂ receptor. We confirmed that the third intracellular loop of the D₂ receptor is a direct contact point for CaM binding using coimmunoprecipitation and a polyHis pull-down assay, and we determined that the D₂-like receptor agonist 7-OH-DPAT increased the colocalization of the D₂ receptor and endogenous CaM in both 293 cells and in primary neostriatal cultures. The N-terminal three or four residues of D₂-IC3 were required for the binding of CaM; mutation of three of these residues in the full-length receptor (I210C/K211C/I212C) decreased the coprecipitation of the D₂ receptor and CaM and also significantly decreased D₂ receptor signaling, without altering the coupling of the receptor to G proteins. Taken together, these findings suggest that binding of CaM to the dopamine D₂ receptor enhances D₂ receptor signaling.     

Mutational Insights into the Roles of Amino Acid Residues in Ligand Binding for Two Closely Related Family 16 Carbohydrate Binding Modules

Carbohydrate binding modules (CBMs) are specialized proteins that bind to polysaccharides and oligosaccharides. Caldanaerobius polysaccharolyticus Man5ACBM16-1/CBM16-2 bind to glucose-, mannose-, and glucose/mannose-configured substrates. The crystal structures of the two proteins represent the only examples in CBM family 16, and studies that evaluate the roles of amino acid residues in ligand binding in this family are lacking. In this study, we probed the roles of amino acids (selected based on CBM16-1/ligand co-crystal structures) on substrate binding. Two tryptophan (Trp-20 and Trp-125) and two glutamine (Gln-81 and Gln-93) residues are shown to be critical in ligand binding. Additionally, several polar residues that flank the critical residues also contribute to ligand binding. The CBM16-1 Q121E mutation increased affinity for all substrates tested, whereas the Q21G and N97R mutants exhibited decreased substrate affinity. We solved CBM/substrate co-crystal structures to elucidate the molecular basis of the increased substrate binding by CBM16-1 Q121E. The Gln-121, Gln-21, and Asn-97 residues can be manipulated to fine-tune ligand binding by the Man5A CBMs. Surprisingly, none of the eight residues investigated was absolutely conserved in CBM family 16. Thus, the critical residues in the Man5A CBMs are either not essential for substrate binding in the other members of this family or the two CBMs are evolutionarily distinct from the members available in the current protein database. Man5A is dependent on its CBMs for robust activity, and insights from this study should serve to enhance our understanding of the interdependence of its catalytic and substrate binding modules.     

Endogenous Inhibitor of Ligand Binding to the Muscarinic Acetylcholine Receptor

An endogenous inhibitor of L-[3H]quinuclinidinyl benzilate binding to the brain muscarinic acetylcholine receptor was identified. [3H]Quinuclinidinyl benzilate binding to rat brain synaptosomes was measured using a filtration assay. The inhibitor was prepared from several calf tissues and was found in highest specific activity in thymus. The loss of binding activity was slow, requiring a 30-40 min preincubation of the synaptosomes with the inhibitor, and reversed by removing the inhibitor by washing the membranes. Scatchard analysis of the binding data showed that the inhibition was noncompetitive resulting from both a decrease in affinity and a decrease in the number of binding sites. Zn2+ was required in low concentrations for this effect. Muscarinic acetylcholine receptor in synaptic membranes and in membranes free of most peripheral membrane proteins was still sensitive to inhibition. Preliminary characterization of the inhibitory molecule showed that it is of low molecular weight, moderately heat-stable, and acidic. The inhibitor was inactivated by reagents that are nonspecific for nucleophiles, but not by reagents specific for primary amine or thiol groups.     

Laminar Distribution of Putative Neurotransmitter Amino Acids and Ligand Binding Sites in the Dog Olfactory Bulb

Coronal sections of frozen dog olfactory bulb have been dissected into four anatomically distinct layers. The laminar distribution of ten amino acids, the dipeptide carnosine, and nine [3H]ligand binding sites in these layers was determined. GABA and tyrosine levels were highest in the mitral cell-granule cell layer, and glutamate levels were slightly elevated in the glomerular layer. The distributions of all other amino acids did not show significant differences across the layers. Carnosine was predominantly localized in the fiber and glomerular layers. With the exception of quinuclidinyl benzilate, the [3H]ligand binding sites showed more discrete distributions. Muscimol, diazepam, kainic acid, and spiroperidol binding were predominantly localized in the mitral cell-granule cell layer, where clonidine binding was at a minimum. Dihydromorphine binding was high in both the fiber and the mitral cell-granule cell layers. Carnosine binding was maximal in the glomerular layer. The implications of these observations with regard to biochemical and neurophysiological data are discussed.     

T Cell Receptor Binding to a pMHCII Ligand Is Kinetically Distinct from and Independent of CD4

Immune recognition of pMHCII ligands by a helper T lymphocyte involves its antigen-specific T cell receptor (TCR) and CD4 coreceptor. We have characterized the binding of both molecules to the same pMHCII. The D10 alphabeta TCR heterodimer binds to conalbumin/I-A(k) with virtually identical kinetics and affinity as the single chain ValphaVbeta domain module (scD10) (Kd = 6-8 microm). The CD4 ectodomain does not alter either interaction. Moreover, CD4 alone demonstrates weak pMHCII binding (Kd = 200 microm), with no discernable affinity for the alphabeta TCR heterodimer. Hence, rather than providing a major contribution to binding energy, the critical role for the coreceptor in antigen-specific activation likely results from transient inducible recruitment of the CD4 cytoplasmic tail-associated lck tyrosine kinase to the pMHCII-ligated TCR complex.     

Internalization of the Hyaluronan Receptor CD44 by Chondrocytes

Chondrocytes express CD44 as a primary receptor for the matrix macromolecule hyaluronan. Hyaluronan is responsible for the retention and organization of proteoglycan within cartilage, and hyaluronan-chondrocyte interactions are important for the assembly and maintenance of the cartilage matrix. Bovine articular chondrocytes were used to study the endocytosis and turnover of CD44 and the effects of receptor occupancy on this turnover. Matrix-intact chondrocytes exhibit approximately a 6% internalization of cell surface CD44 by 4 h. Treatment with Streptomyces hyaluronidase to remove endogenous pericellular matrix increased internalization to approximately 20% of cell surface CD44 at 4 h. This turnover could be partially inhibited by the addition of exogenous hyaluronan to these matrix-depleted chondrocytes. Cell surface biotin-labeled CD44 was internalized by chondrocytes and this internalization was decreased in the presence of hyaluronan. Colocalization of internalized CD44 and fluorescein-labeled hyaluronan in intracellular vesicles correlates with the previous results of receptor-mediated endocytosis pathway for the degradation of hyaluronan by acid hydrolases. Taken together, our results indicate that CD44 is internalized by chondrocytes and that CD44 turnover is modulated by occupancy with hyaluronan.     

Mutagenesis of a Single Amino Acid in the Rat μ-Opioid Receptor Discriminates Ligand Binding

Abstract: To investigate the role of Asp¹¹⁴ in the cloned rat μ-opioid receptor for ligand binding, the charged amino acid was mutated to an asparagine to generate the mutant μ receptor D114N. The wild-type μ receptor and the D114N mutant were then stably expressed in human embryonic kidney 293 cells, and the binding affinities of a series of opioids were investigated. The μ-selective agonists [ d -Ala²,MePhe⁴,Gly-ol⁵]enkephalin and morphine and the endogenous peptides Met-enkephalin and β-endorphin exhibited greatly reduced affinities for the D114N mutant compared with the wild-type μ receptor, as did the potent synthetic agonist etorphine. In contrast to the full agonists, the partial agonists buprenorphine and nalorphine and the antagonists diprenorphine and naloxone bound with similar affinities to the wild-type and D114N mutant μ receptors. The reduced affinities of the full agonists for the D114N mutant did not involve an uncoupling of the receptor from G proteins because methadone and etorphine stimulated the D114N μ receptors to inhibit adenylyl cyclase. Although the Asp¹¹⁴ to Asn¹¹⁴ mutation reduced full-agonist binding, mutation of His²⁹⁷ to Asn²⁹⁷ in the μ receptor did not but, in contrast, did reduce binding affinity of the partial agonist buprenorphine and the antagonist diprenorphine. These results indicate that some partial agonists and antagonists may have different determinants for binding to the μ receptor than do the prototypical full agonists.     

Overexpression of G-Protein-Coupled Receptor 40 Enhances the Mitogenic Response to Epoxyeicosatrienoic Acids

The cytochrome P450 epoxygenase-dependent arachidonic acid metabolites, epoxyeicosatrienoic acids (EETs), are potent survival factors and mitogens for renal epithelial cells, but the molecular identity in the cells that initiates the mitogenic signaling of EETs has remained elusive. We screened kidney cell lines for the expression of G-protein-coupled receptor 40 (GPR40) and found that the porcine renal tubular epithelial cell line LLCPKcl4, which has been previously demonstrated to be sensitive to the mitogenic effect of EETs, expresses higher levels of GPR40 mRNA and protein than the human embryonic kidney cell line HEK293. EETs induced only a weak mitogenic EGFR signaling and mild cell proliferation in HEK293 cells. To determine whether GPR40 expression level is what mediates the mitogenic sensitivity of cells to EETs, we created a human GPR40 (hGPR40) cDNA construct and transfected it into HEK293 cells and picked up a number of stable transfectants. We found that GPR40 overexpression in HEK293 cells indeed significantly enhanced EET-induced cell proliferation and markedly augmented EGFR phosphorylation ERK activation, which were inhibited by the EGFR tyrosine kinase inhibitor, AG1478, or the HB-EGF inhibitor, CRM197. EETs significantly enhanced release of soluble HB-EGF, a natural ligand of EGFR, into the culture medium of hGPR40-transfected HEK293 cells, compared to empty vector-transfected cells. In mouse kidneys, markedly higher level of GPR40 protein was found in the cortex and outer stripe of outer medulla compared to the inner stripe of outer medulla and inner medulla. In situ hybridization confirmed that GPR40 mRNA was localized to a subset of renal tubules in the kidney, including the cortical collecting duct. Thus, this study provides the first demonstration that upregulation of GPR40 expression enhances the mitogenic response to EETs and a relatively high expression level of GPR40 is detected in a subset of tubules including cortical collecting ducts in the mammalian kidney.     

编码流感病毒血凝素基因和CD40L的核酸疫苗抗小鼠流感研究

为了检测表达CD40L的质粒能台作为核酸疫苗佐剂提高A／PR8／34流感病毒血凝素(HA)DNA疫苗的免疫应答,构建了表达鼠CD40L的质粒,行将它与A型流感病毒的HADNA疫苗用电击的方法共同免疫BALB／C小鼠(各30μg),免疫两次,间隔三周,第二次免疫后1周,用致死量流感病毒A／PR8／34攻击。发现与单独免疫30μgHA相比,血清中抗HA的IgG抗体节明显提高,且以IgG2a抗体提高为主,小鼠体重减轻非常少且体重恢复加快。实验结果显示,CD40L能作为流感病毒核酸疫苗佐剂,提高小鼠抗流感病毒攻击的能力。     

编码流感病毒血凝素基因和CD40L的核酸疫苗抗小鼠流感研究

为了检测表达CD40L的质粒能否作为核酸疫苗佐剂提高A/PR8/34流感病毒血凝素(HA)DNA疫苗的免疫应答,构建了表达鼠CD40L的质粒,并将它与A型流感病毒的 HA DNA疫苗用电击的方法共同免疫BALB/C小鼠(各30μg),免疫两次,间隔三周,第二次免疫后1周,用致死量流感病毒A/PR8/34攻击.发现与单独免疫30μg HA相比,血清中抗HA的IgG抗体量明显提高,且以IgG2a抗体提高为主,小鼠体重减轻非常少且体重恢复加快.实验结果显示,CD40L能作为流感病毒核酸疫苗佐剂,提高小鼠抗流感病毒攻击的能力.     

Ligand Binding by Fibroblast Growth Factor Receptors Investigated Using Chimeric Receptor Molecules

Abstract

In order to map in detail the ligand binding sites of fibroblast growth factor receptor 2 (FGFR2) and keratinocyte growth factor receptor (KGFR), we have generated receptor molecules that are chimeric within the membrane proximal sequence that varies between them. The chimeric molecules are found to bind aFGF with a greater than 5-fold difference in affinity, indicating that there is coupling between the chimeric regions with respect to aFGF binding. Further, binding of bFGF and KGF is abolished in the chimeras, showing that the binding site for these ligands requires the whole of the 48- or 50- amino acid variable sequence to be intact. Direct interactions between the different regions exchanged in the chimeras are most probably involved in forming KGF or bFGF binding sites.     

Ligand Binding to CNS Muscarinic Receptor Is Transiently Modified by Convulsant 3-Mercaptopropionic Acid Administration

The administration of convulsant drugs has proven a powerful tool to study experimental epilepsy. We have already reported that the administration of convulsant 3-mercaptopropionic acid (mp) at 150 mg/kg enhances binding affinity of muscarinic antagonist [³H]quinuclidinyl benzilate ([³H]QNB) to certain rat CNS membranes during seizure and postseizure without affecting site number. Results obtained with a 100-mg/kg dose of mp have shown reversible increases in [³H]QNB binding to cerebellum and hippocampus, whereas a delayed response has been found in striatum. Neither a subconvulsant dose nor in vitro addition modifies binding. In order to evaluate preseizure, seizure as well as early (30 min) and late (24 h) postseizure stages, we employed a 50 mg/kg dose and tested [³H]QNB binding to CNS membranes. Changes in binding were as follows (in %): in cerebellum, +37, +86, and +40 at preseizure, seizure and early postseizure stages, respectively, but there was a decrease at late postseizure; in hippocampus, +27 at pre- and seizure stages, but a decrease at early and late postseizure. No changes were found in striatum or cerebral cortex membranes at any stage studied. Saturation curves analysed by Scatchard plots indicated that changes in [³H]QNB binding to cerebellar membranes are attributable to an increase in ligand affinity at seizure, followed by a decrease in binding site number at postseizure. A similar profile was observed for hippocampus except that the decrease in binding site number, though lower than at postseizure, was already evident at seizure stage. Results confirm a region-specific response to the convulsant and transient changes provide an example of neuronal plasticity.     

Modulation of Opioid Receptor Binding by Cis and Trans Fatty Acids

In synaptosomal brain membranes, the addition of oleic acid (cis), elaidic acid (trans), and the cis and trans isomers of vaccenic acid, at a concentration of 0.87 mumol of lipid/mg of protein, strongly reduced the Bmax and, to a lesser degree, the binding affinity of the mu-selective opioid [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol ([3H]DAMGO). At comparable membrane content, the cis isomers of the fatty acids were more potent than their trans counterparts in inhibiting ligand binding and in decreasing membrane microviscosity, both at the membrane surface and in the core. However, trans-vacenic acid affected opioid receptor binding in spite of just marginally altering membrane microviscosity. If the receptors were uncoupled from guanine nucleotide regulatory protein, an altered inhibition profile was obtained: the impairment of KD by the fatty acids was enhanced and that of Bmax reduced. Receptor interaction of the delta-opioid [3H](D-Pen2,D-Pen5)enkephalin was modulated by lipids to a greater extent than that of [3H]DAMGO: saturable binding was abolished by both oleic and elaidic acids. The binding of [3H]naltrexone was less susceptible to inhibition by the fatty acids, particularly in the presence of sodium. In the absence of this cation, however, cis-vaccenic acid abolished the low-affinity binding component of [3H]naltrexone. These findings support the membrane model of opioid receptor sequestration depicting different ionic environments for the mu- and delta-binding sites. The results of this work show distinct modulation of different types and molecular states of opioid receptor by fatty acids through mechanisms involving membrane fluidity and specific interactions with membrane constituents.     

Dimerization of Corticotropin-Releasing Factor Receptor Type 1 Is Not Coupled to Ligand Binding

As described previously, receptor dimerization of G protein-coupled receptors may influence signaling, trafficking, and regulation in vivo. Up to now, most studies aiming at the possible role of receptor dimerization in receptor activation and signal transduction are focused on class A GPCRs. In the present work, the dimerization behavior of the corticotropin-releasing factor receptor type 1 (CRF₁R), which belongs to class B of GPCRs and plays an important role in coordination of the immune response, stress, and learning behavior, was investigated by using fluorescence resonance energy transfer (FRET). For this purpose, we generated fusion proteins of CRF₁R tagged at their C-terminus to a cyan or yellow fluorescent protein, which can be used as a FRET pair. Binding studies verified that the receptor constructs were able to bind their natural ligands in a manner comparable with the wild-type receptor, whereas cAMP accumulation proved the functionality of the constructs. In microscopic studies, a dimerization of the CRF₁R was observed, but the addition of either CRF-related agonists or antagonists did not show any dose-related increase of the observed FRET signal, indicating that the dimer-monomer ratio is not changed on addition of ligand.     

The Uptake and Utilization of Bacteria, Amino Acids and Carbohydrate by the Rumen Ciliate Entodinium longinucleatum in Relation to the Sources of Amino Acids for Protein Synthesis

Entodinium longinucleatum grown in vitro in the presence of bacteria engulfed a wide range of bacterial species at rates of 130–3400 bacteria/h/protozoon (from suspensions of 10 bacteria/ml), but showed a preference for Klebsiella aerogenes and Proteus mirabilis which occurred in the growth medium. Some of the bacteria were digested with release of soluble material into the medium. Free amino acids were incorporated by the protozoa in the presence of chloramphenicol at rates of 5·4–15·1 nmol/h/10⁶ protozoa and approximately 40% of the amino acid-carbon was incorporated into protein. There was no appreciable synthesis of protozoal protein from carbohydrate. Evidence was obtained that the protozoa obtained the amino acids required for growth largely from engulfed bacteria.     

Studies on the Mechanism of Strengthening Fish Gel by Basic Amino Acids

An electro-energizing fermentation (E-E F) method has been developed. In this method, a direct electrical current is applied to a microbial culture to accelerate the reductive metabolism of microorganisms or to impart profitable effects to microbial cells. This E-E F method was applied to l-glutamic acid fermentation by Brevibacterium flavum No. 2247. When glucose was used as a substrate, the addition of 0.01 mm neutral red (NR), redox dye (electron carrier), to the fermentation broth at the beginning of cultivation was effective for l-glutamate (l-Glu) production. A direct current of 200~300 μA/cm² at 1.5 V was applied through out the cultivation of this bacterium. This resulted in about a 10% increase in yield of l-Glu.