Molecular determinants of activation and membrane targeting of phosphoinositol 4-kinase IIbeta

Mammalian cells contain two isoforms of the type II PI4K (phosphoinositol 4-kinase), PI4KIIalpha and beta. These 55 kDa proteins have highly diverse N-terminal regions (approximately residues 1-90) but conserved catalytic domains (approximately from residue 91 to the C-termini). Nearly the entire pool of PI4KIIalpha behaves as an integral membrane protein, in spite of a lack of a transmembrane domain. This integral association with membranes is due to palmitoylation of a cysteine-rich motif, CCPCC, located within the catalytic domain. Although the CCPCC motif is conserved in PI4KIIbeta, only 50% of PI4KIIbeta is membrane-associated, and approximately half of this pool is only peripherally attached to the membranes. Growth factor stimulation or overexpression of a constitutively active Rac mutant induces the translocation of a portion of cytosolic PI4KIIbeta to plasma membrane ruffles and stimulates its activity. Here, we demonstrate that membrane-associated PI4KIIbeta undergoes two modifications, palmitoylation and phosphorylation. The cytosolic pool of PI4KIIbeta is not palmitoylated and has much lower lipid kinase activity than the membrane-associated kinase. Although only membrane-associated PI4KIIbeta is phosphorylated in the unique N-terminal region, this modification apparently does not influence its membrane binding or activity. A series of truncation mutants and alpha/beta chimaeras were generated to identify regions responsible for the isoform-specific behaviour of the kinases. Surprisingly, the C-terminal approx. 160 residues, and not the diverse N-terminal regions, contain the sites that are most important in determining the different solubilities, palmitoylation states and stimulus-dependent redistributions of PI4KIIalpha and beta.     

Molecular determinants of dihydrouridine synthase activity

Dihydrouridine is one of the most abundant modified bases in tRNA. However, little is known concerning the biochemistry of dihydrouridine synthase (DUS) enzymes. To identify molecular determinants that are necessary for DUS activity, we have developed a DUS-complementation assay in Escherichia coli. Using this assay, we have identified amino-acid residues that are critical for the activity of YjbN, an E. coli DUS. We also show that the aq1598 gene product, a putative DUS from Aquifex aeolicus, catalyzes dihydrouridine formation, providing the first biochemical demonstration that A. aeolicus encodes an active DUS.     

Flumazenil and bupivacaine-induced toxicity: inverse agonist type activity

The purpose of this study was to investigate the influence of flumazenil on bupivacaine-induced acute toxicity, 10 groups of mice were previously treated by a 1, 0.5, 0.25 and 0.125 mg/kg single dose of flumazenil or saline 15 minutes before an injection of bupivacaine (50 mg/kg: exp. 1 and 60 mg/kg: exp.2). The convulsant activity, the period of latency to convulse and the induced mortality were assessed in each group. The bupivacaine-induced mortality was increased by flumazenil. Also, the convulsant activity was increased by flumazenil and the period of latency to convulse was proportionally decreased with increasing doses of flumazenil for the two tested doses of bupivacaine.     

Molecular mechanism underlying inverse agonist of angiotensin II type 1 receptor

To delineate the molecular mechanism underlying the inverse agonist activity of olmesartan, a potent angiotensin II type 1 (AT1) receptor antagonist, we performed binding affinity studies and an inositol phosphate production assay. Binding affinity of olmesartan and its related compounds to wild-type and mutant AT1 receptors demonstrated that interactions between olmesartan and Tyr113, Lys199, His256, and Gln257 in the AT1 receptor were important. The inositol phosphate production assay of olmesartan and related compounds using mutant receptors indicated that the inverse agonist activity required two interactions, that between the hydroxyl group of olmesartan and Tyr113 in the receptor and that between the carboxyl group of olmesartan and Lys199 and His256 in the receptor. Gln257 was found to be important for the interaction with olmesartan but not for the inverse agonist activity. Based on these results, we constructed a model for the interaction between olmesartan and the AT1 receptor. Although the activation of G protein-coupled receptors is initiated by anti-clockwise rotation of transmembrane (TM) III and TM VI followed by changes in the conformation of the receptor, in this model, cooperative interactions between the hydroxyl group and Tyr113 in TM III and between the carboxyl group and His256 in TM VI were essential for the potent inverse agonist activity of olmesartan. We speculate that the specific interaction of olmesartan with these two TMs is essential for stabilizing the AT1 receptor in an inactive conformation. A better understanding of the molecular mechanisms of the inverse agonism could be useful for the development of new G protein-coupled receptor antagonists with inverse agonist activity.     

Molecular determinants of human melanocortin-4 receptor responsible for antagonist SHU9119 selective activity

The hypothalamic melanocortin-4 receptor (MC4R), a seven transmembrane G-protein-coupled receptor, plays an important role in the regulation of body weight. The synthetic melanocortin analog SHU9119 has been widely used to characterize the physiological role of MC4R in feeding behavior and energy homeostasis. Previous studies indicated that SHU9119 is an agonist at the melanocortin-1 receptor (MC1R) but an antagonist at the MC4R. However, the molecular basis of the interaction between hMC4R and SHU9119 has not been clearly defined. To gain insight into the molecular determinants of hMC4R in the selectivity of SHU9119 chimeras and mutants hMC1R and hMC4R were expressed in cell lines and pharmacologically analyzed. A region of receptor containing the third transmembrane of hMC4R was found to be required for selective SHU9119 antagonism. Further mutagenesis studies of this region of hMC4R demonstrated that the amino acid residue leucine 133 in the third transmembrane was critical for the selective antagonist activity of SHU9119. The single substitution of leucine 133 to methionine did not affect SHU9119 binding to hMC4R. However, this substitution did convert SHU9119 from an antagonist to an agonist. Conversely, exchange of Met(128) in hMC1R to Leu, the homologous residue 133 of hMC4R, displayed a reduction in SHU9119 binding affinity and potency. This report provides the details of the molecular recognition of SHU9119 antagonism at hMC4R and shows that amino acid Leu(133) of hMC4R plays a key role in melanocortin receptor subtype specificity.     

Molecular Weights of CTLA-4 and CD80 by Sedimentation Equilibrium Ultracentrifugation

Proteins that are heavily glycosylated pose unique challenges in their biophysical characterization. In particular, molecular weight analysis is exacerbated by such glycosylation. For example, glyoproteins are refractory to careful mass spectrum analysis and often give anomalous retention times using size exclusion chromatography. We combine several approaches to characterize the molecular weights of the extracellular domains of the glycoproteins CTLA-4 and CD80 using carbohydrate analysis, electrospray mass spectrometry, size exclusion chromatography, and analytical ultracentrifugation. In addition, we have applied a method described previously, using sedimentation equilibrium analysis to calculate the contribution of carbohydrates to the molecular masses of CTLA-4 and CD80. It is important to understand the oligomeric states of these protein domains because the interaction between these lymphocyte receptors plays an important costimulatory role in the Th-cell antigenic response. It is thought that extracellular interactions between these receptors may regulate both the self-association of these receptor proteins and the oligomeric state of the heterocomplex; this regulation has important consequences for potentiating the signaling mechanism between Th-cells and antigen-presenting cells.     

Molecular determinants of receptor affinity and selectivity of the natural delta-opioid agonist, dermenkephalin

Processing of the polyprotein precursor pro-dermorphin generates two distantly related D-amino acid-containing peptides, dermorphin and dermenkephalin, which are among the most selective high affinity agonists described, respectively, for the mu- and delta-opioid receptors. Dermenkephalin, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2, is a linear, potentially flexible peptide devoid of structural homology with either enkephalins, endorphins, or dynorphins and, as such, represents a useful tool for identifying determinants of high affinity and selective binding of opioids to the delta-receptor. A series of selected dermenkephalin analogs and homologs was investigated for affinity at the mu- and delta-sites in the brain. Whereas dermenkephalin has high affinity and specificity for the delta-opioid receptors, its tetrapeptide amino end, dermenkephalin-[1-4]-NH2 binds almost exclusively at the mu-receptors. Dermorphin, Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2, is only marginally more selective for the u-sites than is dermenkephalin-[1-4]-NH2. Using dermorphin-dermenkephalin peptide hybrids and C-terminal deletion analogs of dermenkephalin, we showed the critical role that the C-terminal residues Met6 and Asp7 play in specifying correct addressing of dermenkephalin toward delta-receptors. The potent mu-deteminant located within the amino end of dermenkephalin is over-whelmed by the powerful delta-directing ability of the carboxy end. The negatively charged side chain of Asp7 makes a significant contribution to the delta-addressing ability of the C-terminal region, a finding consistent with Schwyzer's membrane selection model (Schwyzer, R. (1986) Biochemistry 25, 6335-6342). The Leu residue in position 5 and D-configuration about the alpha-carbon of Met2 were found to be of crucial importance for high affinity binding to delta-receptors. Whereas the Met residue in position 6 in dermenkephalin could safely be oxidized or replaced with D-Met, oxidation of Met2 led to deleterious effects, this analog being 1/100 as potent as dermenkephalin at delta-sites. Overall, the data collected demonstrate that highest levels of selectivity and affinity for the delta-opioid receptors can be achieved with small-sized, potentially flexible, linear peptides and further support the model according to which, in addition to optimum accommodation at the receptor, selection for delta-receptors is reduced by the effective positive charge of the molecule. Dermenkephalin may provide a starting point for the design of agonists and antagonists with nearly total specificity for the delta-sites. Such pharmacological agents could be used to explore the ill-defined physiological role and behavioral actions conveyed by delta-opioid receptors.     

Molecular basis of melanocortin-4 receptor for AGRP inverse agonism

We have investigated receptor structural components of the melanocortin-4 receptor (MC4R) responsible for ligand-dependent inverse agonism. We utilized agouti-related protein (AGRP), an inverse agonist which reduces MC4R basal cAMP production, as a tool to determine the molecular mechanism. We tested a series of chimeric receptors and utilized MC4R and MC1R as templates, in which AGRP is an inverse agonist for MC4R but not for MC1R. Our results indicate that replacements of the extracellular loops 1, 2 and 3 of MC4R with the corresponding regions of MC1R did not affect AGRP inverse agonist activity. However, replacement of the N terminus of MC4R with the same region of MC1R decreases AGRP inverse agonism. Replacement of transmembrane domains 3, 4, 5 and 6 of MC4R with the corresponding regions of MC1R did not affect AGRP inverse agonist activity but mutation of D90A in transmembrane 2 (TM2) and D298A in TM7 abolished AGRP inverse activity. Deletion of the distal MC4R C terminus fails to maintain AGRP mediated reduction in basal cAMP production although it maintains NDP-MSH mediated cAMP production. In conclusion, our results indicate that the N terminus and the distal C terminus of MC4R do appear to play important roles in AGRP inverse agonism but not NDP-MSH mediated receptor activation. Our results also indicate that the residues D90 in TM2 and D298 in TM7 of hMC4R are involved in not only NDP-MSH mediated receptor activation but also AGRP mediated inverse agonism.     

Molecular determinants of permeation through the cation channel TRPV4

We have studied the molecular determinants of ion permeation through the TRPV4 channel (VRL-2, TRP12, VR-OAC, and OTRPC4). TRPV4 is characterized by both inward and outward rectification, voltage-dependent block by Ruthenium Red, a moderate selectivity for divalent versus monovalent cations, and an Eisenman IV permeability sequence. We identify two aspartate residues, Asp(672) and Asp(682), as important determinants of the Ca(2+) sensitivity of the TRPV4 pore. Neutralization of either aspartate to alanine caused a moderate reduction of the relative permeability for divalent cations and of the degree of outward rectification. Neutralizing both aspartates simultaneously caused a much stronger reduction of Ca(2+) permeability and channel rectification and additionally altered the permeability order for monovalent cations toward Eisenman sequence II or I. Moreover, neutralizing Asp(682) but not Asp(672) strongly reduces the affinity of the channel for Ruthenium Red. Mutations to Met(680), which is located at the center of a putative selectivity filter, strongly reduced whole cell current amplitude and impaired Ca(2+) permeation. In contrast, neutralizing the only positively charged residue in the putative pore region, Lys(675), had no obvious effects on the properties of the TRPV4 channel pore. Our findings delineate the pore region of TRPV4 and give a first insight into the possible architecture of its permeation pathway.     

Small molecules with similar structures exhibit agonist, neutral antagonist or inverse agonist activity toward angiotensin II type 1 receptor

Small differences in the chemical structures of ligands can be responsible for agonism, neutral antagonism or inverse agonism toward a G-protein-coupled receptor (GPCR). Although each ligand may stabilize the receptor conformation in a different way, little is known about the precise conformational differences. We synthesized the angiotensin II type 1 receptor blocker (ARB) olmesartan, R239470 and R794847, which induced inverse agonism, antagonism and agonism, respectively, and then investigated the ligand-specific changes in the receptor conformation with respect to stabilization around transmembrane (TM)3. The results of substituted cysteine accessibility mapping studies support the novel concept that ligand-induced changes in the conformation of TM3 play a role in stabilizing GPCR. Although the agonist-, neutral antagonist and inverse agonist-binding sites in the AT(1) receptor are similar, each ligand induced specific conformational changes in TM3. In addition, all of the experimental data were obtained with functional receptors in a native membrane environment (in situ).     

Molecular determinants of ligand binding to the human melanocortin-4 receptor

To elucidate the molecular basis for the interaction of ligands with the human melanocortin-4 receptor (hMC4R), agonist structure-activity studies and receptor point mutagenesis were performed. Structure-activity studies of [Nle(4), D-Phe(7)]-alpha-melanocyte stimulating hormone (NDP-MSH) identified D-Phe7-Arg8-Trp9 as the minimal NDP-MSH fragment that possesses full agonist efficacy at the hMC4R. In an effort to identify receptor residues that might interact with amino acids in this tripeptide sequence 24 hMC4R transmembrane (TM) residues were mutated (the rationale for choosing specific receptor residues for mutation is outlined in the Results section). Mutation of TM3 residues D122 and D126 and TM6 residues F261 and H264 decreased the binding affinity of NDP-MSH 5-fold or greater, thereby identifying these receptor residues as sites potentially involved in the sought after ligand-receptor interactions. By examination of the binding affinities and potencies of substituted NDP-MSH peptides at receptor mutants, evidence was found that core melanocortin peptide residue Arg8 interacts at a molecular level with hMC4R TM3 residue D122. TM3 mutations were also observed to decrease the binding of hMC4R antagonists. Notably, mutation of TM3 residue D126 to alanine decreased the binding affinity of AGRP (87-132), a C-terminal derivative of the endogenous melanocortin antagonist, 8-fold, and simultaneous mutations D122A/D126A completely abolished AGRP (87-132) binding. In addition, mutation of TM3 residue D122 or D126 decreased the binding affinity of hMC4R antagonist SHU 9119. These results provide further insight into the molecular determinants of hMC4R ligand binding.     

Structural determinants of unexpected agonist activity in a retro-peptide analogue of the SDF-1alpha N-terminus

We have synthesised two retro-peptide analogues of the stromal cell derived growth factor 1 (SDF-1alpha) segment known to be critical for CXCR4 receptor binding, corresponding to the sequences HSEFFRCPCRFFESH and HSEFFRGGGRFFESH. We have assayed the ability of these peptides to activate extracellular signal-regulated kinase 1/2 phosphorylation in cells over expressing the SDF-1alpha receptor, finding that the first variant was able to serve as an agonist of CXCR4, whereas the second one was inactive. Finally, by comparing representative solution structures of the two peptides, we have found that the biological response of HSEFFRCPCRFFESH may be ascribed to a beta-beta-type turn motif centred on Phe(4)-Phe(5).     

Molecular targeting of angiogenesis

The majority of pharmacological approaches for the treatment of solid tumors suffer from poor selectivity, thus limiting dose escalation (i.e., the doses of drug which are required to kill tumor cells cause unacceptable toxicities to normal tissues). The situation is made more dramatic by the fact that the majority of anticancer drugs accumulate preferentially in normal tissues rather than in neoplastic sites, due to the irregular vasculature and to the high interstitial pressure of solid tumors. One avenue towards the development of more efficacious and better tolerated anti-cancer drugs relies on the targeted delivery of therapeutic agents to the tumor environment, thus sparing normal tissues. Molecular markers which are selectively expressed in the stroma and in neo-vascular sites of aggressive solid tumors appear to be particularly suited for ligand-based tumor targeting strategies. Tumor blood vessels are accessible to agents coming from the bloodstream, and their occlusion may result in an avalanche of tumor cell death. Furthermore, endothelial cells and stromal cells are genetically more stable than tumor cells and can produce abundant markers, which are ideally suited for tumor targeting strategies. This review focuses on recent advances in the development of ligands for the selective targeting of tumor blood vessels and new blood vessels in other angiogenesis-related diseases.     

Molecular architecture and ligand recognition determinants for T4 RNA ligase

RNA ligase type 1 from bacteriophage T4 (Rnl1) is involved in countering a host defense mechanism by repairing 5'-PO4 and 3'-OH groups in tRNA(Lys). Rnl1 is widely used as a reagent in molecular biology. Although many structures for DNA ligases are available, only fragments of RNA ligases such as Rnl2 are known. We report the first crystal structure of a complete RNA ligase, Rnl1, in complex with adenosine 5'-(alpha,beta-methylenetriphosphate) (AMPcPP). The N-terminal domain is related to the equivalent region of DNA ligases and Rnl2 and binds AMPcPP but with further interactions from the additional N-terminal 70 amino acids in Rnl1 (via Tyr37 and Arg54) and the C-terminal domain (Gly269 and Asp272). The active site contains two metal ions, consistent with the two-magnesium ion catalytic mechanism. The C-terminal domain represents a new all alpha-helical fold and has a charge distribution and architecture for helix-nucleic acid groove interaction compatible with tRNA binding.