Regulation of muscarinic receptor binding by guanine nucleotides and N-ethylmaleimide

The regulation of muscarinic receptor binding by guanine nucleotides and N-ethylmaleimide (NEM) was investigated using the agonist ligand, [³H] cis methyldioxolane ([³H] CD). Characterization studies on rat forebrain homogenates showed that [³H] CD binding was linear with tissue concentration and was unaffected by a change in pH from 5.5 to 8.0. The regional variation in [³H] CD binding in the rat brain correlated generally with [³H] (?)3-quinuclidinyl benzilate ([³H] (?)QNB) binding, although the absolute variation in binding was somewhat less. At a concentration of 100 μM, the GTP analogue, guanyl-5′-yl imidodiphosphate [Gpp(NH)p], caused a 43–77% inhibition of [³H] CD binding in the corpus striatum, ileum, and heart. The results of binding studies using several Gpp(NH)p concentrations demonstrated that the potency of this guanine nucleotide for inhibition of [³H] CD binding was greater in the heart than in the ileum. In contrast to its effects on [³H] CD binding, Gpp(NH)p caused an increase in [³H] (?)QNB binding in the heart heart and ileum and no change in [³H] (?)QNB binding in the corpus striatum. When measured by competitive inhibition of [³H] (?)QNB binding to the longitudinal muscle of the ileum, Gpp(NH)p (100 μM) caused an increase in the IC₅₀ values of a series of agonists in a manner that was correlated with the efficacy of these compounds. The results of binding studies on NEM treated forebrain homogenates revealed an enhancement of [³H] CD binding by NEM.     

Identification of a functional arginine residue involved in coenzyme binding by the NADP-specific glutamate dehydrogenase of Neurospora.

The NADP-specific glutamate dehydrogenase (EC 1.4.1.4) of Neurospora crassa is inhibited by reaction with 1,2-cyclohexanedione which binds to arginine residues. With the 14C-labeled reagent, a peptide was isolated with the sequence: Gly-Gly-Leu-Arg-Leu-His-Pro-Ser-Val-Asn-Leu, corresponding to residues 78 through 88 in the protein. The arginine, residue 81, was present as N7,N8-(1,2-dihydroxycyclohex-1,2-ylene)-arginyl (or DHCH-arginine). Present evidence indicates that this arginine residue resides at or near the nicotinamide binding domain of the enzyme. Similar sequences are present in the bovine liver enzyme (EC 1.4.1.3) and the NAD-specific glutamate dehydrogenase of Neurospora (EC 1.4.1.2).     

Identification of a motif in the carboxyl terminus of CXCR2 that is involved in adaptin 2 binding and receptor internalization

Agonist treatment of cells expressing the chemokine receptor, CXCR2, induces receptor phosphorylation and internalization through a dynamin-dependent mechanism. In the present study, we demonstrate that a carboxyl terminus-truncated mutant of CXCR2 (331T), which no longer undergoes agonist-induced phosphorylation, continues to undergo ligand-induced internalization in HEK293 cells. This mutant receptor exhibits reduced association with beta-arrestin 1 but continues to exhibit association with adaptin 2 alpha and beta subunits. Replacing Leu320-321 and/or Ile323-Leu324 with Ala (LL320,321AA, IL323,324AA, and LLIL320,321,323,324AAAA) in wild-type CXCR2 or 331T causes little change in ligand binding and signaling through Ca(2+) mobilization but greatly impairs the agonist-induced receptor sequestration and ligand-mediated chemotaxis. The LL320,321AA, IL323,324AA, and LLIL320,321,323,324AAAA mutants of CXCR2 exhibit normal binding to beta-arrestin 1 but exhibit decreased binding to adaptin 2alpha and beta. These data demonstrate a role for the LLKIL motif in the carboxyl terminus of CXCR2 in receptor internalization and cell chemotaxis and imply a role for adaptin 2 in the endocytosis of CXCR2.     

Conformational changes in the bacterial SRP receptor FtsY upon binding of guanine nucleotides and SRP

In cotranslational preprotein targeting in Escherichia coli, the signal recognition particle (SRP) binds to the signal peptide emerging from the ribosome and, subsequently, interacts with the signal recognition particle receptor, FtsY, at the plasma membrane. Both FtsY and the protein moiety of the signal recognition particle, Ffh, are GTPases, and GTP is required for the formation of the SRP-FtsY complex. We have studied the binding of GTP/GDP to FtsY as well as the SRP-FtsY complex formation by monitoring the fluorescence of tryptophan 343 in the I box of mutant FtsY. Thermodynamic and kinetic parameters of the FtsY complexes with GDP, GTP, and signal recognition particle are reported. Upon SRP-FtsY complex formation in the presence of GTP, the fluorescence of tryptophan 343 increased by 50 % and was blue-shifted by 10 nm. We conclude that GTP-dependent SRP-FtsY complex formation leads to an extensive conformational change in the I box insertion in the effector region of FtsY.     

Identification of a tubulin binding motif on the P2X2 receptor

To isolate proteins interacting with P2X receptors, GST fusion proteins containing the intracellular C terminal tail of P2X(2), P2X(5), or P2X(7) were used as bait to screen detergent extracts of rat brain synaptosomes. By SDS-PAGE combined with mass spectrometry, two interacting proteins were identified: betaIII tubulin and myelin basic protein. While myelin basic protein bound to all three P2X subunits, betaIII tubulin interacted exclusively with the P2X(2) subunit. The tubulin binding domain could be confined to a proline-rich segment (amino acids 371-412) of the P2X(2) subunit. Our results suggest a role for microtubules in the cellular localisation of the P2X(2) receptor.     

Functional arginine residues involved in coenzyme binding by glutamate dehydrogenases.

Reaction of the NADP-dependent glutamate dehydrogenase of Neurospora with 1,2-cyclohexanedione results in a biphasic loss of enzyme activity. At the end of the rapid phase of the reaction (t1/2 = 1.5 min) the enzyme activity is diminished by approximately 60% with the simultaneous loss of 1 residue of arginine per subunit. After 60 min, the enzyme activity is completely lost with the modification of a total of 2 arginine residues per subunit. Reaction of bovine liver glutamate dehydrogenase with cyclohexanedione causes a rapid loss of approximately 45% of the enzyme activity and modification of about 1.5 residues of arginine per subunit. More prolonged treatment results in reaction of an additional 4 residues of arginine per subunit but is without further effect on the residual activity. The activity of the Neurospora enzyme is not protected by substrate, coenzyme, or a combination of both; however, the activity of the bovine enzyme is partially protected by high levels of NAD or NADP. Although the Km for alpha-ketoglutarate is unchanged by a limited modification of either enzyme with cyclohexanedione, the Km for coenzyme is increased about 2-fold for the Neurospora enzyme and about 1.5-fold for the bovine enzyme. The Ki of the Neurospora dehydrogenase for the competitive inhibitor 2'-monophosphoadenosine-5'-diphosphoribose is unchanged by the enzyme modification, but nicotinamide mononucleotide, a competitive inhibitor for the native Neurospora enzyme, does not inhibit the glutamate dehydrogenase with 1 modified arginine residue. This finding implies that the modified arginine is at or near the nicotinamide binding iste of the enzyme.     

Characterization of the substance P receptor in rat brain cortex membranes and the inhibition of radioligand binding by guanine nucleotides

Rat brain cortex membranes bind to a conjugate of substance P and 125I-labeled Bolton-Hunter reagent, and this binding can be inhibited by a low concentration of substance P (Kd = 1.2 +/- 0.4 X 10(-8) M). This binding is reversible and saturable (0.5 +/- 0.1 pmol of binding sites/mg of protein). Fragments of substance P as small as the carboxyl-terminal hexapeptide can inhibit the binding although their potency decreases with the decrease in the length of the peptides. The binding affinities of smaller peptides or peptides in which the carboxyl-terminal amide or amino acids are removed are drastically reduced. Biologically active analogs of substance P, physalaemin, eledoisin, substance P methyl ester, [D-Ala0]hepta(5-11)substance P, kassinin, and the eledoisin-related hexapeptide also can inhibit the binding. However, the binding is not inhibited by polypeptides structurally unrelated to substance P or by amine hormones/neurotransmitters. The binding affinities of biologically active peptides to rat brain cortex membranes are almost identical with their affinities for rat parotid cells which we previously determined. Furthermore, the recently described substance P antagonist, [D-Pro, D-Trp]substance P, inhibits the binding of the 125I-labeled substance P derivative to brain cortex membranes and to parotid cells equally well. These results suggest that the substance P receptors in the brain cortex and the parotid gland are similar. The brain cortex membrane binding of the 125I-labeled substance P derivative can be inhibited by micromolar concentrations of GTP, GDP, and their analogs. ITP and IDP were less active. Adenine and pyridine nucleotides were inactive.     

Identification of a non-canonical tyrosine-based endocytic motif in an ionotropic receptor

Rapid modulation of the surface number of certain ionotropic receptors is achieved by altering the relative rates of insertion and internalization. These receptors are internalized by a clathrin-mediated pathway; however, a motif that is necessary for endocytosis of ionotropic receptors has not yet been identified. Here, we identified a motif that is required for constitutive and agonist-regulated internalization of the ionotropic P2X(4) receptor. Three amino acids in the C terminus of P2X(4) (Tyr(378), Gly(381), and Leu(382)) compose a non-canonical tyrosine-based sorting signal of the form YXXGL. We found that P2X(4) protein was present in clathrin-coated vesicles isolated from rat brain and that a glutathione S-transferase fusion of the P2X(4) C terminus pulled down the adaptor protein-2 complex from brain extract. Mutation of either the tyrosine-binding pocket of the mu2 subunit of adaptor protein-2 or the YXXGL motif in the receptor C terminus caused a decrease in receptor internalization and a dramatic increase in the surface expression of P2X(4) receptors. The YXXGL motif represents a non-canonical tyrosine-based sorting signal that is necessary for efficient endocytosis of the P2X(4) receptor. Similar motifs are present in other receptors and may be important for the control of their functional expression.     

Identification of a subdomain in the Moloney murine leukemia virus envelope protein involved in receptor binding.

We have mutated amino acids within the receptor-binding domain of Moloney murine leukemia virus envelope in order to identify residues involved in receptor binding. Analysis of mutations in the region of amino acids 81 to 88 indicates that this region is important for specific envelope-receptor interactions. None of the aspartate 84 (D-84) mutants studied bind measurably, although they are efficiently incorporated into particles. D-84 mutants have titers that correspond to the severity of the substitution. This observation suggests that D-84 may provide a direct receptor contact. Mutations in the other charged amino acids in this domain (R-83, E-86, and E-87) yield titers similar to those of wild-type envelope, but the affinity of the mutant envelope in the binding assay is decreased by nonconservative substitutions in parallel to the severity of the change. These other amino acids may either provide secondary receptor contacts or assist in maintaining a structure in the domain that favors efficient binding. We also studied other regions of high hydrophilicity. Our initial characterization indicates that amino acids 106 to 111 and 170 to 188 do not play a major role in receptor binding. Measurements of relative binding affinity and titer indicate that most mutations in the region of amino acids 120 to 131 did not significantly affect receptor binding. However, SU encoded by mutants H123V, R124L, and C131A as well as C81A could not be detected in particles and therefore did not bind measurably. Therefore, the region encompassed by amino acids 81 to 88 appears to be directly involved in receptor binding.     

Search for a dextransucrase minimal motif involved in dextran binding

Fourteen truncated forms of Leuconostoc mesenteroides NRRL B512-F dextransucrase, involving N-, C- or N- plus C-terminal domain truncations were tested for their ability to bind dextrans. The shortest fragment (14kDa molecular weight) that still exhibited a strong interaction with dextran was localized between amino acids N1397 and A1527 of the C-terminal domain (GBD-7) and consists of six YG repeats. With a dissociation constant K(d) of 2.8x10(-9)M, this motif shows a very high affinity for isomaltohexaose and longer dextrans, supporting the proposed role of GBD in polymer formation. The potential application of GBD-7 as an affinity tag onto cheap resins like Sephacryl S300HR for rapid purification was evaluated and is discussed.     

Identification and characterization of a sequence motif involved in nonsense-mediated mRNA decay.

In both prokaryotes and eukaryotes, nonsense mutations in a gene can enhance the decay rate or reduce the abundance of the mRNA transcribed from that gene, and we call this process nonsense-mediated mRNA decay. We have been investigating the cis-acting sequences involved in this decay pathway. Previous experiments have demonstrated that, in addition to a nonsense codon, specific sequences 3' of a nonsense mutation, which have been defined as downstream elements, are required for mRNA destabilization. The results presented here identify a sequence motif (TGYYGATGYYYYY, where Y stands for either T or C) that can predict regions in genes that, when positioned 3' of a nonsense codon, promote rapid decay of its mRNA. Sequences harboring two copies of the motif from five regions in the PGK1, ADE3, and HIS4 genes were able to function as downstream elements. In addition, four copies of this motif can function as an independent downstream element. The sequences flanking the motif played a more significant role in modulating its activity when fewer copies of the sequence motif were present. Our results indicate the sequences 5' of the motif can modulate its activity by maintaining a certain distance between the sequence motif and the termination codon. We also suggest that the sequences 3' of the motif modulate the activity of the downstream element by forming RNA secondary structures. Consistent with this view, a stem-loop structure positioned 3' of the sequence motif can enhance the activity of the downstream element. This sequence motif is one of the few elements that have been identified that can predict regions in genes that can be involved in mRNA turnover. The role of these sequences in mRNA decay is discussed.     

Interaction between the chemotactic cAMP receptor and a detergent-insoluble membrane residue of Dictyostelium discoideum. Modulation by guanine nucleotides

Cells from Dictyostelium discoideum carry chemotactic cAMP receptors on their surface. Kinetic studies have revealed the existence of two slowly dissociating, high affinity receptor forms (SS and S) and one or more fast dissociating, low affinity forms (F) (Van Haastert, P.J.M., and De Wit, R.J.W. (1984) J. Biol. Chem. 259, 13321-13328). We have studied the interaction of these different cAMP-receptor types with a detergent-insoluble membrane residue. Isolated D. discoideum membranes were extracted with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS), which was previously shown to be the only detergent in the presence of which cAMP receptor binding is completely preserved (Janssens, P. M. W., and Van Driel, R. (1986) Biochim. Biophys. Acta 885, 91-101). The protein composition of the CHAPS-insoluble membrane residue appeared to be similar to that of the Triton X-100-insoluble membrane skeleton. Cyclic AMP binding studies revealed a specific association of the slowly dissociating cAMP receptors (SS and S forms) with this CHAPS-insoluble residue. All fast dissociating (F type) receptors were solubilized by CHAPS. GTP induced a transition of 75% of the SS and S receptors to faster dissociating forms. This transition was accompanied by the release of an equal number of receptors from the residue. These effects of GTP required that the cAMP receptor was occupied, and were completely reversible. After removal of the guanine nucleotide SS and S type receptors reappeared, bound to the residue, with a t1/2 of 5-10 min at 0 degrees C. We conclude that a detergent-insoluble membrane residue is involved in signal transduction via the chemotactic cAMP receptor. Both receptor occupation and a guanine nucleotide binding protein control receptor-residue interaction.     

Bovine mitochondrial ribosomes possess a high affinity binding site for guanine nucleotides

Mammalian mitochondrial ribosomes possess a binding site for guanine nucleotides. GTP binds in unit stoichiometry and with high affinity (Kd = 15.3 +/- 2.8 nM) to the small subunit of bovine mitochondrial ribosomes. This binding activity survives high salt washes, indicating that the nucleotide binds to an integral site within this subunit. GDP also binds to the small subunit with high affinity (Kd = 17 +/- 5.8 nm) and in unit stoichiometry. The GTP binding activity can be competed with GDP but not appreciably by other nucleotides, indicating that both GTP and GDP bind specifically and to the same site. The non-hydrolyzable analogs of GTP, guanylyl-5'-imidophosphate, and guanylyl-(beta,gamma-methylene)- diphosphonate also bind to the small subunit, but with reduced affinity. These results indicate that mammalian mitochondrial ribosomes, unlike other ribosomes, are able to interact directly with guanosine triphosphate, suggesting that the bound GTP may be involved in a novel regulatory mechanism in mitochondrial protein synthesis.     

Evolution of glutamate interactions during binding to a glutamate receptor

Glutamate receptors are the predominant mediators of excitatory synaptic signals in the central nervous system and are important in learning and memory as well as in diverse neuropathologies including epilepsy and ischemia. Their primary function is to receive the chemical signal glutamate (1), which binds to an extracellular domain in the receptor, and convert it into an electrical signal through the formation of cation-permeable transmembrane channels. Recently described end-state apo and ligated structures of the ligand-binding domain of a rat glutamate receptor provide a first view of specific molecular interactions between the ligand and the receptor that are central to the allosteric regulation of function in this protein. Yet there is little information on the mechanism and the structures of intermediates (if any) formed during the ligand-binding process. Here we have used time-resolved vibrational spectroscopy to show that the process involves a sequence of interleaved ligand and protein changes that starts with the docking of glutamate at the alpha-carboxylate moiety and ends with the establishment of the interactions between the gamma-carboxylate of glutamate and the protein.     

Identification of a novel ligand binding motif in the transthyretin channel

The design of therapeutic compounds targeting transthyretin (TTR) is challenging due to the low specificity of interaction in the hormone binding site. Such feature is highlighted by the interactions of TTR with diclofenac, a compound with high affinity for TTR, in two dissimilar modes, as evidenced by crystal structure of the complex. We report here structural analysis of the interactions of TTR with two small molecules, 1-amino-5-naphthalene sulfonate (1,5-AmNS) and 1-anilino-8-naphthalene sulfonate (1,8-ANS). Crystal structure of TTR:1,8-ANS complex reveals a peculiar interaction, through the stacking of the naphthalene ring between the side-chain of Lys15 and Leu17. The sulfonate moiety provides additional interaction with Lys15′ and a water-mediated hydrogen bond with Thr119′. The uniqueness of this mode of ligand recognition is corroborated by the crystal structure of TTR in complex with the weak analogue 1,5-AmNS, the binding of which is driven mainly by hydrophobic partition and one electrostatic interaction between the sulfonate group and the Lys15. The ligand binding motif unraveled by 1,8-ANS may open new possibilities to treat TTR amyloid diseases by the elucidation of novel candidates for a more specific pharmacophoric pattern.     

Regulation of neuropeptide Y (NPY) binding by guanine nucleotides in the rat cerebral cortex

The Na+-motive NADH oxidase activity from Vibrio alginolyticus was extracted with octylglucoside and reconstituted into liposomes by dilution. On the addition of NADH, the reconstituted proteoliposomes generated delta psi (inside positive) and delta pH (inside alkaline) in the presence of a proton conductor CCCP, and accumulated Na+ in the presence of valinomycin. These results indicate that the NADH oxidase activity, reconstituted in opposite orientation, leads to the generation of an electrochemical potential of Na+ by the influx of Na+.     

Identification of a nonapeptide motif in the vimentin head domain involved in intermediate filament assembly.

The assembly of soluble vimentin subunits into intermediate filaments (IFs) is dependent on information located in the amino-terminal domain. Using site-directed mutagenesis of a Xenopus laevis vimentin cDNA and an Escherichia coli production system to obtain pure mutated protein, we have identified, in the head domain, a nine amino acid motif (SSYRRIFGG), evolutionarily conserved from amphibia to man, which plays an important role in the orderly formation of IFs. Exchanges in the central di-arginine and in the two aromatic residues interfere with IF assembly of vimentin in vitro: on assembly under standard assembly conditions (160 mM-NaCl) most of the protein is included in dense aggregates, with a variable and minor proportion of IFs, whereas at lower ionic concentrations short and incomplete IF-like structures are formed. The deletion of the whole motif results in a protein that under standard assembly conditions (e.g. 160 mM-NaCl) predominantly and rapidly precipitates into large aggregates of non-IF material, whereas at lower ionic strength (e.g. 50 mM-NaCl) both IFs and dense aggregates are formed simultaneously. Our results show that the mutated protein can assume different forms at the same time and under the same conditions. This motif alone is insufficient for the formation of normal IFs as demonstrated by a mutant in which the motif has been brought closer to the alpha-helical rod domain by deletion of 55 internal amino acid residues. Corresponding observations have been made, by immunofluorescence microscopy, upon transfection of cultured epithelial cells lacking vimentin IFs. The importance of the head domain motif for the assembly and higher-order arrangement of IFs is discussed.