@article{12082, abstract = {Proximity-dependent protein labeling provides a powerful in vivo strategy to characterize the interactomes of specific proteins. We previously optimized a proximity labeling protocol for Caenorhabditis elegans using the highly active biotin ligase TurboID. A significant constraint on the sensitivity of TurboID is the presence of abundant endogenously biotinylated proteins that take up bandwidth in the mass spectrometer, notably carboxylases that use biotin as a cofactor. In C. elegans, these comprise POD-2/acetyl-CoA carboxylase alpha, PCCA-1/propionyl-CoA carboxylase alpha, PYC-1/pyruvate carboxylase, and MCCC-1/methylcrotonyl-CoA carboxylase alpha. Here, we developed ways to remove these carboxylases prior to streptavidin purification and mass spectrometry by engineering their corresponding genes to add a C-terminal His10 tag. This allows us to deplete them from C. elegans lysates using immobilized metal affinity chromatography. To demonstrate the method's efficacy, we use it to expand the interactome map of the presynaptic active zone protein ELKS-1. We identify many known active zone proteins, including UNC-10/RIM, SYD-2/liprin-alpha, SAD-1/BRSK1, CLA-1/CLArinet, C16E9.2/Sentryn, as well as previously uncharacterized potentially synaptic proteins such as the ortholog of human angiomotin, F59C12.3 and the uncharacterized protein R148.3. Our approach provides a quick and inexpensive solution to a common contaminant problem in biotin-dependent proximity labeling. The approach may be applicable to other model organisms and will enable deeper and more complete analysis of interactors for proteins of interest.}, author = {Artan, Murat and Hartl, Markus and Chen, Weiqiang and De Bono, Mario}, issn = {1083-351X}, journal = {Journal of Biological Chemistry}, number = {9}, publisher = {Elsevier}, title = {{Depletion of endogenously biotinylated carboxylases enhances the sensitivity of TurboID-mediated proximity labeling in Caenorhabditis elegans}}, doi = {10.1016/j.jbc.2022.102343}, volume = {298}, year = {2022}, } @article{10117, abstract = {Proximity labeling provides a powerful in vivo tool to characterize the proteome of subcellular structures and the interactome of specific proteins. The nematode Caenorhabditis elegans is one of the most intensely studied organisms in biology, offering many advantages for biochemistry. Using the highly active biotin ligase TurboID, we optimize here a proximity labeling protocol for C. elegans. An advantage of TurboID is that biotin's high affinity for streptavidin means biotin-labeled proteins can be affinity-purified under harsh denaturing conditions. By combining extensive sonication with aggressive denaturation using SDS and urea, we achieved near-complete solubilization of worm proteins. We then used this protocol to characterize the proteomes of the worm gut, muscle, skin, and nervous system. Neurons are among the smallest C. elegans cells. To probe the method's sensitivity, we expressed TurboID exclusively in the two AFD neurons and showed that the protocol could identify known and previously unknown proteins expressed selectively in AFD. The active zones of synapses are composed of a protein matrix that is difficult to solubilize and purify. To test if our protocol could solubilize active zone proteins, we knocked TurboID into the endogenous elks-1 gene, which encodes a presynaptic active zone protein. We identified many known ELKS-1-interacting active zone proteins, as well as previously uncharacterized synaptic proteins. Versatile vectors and the inherent advantages of using C. elegans, including fast growth and the ability to rapidly make and functionally test knock-ins, make proximity labeling a valuable addition to the armory of this model organism.}, author = {Artan, Murat and Barratt, Stephen and Flynn, Sean M. and Begum, Farida and Skehel, Mark and Nicolas, Armel and De Bono, Mario}, issn = {1083-351X}, journal = {Journal of Biological Chemistry}, number = {3}, publisher = {Elsevier}, title = {{Interactome analysis of Caenorhabditis elegans synapses by TurboID-based proximity labeling}}, doi = {10.1016/J.JBC.2021.101094}, volume = {297}, year = {2021}, } @article{8440, abstract = {Mycobacterium tuberculosis can remain dormant in the host, an ability that explains the failure of many current tuberculosis treatments. Recently, the natural products cyclomarin, ecumicin, and lassomycin have been shown to efficiently kill Mycobacterium tuberculosis persisters. Their target is the N-terminal domain of the hexameric AAA+ ATPase ClpC1, which recognizes, unfolds, and translocates protein substrates, such as proteins containing phosphorylated arginine residues, to the ClpP1P2 protease for degradation. Surprisingly, these antibiotics do not inhibit ClpC1 ATPase activity, and how they cause cell death is still unclear. Here, using NMR and small-angle X-ray scattering, we demonstrate that arginine-phosphate binding to the ClpC1 N-terminal domain induces millisecond dynamics. We show that these dynamics are caused by conformational changes and do not result from unfolding or oligomerization of this domain. Cyclomarin binding to this domain specifically blocked these N-terminal dynamics. On the basis of these results, we propose a mechanism of action involving cyclomarin-induced restriction of ClpC1 dynamics, which modulates the chaperone enzymatic activity leading eventually to cell death.}, author = {Weinhäupl, Katharina and Brennich, Martha and Kazmaier, Uli and Lelievre, Joel and Ballell, Lluis and Goldberg, Alfred and Schanda, Paul and Fraga, Hugo}, issn = {0021-9258}, journal = {Journal of Biological Chemistry}, keywords = {Cell Biology, Biochemistry, Molecular Biology}, number = {22}, pages = {8379--8393}, publisher = {American Society for Biochemistry & Molecular Biology}, title = {{The antibiotic cyclomarin blocks arginine-phosphate–induced millisecond dynamics in the N-terminal domain of ClpC1 from Mycobacterium tuberculosis}}, doi = {10.1074/jbc.ra118.002251}, volume = {293}, year = {2018}, } @article{6298, abstract = {Tumor necrosis factor-stimulated gene-6 (TSG-6) is a hyalu-ronan (HA)-binding protein that plays important roles ininflammation and ovulation. TSG-6-mediated cross-linking ofHA has been proposed as a functional mechanism (e.g.for regu-lating leukocyte adhesion), but direct evidence for cross-linkingis lacking, and we know very little about its impact on HA ultra-structure. Here we used films of polymeric and oligomeric HAchains, end-grafted to a solid support, and a combination ofsurface-sensitive biophysical techniques to quantify the bindingof TSG-6 into HA films and to correlate binding to morpholog-ical changes. We find that full-length TSG-6 binds with pro-nounced positive cooperativity and demonstrate that it cancross-link HA at physiologically relevant concentrations. Ourdata indicate that cooperative binding of full-length TSG-6arises from HA-induced protein oligomerization and that theTSG-6 oligomers act as cross-linkers. In contrast, the HA-bind-ing domain of TSG-6 (the Link module) alone binds withoutpositive cooperativity and weaker than the full-length protein.Both the Link module and full-length TSG-6 condensed andrigidified HA films, and the degree of condensation scaled withthe affinity between the TSG-6 constructs and HA. We proposethat condensation is the result of protein-mediated HA cross-linking. Our findings firmly establish that TSG-6 is a potent HAcross-linking agent and might hence have important implica-tions for the mechanistic understanding of the biological func-tion of TSG-6 (e.g.in inflammation).}, author = {Baranova, Natalia and Nilebäck, Erik and Haller, F. Michael and Briggs, David C. and Svedhem, Sofia and Day, Anthony J. and Richter, Ralf P.}, issn = {0021-9258}, journal = {Journal of Biological Chemistry}, number = {29}, pages = {25675--25686}, publisher = {American Society for Biochemistry & Molecular Biology}, title = {{The inflammation-associated protein TSG-6 cross-links hyaluronan via hyaluronan-induced TSG-6 oligomers}}, doi = {10.1074/jbc.m111.247395}, volume = {286}, year = {2011}, } @article{8473, abstract = {β2-microglobulin (β2m), the light chain of class I major histocompatibility complex, is responsible for the dialysis-related amyloidosis and, in patients undergoing long term dialysis, the full-length and chemically unmodified β2m converts into amyloid fibrils. The protein, belonging to the immunoglobulin superfamily, in common to other members of this family, experiences during its folding a long-lived intermediate associated to the trans-to-cis isomerization of Pro-32 that has been addressed as the precursor of the amyloid fibril formation. In this respect, previous studies on the W60G β2m mutant, showing that the lack of Trp-60 prevents fibril formation in mild aggregating condition, prompted us to reinvestigate the refolding kinetics of wild type and W60G β2m at atomic resolution by real-time NMR. The analysis, conducted at ambient temperature by the band selective flip angle short transient real-time two-dimensional NMR techniques and probing the β2m states every 15 s, revealed a more complex folding energy landscape than previously reported for wild type β2m, involving more than a single intermediate species, and shedding new light into the fibrillogenic pathway. Moreover, a significant difference in the kinetic scheme previously characterized by optical spectroscopic methods was discovered for the W60G β2m mutant.}, author = {Corazza, Alessandra and Rennella, Enrico and Schanda, Paul and Mimmi, Maria Chiara and Cutuil, Thomas and Raimondi, Sara and Giorgetti, Sofia and Fogolari, Federico and Viglino, Paolo and Frydman, Lucio and Gal, Maayan and Bellotti, Vittorio and Brutscher, Bernhard and Esposito, Gennaro}, issn = {0021-9258}, journal = {Journal of Biological Chemistry}, keywords = {Cell Biology, Biochemistry, Molecular Biology}, number = {8}, pages = {5827--5835}, publisher = {American Society for Biochemistry & Molecular Biology}, title = {{Native-unlike long-lived intermediates along the folding pathway of the amyloidogenic protein β2-Microglobulin revealed by real-time two-dimensional NMR}}, doi = {10.1074/jbc.m109.061168}, volume = {285}, year = {2010}, } @article{2614, abstract = {Metabotropic glutamate receptors (mGluRs) from group III reduce glutamate release. Because these receptors reduce cAMP levels, we explored whether this signaling pathway contributes to release inhibition caused by mGluRs with low affinity for L-2-amino-4-phosphonobutyrate (L-AP4). In biochemical experiments with the population of cerebrocortical nerve terminals we find that L-AP4 (1 mM) inhibited the Ca2+dependent-evoked release of glutamate by 25%. This inhibitory effect was largely prevented by the pertussis toxin but was insensitive to inhibitors of protein kinase C bisindolylmaleimide and protein kinase A H-89. Furthermore, this inhibition was associated with reduction in N-type Ca2+ channel activity in the absence of any detectable change in cAMP levels. In the presence of forskolin, however, L-AP4 decreased the levels of cAMP. The activation of this additional signaling pathway was very efficient in counteracting the facilitation of glutamate release induced either by forskolin or the β-adrenergic receptor agonist isoproterenol. Imaging experiments to measure Ca2+ dynamics in single nerve terminals showed that L-AP4 strongly reduced the Ca2+ response in 28% of the nerve terminals. Moreover, immunochemical experiments showed that 25-35% of the nerve terminals that were immunopositive to synaptophysin were also immunoreactive to the low affinity L-AP4-sensitive mGluR7. Then, mGluR7 mediates the inhibition of glutamate release caused by 1 mM L-AP4, primarily by a strong inhibition of Ca2+ channels, although high cAMP uncovers the receptor ability to decrease cAMP.}, author = {Millán, Carmelo and Luján, Rafael and Shigemoto, Ryuichi and Sánchez Prieto, José}, issn = {0021-9258}, journal = {Journal of Biological Chemistry}, number = {16}, pages = {14092 -- 14101}, publisher = {American Society for Biochemistry and Molecular Biology}, title = {{The inhibition of glutamate release by metabotropic glutamate receptor 7 affects both [Ca2+]c and cAMP. Evidence for a strong reduction of Ca2+ entry in single nerve terminals}}, doi = {10.1074/jbc.M109044200}, volume = {277}, year = {2002}, } @article{2621, abstract = {The release properties of glutamatergic nerve terminals are influenced by a number of factors, including the subtype of voltage-dependent calcium channel and the presence of presynaptic autoreceptors. Group III metabotropic glutamate receptors (mGluRs) mediate feedback inhibition of glutamate release by inhibiting Ca2+ channel activity. By imaging Ca2+ in preparations of cerebrocortical nerve terminals, we show that voltage-dependent Ca2+ channels are distributed in a heterogeneous manner in individual nerve terminals. Presynaptic terminals contained only N-type (47.5%; conotoxin GVIA-sensitive), P/Q-type (3.9%; agatoxin IVA-sensitive), or both N- and P/Q-type (42.6%) Ca2+ channels, although the remainder of the terminals (6.1%) were insensitive to these two toxins. In this preparation, two mGluRs with high and low affinity for L(+)-2-amino-4-phosphonobutyrate were identified by immunocytochemistry as mGluR4 and mGluR7, respectively. These receptors were responsible for 22.2 and 24.1% reduction of glutamate release, and they reduced the Ca2+ response in 24.4 and 30.3% of the nerve terminals, respectively. Interestingly, mGluR4 was largely (73.7%) located in nerve terminals expressing both N- and P/Q-type Ca2+ channels, whereas mGluR7 was predominantly (69.9%) located in N-type Ca2+ channel-expressing terminals. This specific coexpression of different group III mGluRs and Ca2+ channels may endow synaptic terminals with distinct release properties and reveals the existence of a high degree of presynaptic heterogeneity.}, author = {Millán, Carmelo and Luján, Rafael and Shigemoto, Ryuichi and Sánchez Prieto, José}, issn = {0021-9258}, journal = {Journal of Biological Chemistry}, number = {49}, pages = {47796 -- 47803}, publisher = {American Society for Biochemistry and Molecular Biology}, title = {{Subtype-specific expression of Group III metabotropic glutamate receptors and Ca2+ channels in single nerve terminals}}, doi = {10.1074/jbc.M207531200}, volume = {277}, year = {2002}, } @article{13438, abstract = {ICln is an ion channel identified by expression cloning using a cDNA library from Madin-Darby canine kidney cells. In all organisms tested so far, only one transcript for the ICln protein could be identified. Here we show that two splice variants of the ICln ion channel can be found in Caenorhabditis elegans. Moreover, we show that these two splice variants of the ICln channel protein, which we termed IClnN1 and IClnN2, can be functionally reconstituted and tested in an artificial lipid bilayer. In these experiments, the IClnN1-induced currents showed no voltage-dependent inactivation, whereas the IClnN2-induced currents fully inactivated at positive potentials. The molecular entity responsible for the voltage-dependent inactivation of IClnN2 is a cluster of positively charged amino acids encoded by exon 2a, which is absent in IClnN1. Our experiments suggest a mechanism of channel inactivation that is similar to the “ball and chain” model proposed for the Shaker potassium channel,i.e. a cluster of positively charged amino acids hinders ion permeation through the channel by a molecular and voltage-dependent interaction at the inner vestibulum of the pore. This hypothesis is supported by the finding that synthetic peptides with the same amino acid sequence as the positive cluster can transform the IClnN1-induced current to the current observed after reconstitution of IClnN2. Furthermore, we show that the nematode ICln gene is embedded in an operon harboring two additional genes, which we termed Nx and Ny. Co-reconstitution of Nx and IClnN2 and functional analysis of the related currents revealed a functional interaction between the two proteins, as evidenced by the fact that the IClnN2-induced current in the presence of Nx was no longer voltage-sensitive. The experiments described indicate that the genome organization in nematodes allows an effective approach for the identification of functional partner proteins of ion channels.}, author = {Fürst, Johannes and Ritter, Markus and Rudzki, Jakob and Danzl, Johann G and Gschwentner, Martin and Scandella, Elke and Jakab, Martin and König, Matthias and Oehl, Bernhard and Lang, Florian and Deetjen, Peter and Paulmichl, Markus}, issn = {0021-9258}, journal = {Journal of Biological Chemistry}, keywords = {Cell Biology, Molecular Biology, Biochemistry}, number = {6}, pages = {4435--4445}, publisher = {Elsevier}, title = {{ICln Ion channel splice variants in Caenorhabditis elegans}}, doi = {10.1074/jbc.m107372200}, volume = {277}, year = {2002}, } @article{3928, abstract = {Regulated adhesion of leukocytes to the extracellular matrix is essential for transmigration of blood vessels and subsequent migration into the stroma of inflamed tissues. Although beta(2)-integrins play an indisputable role in adhesion of polymorphonuclear granulocytes (PMN) to endothelium, we show here that beta(1)- and beta(3)-integrins but not beta(2)-integrin are essential for the adhesion to and migration on extracellular matrix molecules of the endothelial cell basement membrane and subjacent interstitial matrix. Mouse wild type and beta(2)-integrin null PMN and the progranulocytic cell line 32DC13 were employed in in vitro adhesion and migration assays using extracellular matrix molecules expressed at sites of extravasation in vivo, in particular the endothelial cell laminins 8 and 10. Wild type and beta(2)-integrin null PMN showed the same pattern of ECM binding, indicating that beta(2)-integrins do not mediate specific adhesion of PMN to the extracellular matrix molecules tested; binding was observed to the interstitial matrix molecules, fibronectin and vitronectin, via integrins alpha(5)beta(1) and alpha(v)beta(3), respectively; to laminin 10 via alpha(6)beta(1); but not to laminins 1, 2, and 8, collagen type I and IV, perlecan, or tenascin-C. PMN binding to laminins 1, 2, and 8 could not be induced despite surface expression of functionally active integrin alpha(6)beta(1), a major laminin receptor, demonstrating that expression of alpha(6)beta(1) alone is insufficient for ligand binding and suggesting the involvement of accessory factors. Nevertheless, laminins 1, 8, and 10 supported PMN migration, indicating that differential cellular signaling via laminins is independent of the extent of adhesion. The data demonstrate that adhesive and nonadhesive interactions with components of the endothelial cell basement membrane and subjacent interstitium play decisive roles in controlling PMN movement into sites of inflammation and illustrate that beta(2)-integrins are not essential for such interactions.}, author = {Sixt, Michael K and Hallmann, Rupert and Wendler, Olaf and Scharffetter Kochanek, Karin and Sorokin, Lydia}, issn = {0021-9258}, journal = {Journal of Biological Chemistry}, number = {22}, pages = {18878 -- 18887}, publisher = {American Society for Biochemistry and Molecular Biology}, title = {{Cell adhesion and migration properties of β2-integrin negative polymorphonuclear granulocytes on defined extracellular matrix molecules. Relevance for leukocyte extravasation}}, doi = {10.1074/jbc.M010898200}, volume = {276}, year = {2001}, } @article{3149, abstract = {The prohormone convertases (PCs) are an evolutionarily ancient group of proteases required for the maturation of neuropeptide and peptide hormone precursors. In Drosophila melanogaster, the homolog of prohormone convertase 2, dPC2 (amontillado), is required for normal hatching behavior, and immunoblotting data indicate that flies express 80- and 75-kDa forms of this protein. Because mouse PC2 (mPC2) requires 7B2, a helper protein for productive maturation, we searched the fly data base for the 7B2 signature motif PPNPCP and identified an expressed sequence tag clone encoding the entire open reading frame for this protein. dPC2 and d7B2 cDNAs were subcloned into expression vectors for transfection into HEK-293 cells; mPC2 and rat 7B2 were used as controls. Although active mPC2 was detected in medium in the presence of either d7B2 or r7B2, dPC2 showed no proteolytic activity upon coexpression of either d7B2 or r7B2. Labeling experiments showed that dPC2 was synthesized but not secreted from HEK-293 cells. However, when dPC2 and either d7B2 or r7B2 were coexpressed in Drosophila S2 cells, abundant immunoreactive dPC2 was secreted into the medium, coincident with the appearance of PC2 activity. Expression and secretion of dPC2 enzyme activity thus appears to require insect cell-specific posttranslational processing events. The significant differences in the cell biology of the insect and mammalian enzymes, with 7B2 absolutely required for secretion of dPC2 and zymogen conversion occurring intracellularly in the case of dPC2 but not mPC2, support the idea that the Drosophila enzyme has specific requirements for maturation and secretion that can be met only in insect cells.}, author = {Hwang, Jae and Siekhaus, Daria E and Fuller, Robert and Taghert, Paul and Lindberg, Iris}, issn = {0021-9258}, journal = {Journal of Biological Chemistry}, number = {23}, pages = {17886 -- 17893}, publisher = {American Society for Biochemistry and Molecular Biology}, title = {{Interaction of Drosophila melanogaster prohormone convertase 2 and 7B2: Insect cell specific processing and secretion}}, doi = {10.1074/jbc.M000032200 }, volume = {275}, year = {2000}, } @article{4179, abstract = {Neurotrophin-3 (NT-3) is a member of the neurotrophin gene family and is highly expressed in the developing rat cerebellum. Here we show that brain-derived neurotrophic factor (BDNF) increased by approximately 10-fold the NT-3 mRNA levels in cultured cerebellar granule neurons isolated from postnatal rats, whereas nerve growth factor (NGF) and NT-3 itself had no effect. The effect of BDNF was additive to that of triiodothyronine (T3), which also increased NT-3 mRNA in these neurons. The drug K252a inhibited the BDNF-mediated stimulation of NT-3 expression, suggesting an involvement of trkB receptors. Nuclear run-on experiments showed that BDNF enhanced NT-3 transcription, whereas the stability of NT-3 mRNA remained unchanged. The data presented are the first demonstration that one neurotrophin regulates the expression of another and provide evidence that NT-3 production in granule neurons is regulated by both BDNF and T3.}, author = {Leingärtner, Axel and Heisenberg, Carl-Philipp J and Kolbeck, Roland and Thoenen, Hans and Lindholm, Dan}, issn = {1083-351X}, journal = {Journal of Biological Chemistry}, number = {2}, pages = {828 -- 830}, publisher = {American Society for Biochemistry and Molecular Biology}, title = {{Brain-derived neurotrophic factor increases neurotrophin-3 expression in cerebellar granule neurons}}, doi = {10.1016/s0021-9258(17)42186-7}, volume = {269}, year = {1994}, } @article{2536, abstract = {A cDNA clone for a new metabotropic glutamate receptor, termed mGluR6, was isolated from a rat retinal cDNA library by cross-hybridization with the previously isolated cDNA clone for a metabotropic glutamate receptor. The cloned mGluR6 subtype consists of 871 amino acid residues and exhibits a structural architecture common to the metabotropic receptor family, possessing a large extracellular domain preceding the seven putative membrane-spanning domains. mGluR6 shows the highest sequence similarity to mGluR4 among the metabotropic receptor subtypes and inhibits the forskolin- stimulated cyclic AMP accumulation in Chinese hamster ovary cells transfected with the cloned cDNA. mGluR6 potently reacts with L-2-amino-4- phosphonobutyrate (L-AP4) and L-serine-O-phosphate, and the potencies of these compounds are one order of magnitude greater than that of L-glutamate. Blot and in situ hybridization analyses indicated that mGluR6 mRNA is restrictedly expressed in the inner nuclear layer of the retina where ON- bipolar cells are distributed. The metabotropic receptor that responds strongly to L-AP4 and L-serine-O-phosphate in ON-bipolar cells is known to mediate glutamate synaptic transmission between photoreceptor cells and ON- bipolar cells. On the basis of the agonist selectivity of mGluR6 and its specific expression in retinal cells, the physiological role of this receptor subtype in the visual system is discussed.}, author = {Nakajima, Yoshiaki and Iwakabe, Hideki and Akazawa, Chihiro and Nawa, Hiroyuki and Shigemoto, Ryuichi and Mizuno, Noboru and Nakanishi, Shigetada}, issn = {0021-9258}, journal = {Journal of Biological Chemistry}, number = {16}, pages = {11868 -- 11873}, publisher = {American Society for Biochemistry and Molecular Biology}, title = {{Molecular characterization of a novel retinal metabotropic glutamate receptor mGluR6 with a high agonist selectivity for L-2-amino-4- phosphonobutyrate}}, doi = {10.1016/S0021-9258(19)50280-0}, volume = {268}, year = {1993}, } @article{2539, abstract = {cDNA clones for four different N-methyl-D-aspartate (NMDA) receptor subunits (NMDAR2A-NMDAR2D) were isolated through polymerase chain reactions followed by molecular screening of a rat brain cDNA library. These subunits are only about 15% identical with the key subunit of the NMDA receptor (NMDAR1) but are highly homologous (~50% homology) with one another. They also commonly possess large hydrophilic domains at both amino- and carboxyl- terminal sides of the four putative transmembrane segments. NMDAR2A and NMDAR2C expressed individually in Xenopus oocytes showed no electrophysiological response to agonists. However, these subunits in combined expression with NMDAR1 markedly potentiated the NMDAR1 activity and produced functional variability in the affinity of agonists, the effectiveness of antagonists, and the sensitivity to Mg2+ blockade. Thus, NMDAR1 is essential for the function of the NMDA receptor, and multiple NMDAR2 subunits potentiate and differentiate the function of the NMDA receptor by forming different heteromeric configurations with NMDAR1. Northern blotting and in situ hybridization analyses revealed that the expressions of individual mRNAs for the NMDAR2 subunits overlap in some brain regions but are also specialized in many other regions. This investigation demonstrates the anatomical and functional differences of the NMDAR2 subunits, which provide the molecular basis for the functional diversity of the NMDA receptor.}, author = {Ishii, Takahiro and Moriyoshi, Koki and Sugihara, Hidemitsu and Sakurada, Kazuhir and Kadotani, Hiroshi and Yokoi, Mineto and Akazawa, Chihiro and Shigemoto, Ryuichi and Mizuno, Noboru and Masu, Masayuki and Nakanishi, Shigetada}, issn = {0021-9258}, journal = {Journal of Biological Chemistry}, number = {4}, pages = {2836 -- 2843}, publisher = {American Society for Biochemistry and Molecular Biology}, title = {{Molecular characterization of the family of the N-methyl-D-aspartate receptor subunits}}, doi = {10.1016/s0021-9258(18)53849-7 }, volume = {268}, year = {1993}, } @article{2533, abstract = {A cDNA clone for a new metabotropic glutamate receptor, mGluR5, was isolated through polymerase chain reaction-mediated DNA amplification by using primer sequences conserved among the metabotropic glutamate receptor (mGluR) family and by the subsequent screening of a rat brain cDNA library. The cloned receptor consists of 1171 amino acid residues and exhibits a structural architecture common to the mGluR family, possessing a large extracellular domain preceding the seven putative membrane-spanning segments. mGluR5 shows the highest sequence similarity to mGluR1 among the mGluR members and is coupled to the stimulation of phosphatidylinositol hydrolysis/ Ca2+ signal transduction in Chinese hamster ovary cells transfected with the cloned cDNA. This receptor also resembles mGluR1 in its agonist selectivity and antagonist responses; the potency rank order of agonists for mGluR5 was determined to be quisqualate > L-glutamate ≥ ibotenate > trans-1-aminocyclopentane-1,3-dicarboxylate. Blot and in situ hybridization analyses indicated that mGluR5 mRNA is widely distributed in neuronal cells of the central nervous system and is expressed differently from mGluR1 mRNA in many brain regions. This investigation thus demonstrates that there is an additional mGluR subtype which closely resembles mGluR1 in its signal transduction and pharmacological properties and is expressed in specialized neuronal cells in the central nervous system.}, author = {Abe, Takaaki and Sugihara, Hidemitsu and Nawa, Hiroyuki and Shigemoto, Ryuichi and Mizuno, Noboru and Nakanishi, Shigetada}, issn = {0021-9258}, journal = {Journal of Biological Chemistry}, number = {19}, pages = {13361 -- 13368}, publisher = {American Society for Biochemistry and Molecular Biology}, title = {{Molecular characterization of a novel metabotropic glutamate receptor mGluR5 coupled to inositol phosphate/Ca2+ signal transduction}}, doi = {10.1016/S0021-9258(18)42219-3}, volume = {267}, year = {1992}, } @article{2480, abstract = {Functional cDNA clones for rat neuromedin K receptor were isolated from a rat brain cDNA library by cross-hybridization with the bovine substance K recepor cDNA. Injection of the mRNA synthesized in vitro from the cloned cDNA into Xenopus oocytes elicited electrophysiological responses to tachykinins, with the most potent sensitivity being to neuromedin K. Ligand-binding displacement in membranes of mammalian COS cells transfected with the cDNA indicated the rank order of affinity of the receptor to tachykinins; neuromedin K > substance K > substance P. The hybridization analysis showed that the neuromedin K receptor mRNA is expressed in both the brain and the peripheral tissues at different levels. The rat neuromedin K receptor consists of 452 amino acid residues and belongs to the family of G protein-coupled receptors, which are thought to have seven transmembrane domains. The sequence comparison of the rat neuromedin K, substance P, and substance K receptors revealed that these receptors are highly conserved in the seven transmembrane domains and the cytoplasmic sides of the receptors. They also show some structural characteristics, including the common presence of histidine residues in transmembrane segments V and VI and the difference in the numbers and distributions of serine and threonine residues as possible phosphorylation sites in the cytoplasmic regions. This paper thus presents the first comprehensive analysis of the molecular nature of the multiple peptide receptors that exhibit similar but pharmacologically distinguishable activities.}, author = {Shigemoto, Ryuichi and Yokota, Yoshifumi and Tsuchida, Kunihiro and Nakanishi, Shigetada}, issn = {1083-351X}, journal = {Journal of Biological Chemistry}, number = {2}, pages = {623 -- 628}, publisher = {American Society for Biochemistry and Molecular Biology}, title = {{Cloning and expression of a rat neuromedin K receptor cDNA}}, doi = {10.1016/s0021-9258(19)40095-1 }, volume = {265}, year = {1990}, } @article{2525, abstract = {This paper describes the amino acid sequence of the rat substance P receptor and its comparison with that of the rat substance K receptor on the basis of molecular cloning and sequence analysis. From a rat brain cDNA library constructed with an RNA expression vector, we identified a cDNA mixture containing a functional substance P receptor cDNA by examining electrophysiologically a receptor expression following injection of the mRNAs synthesized in vitro into Xenopus oocytes. A receptor cDNA clone was then isolated by cross-hybridization with the bovine substance K receptor DNA. The clone was confirmed by selective binding of substance P to the cloned receptor expressed in mammalian COS cells. The deduced amino acid sequence (407 amino acid residues) possesses seven putative membrane spanning domains and shows a sequence similarity to the members of G-protein-coupled receptors. The rat substance P and substance K receptor are very similar in both size and amino acid sequences, particularly in the putative transmembrane similarity is in marked contrast to the sequence divergence in the amino- and carboxyl-terminal regions and the third cytoplasmic loop. The observed sequence similarytity and divergence would thus contribute to the expression of similar but pharmacological regions and the first and second cytoplasmic loops. This distinguishable activities of the two tachykinin receptors.}, author = {Yokota, Yoshifumi and Sasai, Yoshiki and Tanaka, Kohichi and Fujiwara, Tsutomu and Tsuchida, Kunihiro and Shigemoto, Ryuichi and Kakizuka, Akira and Ohkubo, Hiroaki and Nakanishi, Shigetada}, issn = {1083-351X}, journal = {Journal of Biological Chemistry}, number = {30}, pages = {17649 -- 17652}, publisher = {American Society for Biochemistry and Molecular Biology}, title = {{Molecular characterization of a functional cDNA for rat substance P receptor}}, doi = {doi.org/10.1016/S0021-9258(19)84619-7}, volume = {264}, year = {1989}, }