Chimeric yeast G-protein α subunit harboring a 37-residue C-terminal gustducin-specific sequence is functional in Saccharomyces cerevisiae.

Despite many recent studies of G-protein-coupled receptor (GPCR) structures, it is not yet well understood how these receptors activate G proteins. The GPCR assay using baker's yeast, Saccharomyces cerevisiae, is an effective experimental model for the characterization of GPCR-Gα interactions. Here, using the yeast endogenous Gα protein (Gpa1p) as template, we constructed various chimeric Gα proteins with a region that is considered to be necessary for interaction with mammalian receptors. The signaling assay using the yeast pheromone receptor revealed that the chimeric Gα protein harboring 37 gustducin-specific amino acid residues at its C-terminus (GPA1/gust37) maintained functionality in yeast. In contrast, GPA1/gust44, a variant routinely used in mammalian experimental systems, was not functional.

Characterization of the tritium-labeled analog of L-threo-beta-benzyloxyaspartate binding to glutamate transporters.

L-Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Termination of glutamate receptor activation and maintenance of low extracellular glutamate concentrations are primarily achieved by glutamate transporters (excitatory amino acid transporters 1-5, EAATs1-5) located on both the nerve endings and the surrounding glial cells. To identify the physiological roles of each subtype, subtype-selective EAAT ligands are required. In this study, we developed a binding assay system to characterize EAAT ligands for all EAAT subtypes. We recently synthesized novel analogs of threo-beta-benzyloxyaspartate (TBOA) and reported that they blocked glutamate uptake by EAATs 1-5 much more potently than TBOA. The strong inhibitory activity of the TBOA analogs suggested that they would be suitable to use as radioisotope-labeled ligands, and we therefore synthesized a tritiated derivative of (2S,3S)-3-{3-[4-ethylbenzoylamino]benzyloxy}aspartate ([3H]ETB-TBOA). [3H]ETB-TBOA showed significant high-affinity specific binding to EAAT-transfected COS-1 cell membranes with each EAAT subtype. The Hill coefficient for the Na+-dependence of [3H]ETB-TBOA binding revealed a single class of noncooperative binding sites for Na+, suggesting that Na+ binding in the ligand binding step is different from Na+ binding in the substrate uptake process. The binding was displaced by known substrates and blockers. The rank order of inhibition by these compounds was consistent with glutamate uptake assay results reported previously. Thus, the [3H]ETB-TBOA binding assay will be useful to screen novel EAAT ligands for all EAAT subtypes.

Characteristic expression patterns of allatostatin-like peptide, FMRFamide-related peptide, orcokinin, tachykinin-related peptide, and SIFamide in the olfactory system of crayfish Procambarus clarkii.

The olfactory system plays important roles in various crustacean behaviors. Despite numerous studies on different aspects of the olfactory neural pathway, only the decapod-tachykinin-related peptide (decapod-TRP) has been identified as a neuromodulator in this processing to date. To establish the functions of other related neuropeptides, we initially performed cDNA cloning of FMRFamide-related peptide (FaRP) and allatostatin (AST)-like peptide from the crayfish Procambarus clarkii, followed by in situ hybridization (ISH) analysis of these peptides, along with decapod-TRP, orcokinin, and crustacean-SIFamide. Cloned FaRP cDNA encodes seven copies of C-terminal RN(F/Y)LRFamide-containing peptide, whereas AST-like peptide cDNA comprises 29 copies of AST-like peptide (-YXFGLamide) and three additional putative peptides. ISH analysis of the brain revealed specific expression of crustacean-SIFamide mRNA in most projection neurons (cell cluster 10), and predominant localization of other mRNAs to interneurons. The data suggest that the crustacean-SIFamide neuropeptide is involved in output of the deutocerebrum to the protocerebrum. Double-fluorescence ISH data further disclose that, in cluster 9, orcokinin is coexpressed in decapod-TRP-specific interneurons, whereas AST-like peptide-containing cells do not overlap with orcokinin-expressing cells. On the other hand, FaRP-expressing cells overlap with both orcokinin- and AST-like peptide-specific cells. In cluster 11, where signals for AST-like peptide are absent, a number of interneurons express both decapod-TRP and orcokinin, emphasizing a close relationship between these two factors with regard to olfactory processing, and possibly tactile and/or visual sensory systems. These characteristic expression patterns of neuropeptides support their distinct involvement in the modulation of olfactory processing.

APSGFLGMRamide is a unique tachykinin-related peptide in crustaceans.

We report here the identification of a tachykinin-related peptide (TRP) in crustaceans. Direct MALDI-TOF MS with brain slices of the crayfish Procambarus clarkii indicated the presence of a peptide having an MS number of around 934. Quadrupole orthogonal acceleration time-of-flight (Q-TOF) MS/MS analysis implied the sequence to be APSGFLGMRamide, identical to that of CabTRP Ia, isolated previously from the crab Cancer borealis, and Pev-tachykinin, from the shrimp Penaeus vannamei. The peptide has been shown to be myoactive in the cockroach hindgut, but the structure of its precursor protein had not been elucidated. A cDNA encoding crayfish preproTRP was cloned, revealing that the 225-residue protein contains seven identical copies of the peptide APSGFLGMRamide. This is unique because TRPs identified in other invertebrates were known to exist in several related forms in each species. The conserved structure of TRP in crustaceans was confirmed by cloning preproTRP from the spiny lobster Panulirus interruptus. RT-PCR and Northern blotting analyses suggested that the crayfish preproTRP mRNA is expressed throughout the nervous system, and in situ hybridization studies of the brain revealed that the transcript predominantly localizes to cell clusters 11 (dorsal lateral cells) and 9 (ventral lateral cells).

Prepro-tachykinin gene expression in the brain of the honeybee Apis mellifera.

We have recently identified a tachykinin-related peptide (AmTRP) from the mushroom bodies (MBs) of the brain of the honeybee Apis mellifera L. by using direct matrix-assisted laser desorption/ionization with time-of-flight mass spectometry and have isolated its cDNA. Here, we have examined prepro-AmTRP gene expression in the honeybee brain by using in situ hybridization. The prepro-AmTRP gene is expressed predominantly in the MBs and in some neurons located in the optic and antennal lobes. cDNA microarray studies have revealed that AmTRP expression is enriched in the MBs compared with other brain regions. There is no difference in AmTRP-expressing cells among worker, queen, and drone brains, suggesting that the cell types that express the prepro-AmTRP gene do not change according to division of labor, sex, or caste. The unique expression pattern of the prepro-AmTRP gene suggests that AmTRPs function as neuromodulators in the MBs of the honeybee brain.

Identification of GYRKPPFNGSIFamide (crustacean-SIFamide) in the crayfish Procambarus clarkii by topological mass spectrometry analysis.

A new concept relating to the purification protocol for biological proteins and peptides has been designed as "topological mass spectrometry analysis," in combination with MALDI-TOF MS using slices of tissues, chromatographic purification from the extract of tissues, molecular cloning for the determination of the precursor structure, and capillary LC-MS/MS analysis for elucidation of its posttranslational modifications. In an actual application, we identified an alpha-amidated neuropeptide from the red swamp crayfish (Procambarus clarkii) brain. Initially, an MS number of around m/z 1382 was found by the direct MALDI-TOF MS analysis with slices of the accessory lobe of the brain. After two steps of reversed-phase HPLC separation with brain extract, the structure of a 1381 Da peptide was sequenced to the GYRKPPFNGSIFamide (named crustacean-SIFamide). Subsequently, the cDNA has been characterized and encodes a 76 amino acid precursor protein that contains a signal sequence, one copy of GYRKPPFNGSIFG and one additional peptide. The RT-PCR analysis implied that the mRNA of the neuropeptide was expressed throughout the nervous system of the crayfish. Furthermore, immunostaining demonstrated that the neuropeptide is distributed in the olfactory lobe, accessory lobe, olfactory globular tract, and olfactory lobe cells. In addition, database searches revealed that there are homologous sequences of the AYRKPPFNGSIFamide in the genome library of fruitfly Drosophila melanogaster and AYRKPPFNGSLFamide isolated from the grey fleshfly Neobellieria bullata, and GYRKPPFNGSIFamide isolated from the giant tiger prawn Penaeus monodon. These results suggested that the neuropeptide family might be widely distributed in arthropods and plays a significant role in the nervous system.

Cloning and expression of a cDNA for a putative G protein-coupled receptor from the hepatopancreas of the crayfish, Procambarus clarkii.

The authors cloned a novel cDNA encoding a putative G protein-coupled receptor (GPCR) from the hepatopancreas of the crayfish, Procambarus clarkii, using a screening approach with synthetic oligonucleotides. The oligonucleotides were designed homologous to the transmembrane spanning domain III of previously cloned receptors from various living organisms. Sequence analysis revealed that one of the positive clones contained a cDNA insertion of 3489 bp representing the mRNA coding for a part of a putative GPCR (termed HP1R). The clone was truncated at the 5' end. The long 3'-untranslated regions (UTR) of 2446 bp contained three typical AATAAA censensus sequences for mRNA polyadenylation followed by the poly(A) tail at the 3' end. To obtain a full-length cDNA clone, rapid amplification of the 5' cDNA ends (5' RACE) technique was then applied. Sequence analysis revealed that the full-length cDNA clone had an open-reading frame of 1116 bp with a 103-bp 5'-UTR. The predicted amino acid sequence of HP1R was 372 residues long. Hydropathicity analysis of HP1R suggested the presence of seven transmembrane domains. The database search revealed that the predicted sequence is most closely related to probable GPCR AH9.1 from Caenorhabditis elegans (27% identity, 48% homology). In addition, HP1R has lower homologies with receptors for somatostatin, opioid, dopamine, adrenalin, and so on. The similarity implied that HP1R is a new member of putative GPCRs whose endogenous ligands were unknown. In addition, RT-PCR analysis suggested that the transcript was expressed predominantly in the hepatopancreas but poorly in the muscle or brain.