Characterization of a family of endogenous neuropeptide ligands for the G protein-coupled receptors GPR7 and GPR8 -- Data Supplement - Supporting Information -- Proceedings of the National Academy of Sciences

Supporting information for Tanka et al. (2003) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0837789100

Supporting Methods
Melanophore Pigment Aggregation Assay.
Culture of Xenopus laevis melanophores in Xenopus fibroblast-conditioned media, and DNA transfections by electroporation were done as described (1). Briefly, human GPR7 or GPR8 cDNA subcloned in pcDNA3.1 (+) expression vector (Invitrogen) was transfected into the β2-7 cell line. After 48 h, absorbance at 650 nm was measured on a 96-well microplate reader (Molecular Devices Vmax) before (Ai) and after (Af) incubation for 1 h with HPLC fractions or synthetic ligands. The equation (1-Af/Ai) was used to quantify melanosome aggregation, which is an index of the Gi-coupled signaling (2). Given the similarity of GPR7/GPR8 with opioid and somatostatin receptors (3), we examined the potential crossreactivity among these neuropeptide systems using the melanophore assay. Expression vectors for rat μ-, κ-, and δ-opioid receptors, and human somatostatin receptor (SSTR) 4 and 5 (4), were used. Synthetic somatostatin and opioid peptides were purchased from Bachem. We detected no crossreactivity of synthetic human neuropeptide B (NPB) or neuropeptide W (NPW) on rat μ-, κ-, or δ-opioid receptor, or human SSTR4 or SSTR5. Conversely, somatostatins-14 and -28, nociceptin, α- and β-endorphins, Leu- and Met-enkephalins. and dynorphin A showed no detectable activity on human GPR7 or GPR8 (data not shown). Purification of NPB.
The acid extraction of frozen bovine hypothalami (≈600 g, Pel-Freez) was performed as described (5, 6). The extract was loaded onto a C18 reverse-phase HPLC column, Vydac 218TP1022 (22 mm × 250 mm), preequilibrated with 0.1% trifluoroacetic acid (TFA). Peptides were eluted with a 100-min linear gradient of 15–45% acetonitrile (CH₃CN) in 0.1% TFA at a flow rate of 10 ml/min. Fractions were collected at 1-min intervals, and 0.5% of each fraction was subjected to the melanophore assay. Subsequent purification steps used a Vydac diphenyl column, a TosoHaas TSK-ODS120T column, a TosoHaas TSK-CN 80Ts column, an Alltech mixed-mode C18/Cation column and a Pharmacia μRPC C2/C18 column, all with 0.1% TFA /CH₃CN mobile phase. Purified peptide was subjected to the matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS, Voyager-DE, Applied Biosystems). The N-terminal amino acid sequence of the purified peptide was determined by an ABI 494 Protein Sequencer (Applied Biosystems). Cloning of NPB and NPW cDNAs.
Mouse prepro-NPB cDNA was cloned by PCR to amplify the translational open reading frame from the EST cDNA (AI430753) using the oligonucleotides 5'-AGCTCCATGGCCCGGTGTA-3' and 5'-TTCAGGGCTCAGGTGCTCT-3'. To obtain the full coding region of human orthologue cDNA, PCR was performed on Marathon-Ready cDNA (CLONTECH) with a following pair of primers: 5'-GGAATTCCATGGCCCGGTCCGCGACA-3' and 5'-GCTCTAGAGCAGGAGAGGTCCGGGCTCA-3', designed based on the Human Genome Database (AC069004). For the bovine prepro-NPB cDNA, PCR was performed on bovine spinal cord cDNA, which was generated using Marathon cDNA Adaptor kit, with 5'-AC(A/G)CTGG(C/T)GGCCGC(C/T)GCCCTGGCGCTG-3' and the AP2 primer. We also found rat, Xenopus, and zebrafish NPB orthologue cDNAs in the EST database (BG376816, AL640188, and BM571370, respectively). cDNAs of a paralogous peptide (NPW) were also found (human: AI500303, and rat: BQ134951) in the EST database. To obtain a full-length mouse prepro-NPW cDNA, 5'-RACE and 3'-RACE were performed by using poly(A)-rich RNA from mouse lung. Specific primers 5'-GACTTGCTGCAGGAACCATGG-3' and 5'-CCCAGTTCTTGTCCTAACCCT-3' were designed based on the mouse genome database (AC021445), and used for 5'-RACE and 3'-RACE, respectively. For cloning human prepro-NPW cDNA, specific primers: 5'-GGGCAGGCCAAGGCGGTTTGCTGC-3' and 5'-CTGCTCATGGGGCTGCGTCGCTCA-3' were used for 5'-RACE and 3'-RACE, respectively. Peptide Synthesis and Structural Determination.
All peptides were synthesized by a standard Fmoc solid-phase synthesis method (7) on an ABI 431A peptide synthesizer (Applied Biosystems) and purified by preparative reverse-phase HPLC. Molecular weights were confirmed by the MALDI-TOF mass spectrometric analyses (REFLEX III, Bruker, Fremont, CA). 5-Br-DL-tryptophan (Trp) and 6-Br-DL-Trp were purchased from Sigma and Biosynth AG (Switzerland), respectively, and used for peptide synthesis after Fmoc-derivatization. Brominated d- or l-Trp-containing peptides were separated on a preparative reverse-phase HPLC, and l-Trp-containing peptides were identified by susceptibility to aminopeptidase-M. Cytoplasmic Ca²⁺ Transient Assay.
Ltk- cells were stably cotransfected with the human GPR7 cDNA and the Gqi chimeric G protein α subunit cDNA (8). Cells were incubated in the loading buffer (20 mM HEPES-Hanks, pH 7.4, containing 10 mM glucose, 0.1% BSA, and 4 µM Fura-2/AM) for 2 h at 16°C. Cytoplasmic calcium levels were monitored by a CAF-110 fluorescence spectrophotometer (JASCO) as described (9). In Situ
Hybridization. Sectional in situ hybridization was performed as described (10). A 0.4-kb EcoRI-EagI fragment of the mouse NPB cDNA, a 0.45-kb coding region of the mouse NPW cDNA spanning from the mature peptide to the stop codon, and a 0.44-kb coding region of the mouse GPR7 cDNA between the second to fifth transmembrane regions were subcloned into pBluescript (Stratagene). Antisense and sense riboprobes were generated with T7 and T3 phage RNA polymerases, respectively, using the Maxiscript kit (Ambion) in the presence of ³⁵S-CTP and ³⁵S-UTP. Sense riboprobes produced no signals above background. Experimental Animals.
Male C57/BL6 mice (23–25 g, Charles River Breeding Laboratories) and male Wister rats (240–260 g, Charles River Breeding Laboratories) were housed under controlled lighting (12 h light/dark cycle; light on 08:00–20:00) and temperature (22ºC). Food (standard chow pellets) and water were available ad libitum. All experimental procedures involving animals were approved by the Institutional Animal Care and Research Advisory Committee (IACRAC) of the University of Texas Southwestern Medical Center at Dallas, and were in accordance with National Institutes of Health guidelines. Intracerebroventricular Cannulation.
Animals were anesthetized with pentobarbital (50 mg/kg, i.p.), and positioned in a stereotaxic frame (Kopf Instruments, Tujunga CA). A guide cannula was implanted into the lateral ventricle of mouse (0.2-mm posterior, 1-mm right lateral, 2.5-mm ventral from bregma) or rat (0.5-mm posterior, 1.3-mm left lateral, 4.3-mm ventral from bregma) under sterile conditions. Animals were then housed for a recovery period of at least 7 days. The position and patency of cannula was verified by the injection of human neuropeptide Y (0.3 nmol) to test for a positive orexigenic response. Food Intake.
Three nmol or 10 nmol of nonbrominated synthetic rat NPB in 3 μl of sterile water was injected in bolus into the ventricle of the mouse. In a subset of experiments, 0.3 nmol of corticotropin-releasing factor (CRF) was preadministered into the ventricle 15 min before the NPB injection. Intracerebroventricular injection was performed at the end of light phase (20:00) and the changes in food weight in the dark phase were measured at 22:00, 24:00, and 08:00 next morning, as described (5). Locomotion Analysis.
Three nanomol of nonbrominated synthetic rat NPB in 5 μl of sterile water was injected in bolus into the lateral ventricle. After injection, rats were placed in a square field (36 × 36 cm) novel to the animals, which has 15 × 15 infrared beam arrays with 2.4-cm intervals. The distance traveled was monitored for 2 h in both light and dark phases. Analgesic Effects.
For both the paw flick test and formalin test, 3 nmol of synthetic rat NPB in 5 μl of sterile water was injected in bolus into the lateral ventricle of rats during the light phase. For the paw flick test, rats were acclimatized to a clear container for 20 min after the intracerebroventricular injection of nonbrominated synthetic rat NPB. Then, a heat-producing narrow beam of light was directed at a hindpaw from the bottom. Latency (sec) for flicking paw out of the heat-producing beam was measured. In experiments with chemical inflammation, carrageenan (2 mg/150 μl) was injected into a hindpaw 3 h before the paw flick test. For the formalin test, 50 μl of 5% formalin was injected subcutaneously into the planter surface of the right hindpaw 10 min after the intracerebroventricular injection of brominated or nonbrominated synthetic rat NPB. Rats were immediately placed on a polycarbonate box for behavioral observation. The time (sec) spent for licking or biting the formalin-injected paw per 5-min intervals was recorded.

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