Journal: Journal of Biological Chemistry

Article Title: Identification of Relaxin-3/INSL7 as an Endogenous Ligand for the Orphan G-protein-coupled Receptor GPCR135

doi: 10.1074/jbc.m308995200

Figure Lengend Snippet: FIG. 1. Identification and charac- terization of GPCR135 ligand activity in rat brain extracts. A, GPCR135 li- gand activity in different rat tissues. Ex- tracted rat tissues at the dilutions shown were used as ligands in the GTPS bind- ing assay using human GPCR135 ex- pressing cell membrane. B, molecular weight characterization of GPCR135 li- gand from rat brain extract. Crude rat brain ethanol/HCl extract was run through a HPLC gel filtration column. Fractions were collected and assayed for GPCR135 ligand activity in GTPS bind- ing assays using human GPCR135 ex- pressing cell membranes. In a parallel ex- periment, peptides and nucleotides with known molecular weights were run using the same conditions to serve as the molec- ular mass standards.

Article Snippet: Chinese hamster ovary cells stably expressing GPCR135 and control Chinese hamster ovary cells were stimulated with buffer, 200 nM relaxin-3, 5 M forskolin, or 5 M forskolin plus 200 nM relaxin-3. cAMP from the treated cells was extracted and measured using cAMP flash plates (PerkinElmer Life Sciences).

Techniques: Activity Assay, Membrane, Molecular Weight, Filtration

Journal: Journal of Biological Chemistry

Article Title: Identification of Relaxin-3/INSL7 as an Endogenous Ligand for the Orphan G-protein-coupled Receptor GPCR135

doi: 10.1074/jbc.m308995200

Figure Lengend Snippet: FIG. 2. Purification and characterization of porcine GPCR135 ligand. After extraction, fractionation, two rounds of ion exchange, and two rounds of reverse phase chromatographies, homogeneous peptide was obtained. A, the final RP-HPLC. Fractions 39–40 showed ligand activity for GPCR135 as demonstrated in GTPS binding assays. B, GTPS binding assays for GPCR135 ligand activity in the RP-HPLC fractions. C, the porcine GPCR135 ligand in fraction 39-40 was subjected to amino acid sequence analysis. The resulting amino acid sequences are aligned to human, mouse, and rat relaxin-3. All cysteine residues are highlighted in bold letters. The three amino acids in porcine GPCR135 ligand that differ from human relaxin-3 are underlined.

Article Snippet: Chinese hamster ovary cells stably expressing GPCR135 and control Chinese hamster ovary cells were stimulated with buffer, 200 nM relaxin-3, 5 M forskolin, or 5 M forskolin plus 200 nM relaxin-3. cAMP from the treated cells was extracted and measured using cAMP flash plates (PerkinElmer Life Sciences).

Techniques: Purification, Extraction, Fractionation, Activity Assay, Binding Assay, Sequencing

Journal: Journal of Biological Chemistry

Article Title: Identification of Relaxin-3/INSL7 as an Endogenous Ligand for the Orphan G-protein-coupled Receptor GPCR135

doi: 10.1074/jbc.m308995200

Figure Lengend Snippet: FIG. 3. Expression, purification, and characterization of recombinant human relaxin-3. A, schematic diagram of the predicted amino acid sequence for the human relaxin-3 expression construct, relaxin-3RR. Arrows indicate an enterokinase cleavage site (DDDDK) and two furin cleavage sites (RWRR and RGRR). The amino acid R with the star (*) indicates the mutation made from the natural sequence of RGSR to RGRR, an artificial furin site in the construct. The numbers indicate the corresponding positions of the amino acid in the natural human relaxin-3 pre-propeptide. B, RP-HPLC analysis of purified recombinant human relaxin-3. The purified human relaxin-3 was run in a C-18 RP-HPLC analytical column in 0.1% trifluoroacetic acid with an acetonitrile (ACN) gradient. C, fractions from B were assayed for GPCR135 ligand activity in GTPS binding assays using GPCR135 expressing cell membranes.

Article Snippet: Chinese hamster ovary cells stably expressing GPCR135 and control Chinese hamster ovary cells were stimulated with buffer, 200 nM relaxin-3, 5 M forskolin, or 5 M forskolin plus 200 nM relaxin-3. cAMP from the treated cells was extracted and measured using cAMP flash plates (PerkinElmer Life Sciences).

Techniques: Expressing, Purification, Recombinant, Sequencing, Construct, Mutagenesis, Activity Assay, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Identification of Relaxin-3/INSL7 as an Endogenous Ligand for the Orphan G-protein-coupled Receptor GPCR135

doi: 10.1074/jbc.m308995200

Figure Lengend Snippet: FIG. 4. Characterization of GPCR135 using 125I-relaxin-3 as the radioligand. A, saturation isotherm binding for 125I-relaxin-3 and GPCR135. GPCR135 expressing cell membranes were used in binding assays with different concentrations of 125I-relaxin-3 as the radioligand. Nonspecific binding was determined using the same conditions in the presence of 5 M unlabeled relaxin-3. B, competition binding analysis of GPCR135. GPCR135 expressing cell membranes were used to perform radioligand binding assays with 100 pM 125I-relaxin-3 as the radioligand. Different unlabeled peptides at various concentrations were used as competitors.

Article Snippet: Chinese hamster ovary cells stably expressing GPCR135 and control Chinese hamster ovary cells were stimulated with buffer, 200 nM relaxin-3, 5 M forskolin, or 5 M forskolin plus 200 nM relaxin-3. cAMP from the treated cells was extracted and measured using cAMP flash plates (PerkinElmer Life Sciences).

Techniques: Binding Assay, Expressing

Journal: Journal of Biological Chemistry

Article Title: Identification of Relaxin-3/INSL7 as an Endogenous Ligand for the Orphan G-protein-coupled Receptor GPCR135

doi: 10.1074/jbc.m308995200

Figure Lengend Snippet: FIG. 5. Functional characterization of GPCR135 using relaxin-3 and related peptides as ligands. A, relaxin-3 stimulates 35S-GTPS binding in GPCR135 expressing cells. Different peptides were added at various concentrations to the human GPCR135 expressing cell membranes to stimulate GTPS incorporation. The specific 35S-GTPS incorporation was obtained by subtracting counts without ligand from the counts with ligand. B, inhibition of forskolin-stimulated cAMP production by relaxin-3. Chinese hamster ovary cells stably expressing GPCR135 and control Chinese hamster ovary cells were stimulated with buffer, 200 nM relaxin-3, 5 M forskolin, or 5 M forskolin plus 200 nM relaxin-3. cAMP from the treated cells was extracted and measured using cAMP flash plates (PerkinElmer Life Sciences). C, dose response of relaxin-3 inhibition of cAMP production in GPCR135 expressing cells. Chinese hamster ovary cells stably expressing GPCR135 were stimulated with different concentrations of peptides, including relaxin-3, at various concentrations. Forskolin was then added to all samples at a final concentration of 5 M. cAMP from the stimulated cells was extracted and measured using cAMP flash plates. D, relaxin-3 stimulates Ca2 mobilization in HEK293 cells co-expressing GPCR135 and Gqi5. HEK293 cells, either mock transfected (293), transfected with Gqi5 (293/Gqi5), human GPCR135 (GPCR135), or co-transfected with human GPCR135 and Gqi5 (GPCR135/Gqi5), were used for Ca2 mobilization assays. Relaxin-3 stimulated intracellular Ca2 mobilization was monitored by FLIPR.

Article Snippet: Chinese hamster ovary cells stably expressing GPCR135 and control Chinese hamster ovary cells were stimulated with buffer, 200 nM relaxin-3, 5 M forskolin, or 5 M forskolin plus 200 nM relaxin-3. cAMP from the treated cells was extracted and measured using cAMP flash plates (PerkinElmer Life Sciences).

Techniques: Functional Assay, Binding Assay, Expressing, Inhibition, Stable Transfection, Control, Concentration Assay, Transfection

Journal: Journal of Biological Chemistry

Article Title: Identification of Relaxin-3/INSL7 as an Endogenous Ligand for the Orphan G-protein-coupled Receptor GPCR135

doi: 10.1074/jbc.m308995200

Figure Lengend Snippet: FIG. 6. A, RT-PCR detection of GPCR135 and relaxin-3 mRNA expression profiles in different human tissues. The PCR products were run in 2% agarose gels, stained with ethidium bromide, and visualized under UV irradiation. In parallel, PCRs amplifying human -actin cDNA served as the controls. B–E, coronal brain sections showing expression of GPCR135 and relaxin-3 mRNA by in situ hybridization. B, autoradiogram shows that GPCR135 mRNA distribution is distinct in the paraventricular nucleus (PVN) and supraoptic nucleus (SON). C, brightfield photomicrograph of the paraventricular nucleus showing expression of GPCR135 mRNA. Strong hybridization signal is seen as dark grains. Magnification 20. D, relaxin-3 mRNA distribution in the central gray and nucleus incertus. E, brightfield photomicrograph of central gray (CG) and nucleus incertus (NI) showing expression of relaxin-3 mRNA. Magnification 2. B and D in the figure are pseudocolor images from autoradiograms processed on the Fuji Film Bio-Imaging Analyzer System. Colors represent relative levels of hybridization densities with the rank order of red yellow green blue black.

Article Snippet: Chinese hamster ovary cells stably expressing GPCR135 and control Chinese hamster ovary cells were stimulated with buffer, 200 nM relaxin-3, 5 M forskolin, or 5 M forskolin plus 200 nM relaxin-3. cAMP from the treated cells was extracted and measured using cAMP flash plates (PerkinElmer Life Sciences).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Staining, Irradiation, In Situ Hybridization, Hybridization, Imaging