Journal: The Journal of Biological Chemistry

Article Title: The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation *

doi: 10.1074/jbc.M113.499640

Figure Lengend Snippet: Boxshade alignment of LDLa module sequences from RXFP1 receptors from various mammalian species in comparison with the sequence of LB2 of the human LDLr. The conserved cysteine residues are highlighted in red, conserved amino acids are highlighted in black, and conservative amino acid substitutions are shaded.

Article Snippet: Hence, the DNA sequence encoding the LB2 module with introduced RXFP1 LDLa residues and flanking EcoRI restriction sites was purchased from GenScript and cloned into the original pcDNA3.1/Zeo construct ( 19 ).

Techniques: Comparison, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation *

doi: 10.1074/jbc.M113.499640

Figure Lengend Snippet: RXFP1 variants and their corresponding mutation as well as the template DNA and primers used in QuikChange site-directed mutagenesis

Article Snippet: Hence, the DNA sequence encoding the LB2 module with introduced RXFP1 LDLa residues and flanking EcoRI restriction sites was purchased from GenScript and cloned into the original pcDNA3.1/Zeo construct ( 19 ).

Techniques: Mutagenesis, Construct, Sequencing, Residue

Journal: The Journal of Biological Chemistry

Article Title: The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation *

doi: 10.1074/jbc.M113.499640

Figure Lengend Snippet: H2 relaxin-induced cAMP response of RXFP1-LB2 (A) and G12del-RXFP1-LB2 and S34A-RXFP1-LB2 (B) compared with RXFP1. cAMP activity is expressed as the percentage of the 5 μm forskolin-stimulated response for each receptor and has been normalized for cell surface expression. Symbols represent means and vertical bars (not visible) represent S.E. of triplicate determinations from at least three independent experiments.

Article Snippet: Hence, the DNA sequence encoding the LB2 module with introduced RXFP1 LDLa residues and flanking EcoRI restriction sites was purchased from GenScript and cloned into the original pcDNA3.1/Zeo construct ( 19 ).

Techniques: Activity Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation *

doi: 10.1074/jbc.M113.499640

Figure Lengend Snippet: Pooled activity data for RXFP1 mutants compared with wild type receptor Competition binding data showing the pIC 50 values, receptor expression data showing the cell surface expression, and cAMP activity data showing the pEC 50 values as well as percent forskolin-stimulated maximum responses of RXFP1 and all its variants used in this study. NA, no activity.

Article Snippet: Hence, the DNA sequence encoding the LB2 module with introduced RXFP1 LDLa residues and flanking EcoRI restriction sites was purchased from GenScript and cloned into the original pcDNA3.1/Zeo construct ( 19 ).

Techniques: Activity Assay, Binding Assay, Expressing, Construct

Journal: The Journal of Biological Chemistry

Article Title: The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation *

doi: 10.1074/jbc.M113.499640

Figure Lengend Snippet: H2 relaxin-induced cAMP response of SLGYFP-RXFP1-LB2, NITK-RXFP1-LB2, LLH-RXFP1-LB2, and NGVD-RXFP1-LB2 (A) and SLGYFP-RXFP1-LB2, NITK-RXFP1-LB2, SLGYFP NITK-RXFP1-LB2, and SLGYFP NITK C-term-RXFP1-LB2 (B) compared with RXFP1. cAMP activity is expressed as the percentage of the 5 μm forskolin-stimulated response for each receptor and has been normalized for cell surface expression. Symbols represent means and vertical bars (not visible) represent S.E. of triplicate determinations from at least three independent experiments.

Article Snippet: Hence, the DNA sequence encoding the LB2 module with introduced RXFP1 LDLa residues and flanking EcoRI restriction sites was purchased from GenScript and cloned into the original pcDNA3.1/Zeo construct ( 19 ).

Techniques: Activity Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation *

doi: 10.1074/jbc.M113.499640

Figure Lengend Snippet: 1H,15N HSQC of 1 mm13C,15N-labeled SLGYFP NITK-LB2 in 50 mm imidazole, 10 mm CaCl2, 10% D2O, pH 6. 0 and 25 °C acquired at 800 MHz. Assignments of observed peptide and side chain NH resonances are indicated. Numbering is based on the chimeric LB2 module in Protein Data Bank code 2M7P.

Article Snippet: Hence, the DNA sequence encoding the LB2 module with introduced RXFP1 LDLa residues and flanking EcoRI restriction sites was purchased from GenScript and cloned into the original pcDNA3.1/Zeo construct ( 19 ).

Techniques: Labeling

Journal: The Journal of Biological Chemistry

Article Title: The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation *

doi: 10.1074/jbc.M113.499640

Figure Lengend Snippet: Input for the structure calculation and characterization of the bundle of 20 CYANA conformers of SLYFP NITK-LB2 (residues 1–42) ali, aliphatic; aro, aromatic.

Article Snippet: Hence, the DNA sequence encoding the LB2 module with introduced RXFP1 LDLa residues and flanking EcoRI restriction sites was purchased from GenScript and cloned into the original pcDNA3.1/Zeo construct ( 19 ).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation *

doi: 10.1074/jbc.M113.499640

Figure Lengend Snippet: A, ensemble of the 20 lowest energy structures of SLYFP NITK-LB2. Structures are represented as an overlay of the backbone structures in MOLMOL that overlay with an r.m.s.d. of 0.35 Å between residues 6 and 42. The side chains from the two “add-back” regions SLYFP and NITK have been represented as lines. B, overlay of SLYFP NITK-LB2 (blue) onto the structure of RXFP1 LDLa (orange) (Protein Data Bank code 2JM4). The side chains of Leu-7, Tyr-9, and Lys-17 overlay with good agreement. The overall r.m.s.d. of the two structures between residues 6 and 42 is 1.68 Å.

Article Snippet: Hence, the DNA sequence encoding the LB2 module with introduced RXFP1 LDLa residues and flanking EcoRI restriction sites was purchased from GenScript and cloned into the original pcDNA3.1/Zeo construct ( 19 ).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation *

doi: 10.1074/jbc.M113.499640

Figure Lengend Snippet: Overlay of the mean structure of SLYFP NITK-LB2 (blue), RXFP1 LDLa (orange), and LB2 (pink) represented as a cartoon model. The side chains of Lys-17 and equivalent Arg in LB2 are represented as sticks in addition to Asn-33 (RXFP1) and the equivalent Asn-32 from LB2. The overlay demonstrates that the chimera is more “RXFP1”-like despite the LB2 scaffold.

Article Snippet: Hence, the DNA sequence encoding the LB2 module with introduced RXFP1 LDLa residues and flanking EcoRI restriction sites was purchased from GenScript and cloned into the original pcDNA3.1/Zeo construct ( 19 ).

Techniques: