Abstract
The relaxin-like factor (RLF) is thought to be responsible for the intra-abdominal migration of the testis during mammalian development. Our latest studies of RLF and LGR8 have revealed that the N-terminal region of the A chain is not required for receptor binding but is indispensable for cyclic AMP generation. RLF derivatives with six residues deleted from the N terminus of the A chain are active, whereas further truncation, up to the first A chain cysteine (A-10), yields tightly binding ligands devoid of signaling activity. These derivatives are specific competitive inhibitors (RLFi) of RLF. Although receptor binding is dependent upon B chain residues, the N-terminal region of the A chain is a generic trigger of the trans-membrane signaling activity.
Highlights
The relaxin-like factor (RLF),1 the product of the Insl3 gene (1), appears to be crucial for testicular descent in human neonates
Our latest studies of RLF and LGR8 have revealed that the N-terminal region of the A chain is not required for receptor binding but is indispensable for cyclic AMP generation
Receptor binding is dependent upon B chain residues, the N-terminal region of the A chain is a generic trigger of the trans-membrane signaling activity
Summary
Materials—Fmoc amino acid derivatives were purchased from either Advanced ChemTech (Louisville, KY) or Novabiochem. Each RLF chain was purified by reversed-phase HPLC on a Rainin C18-column (41.4 mm ϫ 250 mm) using 0.1% trifluoroacetic acid in water (solvent A) and 0.1% trifluoroacetic acid in 83% acetonitrile (solvent B) at a flow rate of 40 ml/min. The methionine sulfoxide (B-5) (1.25 mol) was reduced with 800 l of 50 mM ammonium iodide in 90% trifluoroacetic acid for 15 min on ice. The reaction was quenched by addition of 10 mM ascorbic acid in water (5 ml), and the peptide was isolated by gel filtration on Sephadex G25 sf in 1 M acetic acid, followed by HPLC purification on a Jupiter C18-column. The reaction was quenched by the addition of 50 l of 0.1% trifluoroacetic acid in water and was subjected to analytical HPLC on Aquapore 300 using a 40-min linear gradient from 0 to 40% B. Assays were performed in duplicate, and 2–4 independent assays were averaged (Ϯ S.E.)
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