Solid-phase synthesis of europium-labeled human INSL3 as a novel probe for the study of ligand-receptor interactions.
An efficient solid-phase synthesis protocol has been developed which, together with regioselective sequential formation of the three disulfide bonds, enabled the preparation of specifically monolanthanide (europium)-labeled human insulin-like peptide 3 (INSL3) for the study of its interaction with its G-protein-coupled receptor, RXFP2, via time-resolved fluorometry. A commercially available chelator, diethylene triamine pentaacetic acid (DTPA), was coupled to the N-terminus of the INSL3 A-chain on the solid phase, and then a coordination complex between europium ion and DTPA was formed using EuCl 3 to protect the chelator from production of an unidentified adduct during subsequent combination of the A- and B-chains. The labeled peptide was purified in high yield using high-performance liquid chromatography with nearly neutral pH buffers to prevent the liberation of Eu (3+) from the chelator. Using time-resolved fluorometry, saturation binding assays were undertaken to determine the binding affinity (p K d) of labeled INSL3 for RXFP2 in HEK-293T cells stably expressing RXFP2. The dissociation constant of DTPA-labeled INSL3 (9.05 +/- 0.03, n = 3) that was obtained from saturation binding experiments was comparable to that of (125)I-labeled INSL3 (9.59 +/- 0.09, n = 3). The receptor binding affinity (p K i) of human INSL3 was determined to be 9.27 +/- 0.06, n = 3, using Eu-DTPA-INSL3 as a labeled ligand, which again is similar to that obtained when (125)I-INSL3 was used as labeled ligand (9.34 +/- 0.02, n = 4). This novel lanthanide-coordinated, DTPA-labeled INSL3 has excellent sensitivity, stability, and high specific activity, properties that will be particularly beneficial in high-throughput screening of INSL3 analogues in structure-activity studies.
Howard Florey Institute, School of Chemistry, Department of Pharmacology, The University of Melbourne, Victoria 3010, Australia.
Fazel
F
Shabanpoor
Richard A
RA
Hughes
Ross A D
RA
Bathgate
Suode
S
Zhang
Denis B
DB
Scanlon
Feng
F
Lin
Mohammed Akhter
MA
Hossain
Frances
F
Separovic
John D
JD
Wade
D000595:Amino Acid Sequence; D002614:Chelating Agents / Q000737:chemistry; D002942:Circular Dichroism; D005063:Europium / Q000737:chemistry*; D006801:Humans; D007328:Insulin / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D004369:Pentetic Acid / Q000737:chemistry; D011485:Protein Binding; D011506:Proteins / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*; D013237:Stereoisomerism; D013379:Substrate Specificity
D002614:Chelating Agents; D007328:Insulin; C084797:Leydig insulin-like protein; D008024:Ligands; D011506:Proteins; C472867:RXFP2 protein, human; D043562:Receptors, G-Protein-Coupled; D005063:Europium; D004369:Pentetic Acid