Synthesis, conformation, receptor binding and biological activities of monobiotinylated human insulin-like peptide 3.
Biotin-avidin immobilization has been routinely used as a tool to study peptide-receptor and peptide-antibody interactions. Biotinylated peptides can also be employed to localize cells that express the peptides' receptor, and to analyse ligand-receptor binding. Insulin-like peptide 3 (INSL3) is a peptide hormone which contains A- and B-chains connected by two disulphide bonds and plays a role in testicular descent during sexual development. In order to study the interaction of INSL3 with its receptor LGR8, a G protein-coupled receptor, we chemically synthesized Nalpha-mono-biotinylated human INSL3 (B-hINSL3) and compared it structurally and biologically with hINSL3. Both peptides exhibited similar, but high, receptor binding affinities on human foetal kidney fibroblast 293T cells transfected human LGR8 based on a competition radioreceptor assay with 33P-labelled relaxin H2 (B33). The modified B-hINSL3 showed full biological activity as determined by the stimulation of gubernacular cell proliferation. The labelled B-hINSL3 contains a higher alpha-helix content, and this increased helical structure is accompanied by an increase in ability to stimulate cAMP accumulation in 293T cells expressing LGR8. Our results suggest that the N-terminal region of the A-chain is not involved in the interaction of INSL3 with its receptor. However, the introduction of biotin onto the N-terminus of the A-chain promoted conformational stability which, in turn, permitted better receptor activation.
Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Victoria 3010, Australia.
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Tomiyama
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D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D019921:Biotinylation; D001973:Bromodeoxyuridine / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000096:biosynthesis; D004247:DNA / Q000378:metabolism; D005347:Fibroblasts; D006801:Humans; D007328:Insulin; D008297:Male; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology*; D051381:Rats; D043562:Receptors, G-Protein-Coupled; D018000:Receptors, Peptide / Q000187:drug effects* / Q000378:metabolism*; D012065:Relaxin / Q000378:metabolism
D007328:Insulin; C084797:Leydig insulin-like protein; D010455:Peptides; D011506:Proteins; C095068:RLN2 protein, human; D043562:Receptors, G-Protein-Coupled; D018000:Receptors, Peptide; C033395:relaxin receptors; C047086:cyclic adenosine 3',5'-diphosphate; D012065:Relaxin; D004247:DNA; D000242:Cyclic AMP; D001973:Bromodeoxyuridine