Synthesis,conformation, receptor binding and biological activities of monobiotinylated human insulin‐like peptide 3*

Abstract: 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 N^α‐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 α‐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.