Fig. 4.

Binding properties of OS₂ and its mutants to M- and N-type receptors measured by competition assays with iodinated WT OS₂. Competition experiments between ¹²⁵I-OS₂ and unlabeled WT recombinant OS₂ (*), the mutant H48Q (▯) and the C-terminal chimera (↓) for binding to (A) the cloned rabbit M-type receptor expressed in HEK 293 cells, (B) N-type receptors from mouse brain membranes, and (C) N-type like receptors from HeLa P4 cell membranes. Membranes were incubated with ¹²⁵I-OS₂ and various concentrations of unlabeled competitors. Results are expressed as percentage of the ¹²⁵I-OS₂ specific binding measured in the absence of competitor. The membrane concentration was adjusted to obtain a specific binding of 10–20% of the total radiolabeled ligand added (55 pM). The non specific binding was measured in the presence of 100 nM unlabeled OS₂ and was below 10% of total binding.