Fret Analysis using Sperm-Activating Peptides Tagged with Fluorescent Proteins Reveals that Ligand-Binding Sites Exist as Clusters

Abstract

Cells can communicate even when they're far from each other; this is critical for gametes from species that reproduce through external fertilization. In sea urchins, the molecules used by egg to attract sperm are the sperm-activating peptides (SAPs), which diffuse from the external layer of eggs into the sea water. The decapeptide speract was the first SAP described and its function has been studied by purified speract or using chemically synthesized analogues. This work attempts to prepare a recombinant speract analogue with new characteristics that could allow us to investigate unknown aspects about the speract-induced signaling pathway and the interaction with its receptor. To this end, speract was fused to three fluorescent proteins (FP; FP-speract) differing in color: cyan (eCFP), yellow (mVenus) and a large Stokes shift yellow (mAmetrine) and its activity was evaluated. First, using mAmetrine-speract for competitive binding assays, we determined that the affinity of FP-speract is only 7.6-fold smaller than speract, even when the FP is twenty times larger than the decapeptide. We also tested the ability of eCFP-speract to induce sperm physiological responses by measuring membrane potential, intracellular Ca2+ concentration and intracellular pH and neither showed significant differences between 10 nM speract and 10 nM eCFP-speract. Another important feature of our analogue is that it maintains its fluorescence when is bound to its receptor. Finally and most importantly, we used eCFP-speract and mVenus-speract as a pair for Fluorescence Resonance Energy Transfer (FRET) assays and obtained positive signal upon receptor binding. This result, strongly suggests that the speract receptor exists as an oligomer (at least as a dimer) or that a single receptor has multiple binding sites. This property may explain positive and/or negative cooperatively of SAP-receptor interactions as previously reported.