FRET analysis using sperm-activating peptides tagged with fluorescent proteins reveals that ligand-binding sites exist as clusters

Author:

Arcos-Hernández César1,Romero Francisco1,Sánchez-Guevara Yoloxochitl1,Beltrán Carmen1,Nishigaki Takuya1

Affiliation:

1. Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (IBT-UNAM), Av. Universidad 2001, Col. Chamilpa, Cuernavaca, Mor. 62210, Mexico

Abstract

ABSTRACT Long-range cellular communication between the sperm and egg is critical for external fertilization. Sperm-activating peptides (SAPs) are diffusible components of the outer layer of eggs in echinoderms, and function as chemoattractants for spermatozoa. The decapeptide named speract is the best-characterized sea urchin SAP. Biochemical and physiological actions of speract have been studied with purified or chemically synthesized peptides. In this work, we prepared recombinant speract fused to a fluorescent protein (FP; FP-speract) using three color variants: a cyan (eCFP), a yellow (mVenus) and a large Stokes shift yellow (mAmetrine) FP. Although these fluorescence tags are 20 times larger than speract, competitive binding experiments using mAmetrine-speract revealed that this FP-speract has binding affinity to the receptor that is comparable (7.6-fold less) to that of non-labeled speract. Indeed, 10 nmol l−1 eCFP-speract induces physiological sperm responses such as membrane potential changes and increases in intracellular pH and Ca2+ concentrations similar to those triggered by 10 nmol l−1 speract. Furthermore, FP-speract maintains its fluorescence upon binding to its receptor. Using this property, we performed fluorescence resonance energy transfer (FRET) measurements with eCFP-speract and mVenus-speract as probes and obtained a positive FRET signal upon binding to the receptor, which suggests that the speract receptor exists as an oligomer, at least as a dimer, or alternatively that a single speract receptor protein possesses multiple binding sites. This property could partially account for the positive and/or negative cooperative binding of speract to the receptor.

Funder

Dirección General de Asuntos del Personal Académico - Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica

Consejo Nacional de Ciencia y Tecnología

Publisher

The Company of Biologists

Subject

Insect Science,Molecular Biology,Animal Science and Zoology,Aquatic Science,Physiology,Ecology, Evolution, Behavior and Systematics

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