title: FRET analysis using sperm-activating peptides tagged with fluorescent proteins reveals that ligand-binding sites exist as clusters.
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 Ca(2+) 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.
authors.affiliation: 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.
authors.forename: César
authors.initials: C
authors.lastname: Arcos-Hernández
authors.affiliation: 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.
authors.forename: Francisco
authors.initials: F
authors.lastname: Romero
authors.affiliation: 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.
authors.forename: Yoloxochitl
authors.initials: Y
authors.lastname: Sánchez-Guevara
authors.affiliation: 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.
authors.forename: Carmen
authors.initials: C
authors.lastname: Beltrán
authors.affiliation: 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 takuya@ibt.unam.mx.
authors.forename: Takuya
authors.initials: T
authors.lastname: Nishigaki
mesh_terms: D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D005260:Female; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000235:genetics; D007700:Kinetics; D008297:Male; D008564:Membrane Potentials; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012617:Sea Urchins / Q000502:physiology*; D013050:Spectrometry, Fluorescence; D013094:Spermatozoa / Q000502:physiology
chemical_list: D005456:Fluorescent Dyes; D009842:Oligopeptides; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C035027:egg surface sperm receptor; C527701:enhanced cyan fluorescent protein; D049452:Green Fluorescent Proteins; C028364:speract; D002118:Calcium
keywords: Fluorescence resonance energy transfer; Receptor; Recombinant peptide ligand; Speract