title: Electroporation-aided DNA immunization generates polyclonal antibodies against the native conformation of human endothelin B receptor.
abstract: Endothelin B receptor (ET(B)R) is a G protein-coupled receptor (GPCR) specific for endothelin peptides (including endothelin-1, ET1), which mediates a variety of key physiological functions in normal tissues, such as modulation of vasomotor tone, tissue differentiation, or cell proliferation. Moreover, ET(B)R, overexpressed in various cancer cells including melanoma, has been implicated in the growth and progression of tumors, as well as in controlling T cell homing to tumors. To gather information on receptor structure and function, antibodies are generally considered choice molecular probes, but generation of such reagents against the native conformation of GPCRs is a real technical challenge. Here, we show that electroporation-aided genetic immunization, coupled to cardiotoxin pretreatment, is a simple and very efficient method to raise large amounts of polyclonal antibodies highly specific for native human ET(B)R (hET(B)R), as assessed by both flow cytometry analysis of different stably transfected cell lines and a new and rapid cell-based enzyme-linked immunosorbent assay that we also describe. The antibodies recognized two major epitopes on hET(B)R, mapped within the N-terminal extracellular domain. They were used to reveal hET(B)R on membranes of three different human melanoma cell lines, by flow cytometry and confocal microscopy, a method that we show is more relevant than mRNA polymerase chain reaction in assessing receptor expression. In addition, ET-1 partially competed with antibodies for receptor binding. The strategy described here, thus, efficiently generated new immunological tools to further analyze the role of ET(B)R under both normal and pathological conditions, including cancers. Above all, it can now be used to raise monoclonal antibodies against hET(B)R and, more generally, against GPCRs that constitute, by far, the largest reservoir of potential pharmacological targets.
authors.affiliation: CEA, iBiTecS, SPI, Laboratoire d'Ingénierie des Anticorps pour la Santé (LIAS), Gif sur Yvette, France.
authors.forename: Bertrand
authors.initials: B
authors.lastname: Allard
authors.forename: Fabienne
authors.initials: F
authors.lastname: Priam
authors.forename: Frédérique
authors.initials: F
authors.lastname: Deshayes
authors.forename: Frédéric
authors.initials: F
authors.lastname: Ducancel
authors.forename: Didier
authors.initials: D
authors.lastname: Boquet
authors.forename: Anne
authors.initials: A
authors.lastname: Wijkhuisen
authors.forename: Jean-Yves
authors.initials: JY
authors.lastname: Couraud
mesh_terms: D000818:Animals; D000917:Antibody Formation / Q000276:immunology*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000276:immunology*; D017931:DNA Primers / Q000235:genetics; D018274:Electroporation / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D018604:Epitope Mapping; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007114:Immunization / Q000379:methods*; D051379:Mice; D018613:Microscopy, Confocal; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D044023:Receptor, Endothelin B / Q000235:genetics / Q000276:immunology*
chemical_list: D017931:DNA Primers; D044023:Receptor, Endothelin B; D004247:DNA