title: Methods to immobilize GPCR on the surface of SPR sensors.
abstract: The G protein-coupled receptors (GPCRs) form one of the largest membrane receptor families. The nature of the ligands that interact with these receptors is highly diverse; they include light, peptides and hormones, neurotransmitters, and small molecular weight compounds. The GPCRs are involved in a wide variety of physiological processes and thus hold considerable therapeutic potential.GPCR function is usually determined in cell-based assays, whose complexity nonetheless limits their use. The use of alternative, cell-free assays is hampered by the difficulties in purifying these seven-transmembrane domain receptors without altering their functional properties. Several methods have been proposed to immobilize GPCR on biosensor surfaces which use antibodies or avidin-/biotin-based capture procedures, alone or with reconstitution of the GPCR physiological microenvironment. Here we propose a method for GPCR immobilization in their native membrane microenvironment that requires no manipulation of the target receptor and maintains the many conformations GPCR can adopt in the cell membrane.
authors.affiliation: Department of Immunology and Oncology, Centro Nacional de Biotecnología (CNB/CSIC), Darwin 3, Campus de Cantoblanco, Madrid, 28049, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Martínez-Muñoz
authors.forename: Rubén
authors.initials: R
authors.lastname: Barroso
authors.forename: Anabel Guedán
authors.initials: AG
authors.lastname: Paredes
authors.forename: Mario
authors.initials: M
authors.lastname: Mellado
authors.forename: José Miguel
authors.initials: JM
authors.lastname: Rodríguez-Frade
mesh_terms: D002232:Carbocyanines; D005456:Fluorescent Dyes; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D055767:Immobilized Proteins / Q000235:genetics* / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D019718:Receptors, CXCR4 / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods; D014162:Transfection; D018116:Vesiculovirus / Q000235:genetics* / Q000378:metabolism; D014771:Virion; D019065:Virus Assembly / Q000235:genetics
chemical_list: C527276:CXCR4 protein, human; D002232:Carbocyanines; D005456:Fluorescent Dyes; D055767:Immobilized Proteins; D019718:Receptors, CXCR4; D011994:Recombinant Proteins; C089187:cyanine dye 3; D049452:Green Fluorescent Proteins