title: Characterization of 5-HT₁A receptors and their complexes with G-proteins in budded baculovirus particles using fluorescence anisotropy of Bodipy-FL-NAN-190.
abstract: Bodipy-FL-NAN-190 was found to be well suited for characterization of ligand binding to 5-HT1A receptors expressed in budded baculovirus particles, as binding is accompanied by large increases in fluorescence intensity and anisotropy. This ligand appears to bind rapidly (t1/2,ass<1 min), reversibly (t1/2,diss∼6 min) and has high affinity (Kd=0.30 ± 0.13 nM). This fluorescence anisotropy assay based on Bodipy-FL-NAN-190 binding to baculovirus particles was also a suitable assay system for the pharmacological characterization of non-labelled serotonergic ligands, as well as being sensitive to the presence of G-proteins and guanine nucleotides. Coexpression of αi subunits of human G-proteins in baculovirus particles resulted in the appearance of significantly greater proportion of nucleotide sensitive high affinity agonist binding sites. There were no significant differences between αi1 and αi3 subtypes, while ligand binding in the presence of αi2 had higher sensitivity to GDP and Mn(2+).
authors: University of Tartu, Institute of Chemistry, Ravila 14a, 50411 Tartu, Estonia.
authors: Lauri
authors: L
authors: Tõntson
authors: University of Tartu, Institute of Chemistry, Ravila 14a, 50411 Tartu, Estonia; Competence Centre on Reproductive Medicine & Biology, Tiigi 61b, 50410 Tartu, Estonia.
authors: Sergei
authors: S
authors: Kopanchuk
authors: University of Tartu, Institute of Chemistry, Ravila 14a, 50411 Tartu, Estonia; Competence Centre on Reproductive Medicine & Biology, Tiigi 61b, 50410 Tartu, Estonia. Electronic address: ago.rinken@ut.ee.
authors: Ago
authors: A
authors: Rinken
mesh_terms: D016367:Baculoviridae / Q000378:metabolism*; D001896:Boron Compounds / Q000737:chemistry*; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D011485:Protein Binding; D044282:Receptor, Serotonin, 5-HT1A / Q000378:metabolism*
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D005456:Fluorescent Dyes; D044282:Receptor, Serotonin, 5-HT1A; D019204:GTP-Binding Proteins
keywords: 5-HT(1A) receptors; Bodipy-FL-NAN-190; Budded baculovirus particles; Fluorescence anisotropy; Manganese