title: Analysis of high-affinity assembly for AMPA receptor amino-terminal domains.
abstract: Analytical ultracentrifugation (AUC) and steady-state fluorescence anisotropy were used to measure the equilibrium dissociation constant (Kd) for formation of dimers by the amino-terminal domains (ATDs) of the GluA2 and GluA3 subtypes of AMPA receptor. Previous reports on GluA2 dimerization differed in their estimate of the monomer-dimer Kd by a 2,400-fold range, with no consensus on whether the ATD forms tetramers in solution. We find by sedimentation velocity (SV) analysis performed using absorbance detection a narrow range of monomer-dimer Kd values for GluA2, from 5 to 11 nM for six independent experiments, with no detectable formation of tetramers and no effect of glycosylation or the polypeptide linker connecting the ATD and ligand-binding domains; for GluA3, the monomer-dimer Kd was 5.6 µM, again with no detectable tetramer formation. For sedimentation equilibrium (SE) experiments, a wide range of Kd values was obtained for GluA2, from 13 to 284 nM, whereas for GluA3, the Kd of 3.1 µM was less than twofold different from the SV value. Analysis of cell contents after the ∼1-week centrifuge run by silver-stained gels revealed low molecular weight GluA2 breakdown products. Simulated data for SE runs demonstrate that the apparent Kd for GluA2 varies with the extent of proteolysis, leading to artificially high Kd values. SV experiments with fluorescence detection for GluA2 labeled with 5,6-carboxyfluorescein, and fluorescence anisotropy measurements for GluA2 labeled with DyLight405, yielded Kd values of 5 and 11 nM, consistent with those from SV with absorbance detection. However, the sedimentation coefficients measured by AUC using absorbance and fluorescence systems were strikingly different, and for the latter are not consistent with hydrodynamic protein models. Thus, for unknown reasons, the concentration dependence of sedimentation coefficients obtained with fluorescence detection SV may be unreliable, limiting the usefulness of this technique for quantitative analysis.
authors.affiliation: Laboratory of Cellular Imaging and Macromolecular Biophysics, Bioengineering and Physical Science Shared Resource, Section on Biophotonics, The National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, USA.
authors.forename: Huaying
authors.initials: H
authors.lastname: Zhao
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Berger
authors.forename: Patrick H
authors.initials: PH
authors.lastname: Brown
authors.forename: Janesh
authors.initials: J
authors.lastname: Kumar
authors.forename: Andrea
authors.initials: A
authors.lastname: Balbo
authors.forename: Carrie A
authors.initials: CA
authors.lastname: May
authors.forename: Ernesto
authors.initials: E
authors.lastname: Casillas
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Laue
authors.forename: George H
authors.initials: GH
authors.lastname: Patterson
authors.forename: Mark L
authors.initials: ML
authors.lastname: Mayer
authors.forename: Peter
authors.initials: P
authors.lastname: Schuck
mesh_terms: D002478:Cells, Cultured; D005454:Fluorescence Polarization; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D055503:Protein Multimerization; D018091:Receptors, AMPA / Q000737:chemistry* / Q000378:metabolism; D014461:Ultracentrifugation
chemical_list: D018091:Receptors, AMPA