title: Interaction between the D2 dopamine receptor and neuronal calcium sensor-1 analyzed by fluorescence anisotropy.
abstract: Neuronal calcium sensor-1 (NCS-1) is a small calcium binding protein that plays a key role in the internalization and desensitization of activated D2 dopamine receptors (D2Rs). Here, we have used fluorescence anisotropy (FA) and a panel of NCS-1 EF-hand variants to interrogate the interaction between the D2R and NCS-1. Our data are consistent with the following conclusions. (1) FA titration experiments indicate that at low D2R peptide concentrations calcium-loaded NCS-1 binds to the D2R peptide in a monomeric form. At high D2R peptide concentrations, the FA titration data are best fit by a model in which the D2R peptide binds two NCS-1 monomers sequentially in a cooperative fashion. (2) Competition FA experiments in which unlabeled D2R peptide was used to compete with labeled peptide for binding to NCS-1 shifted titration curves to higher NCS-1 concentrations, suggesting that the binding of NCS-1 to the D2R is highly specific and that binding occurs in a cooperative fashion. (3) N-Terminally myristoylated NCS-1 dimerizes in a calcium-dependent manner. (4) Co-immunoprecipitation experiments in HEK-293 confirm that NCS-1 can oligomerize in cell lysates and that oligomerization is dependent on calcium binding and requires functionally intact EF-hand domains. (5) Ca(2+)/Mg(2+) FA titration experiments revealed that NCS-1 EF-hands 2-4 (EF2-4) contributed to binding with the D2R peptide. EF2 appears to have the highest affinity for Ca(2+), and occupancy of this site is sufficient to promote high-affinity binding of the NCS-1 monomer to the D2R peptide. Magnesium ions may serve as a physiological cofactor with calcium for NCS-1-D2R binding. Finally, we propose a structural model that predicts that the D2R peptide binds to the first 60 residues of NCS-1. Together, our results support the possibility of using FA to screen for small molecule drugs that can specifically block the interaction between the D2R and NCS-1.
authors.affiliation: Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Woll
authors.forename: Dan A
authors.initials: DA
authors.lastname: De Cotiis
authors.forename: Maria C
authors.initials: MC
authors.lastname: Bewley
authors.forename: Diana M
authors.initials: DM
authors.lastname: Tacelosky
authors.forename: Robert
authors.initials: R
authors.lastname: Levenson
authors.forename: John M
authors.initials: JM
authors.lastname: Flanagan
mesh_terms: D016880:Anisotropy; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008027:Light; D008274:Magnesium / Q000737:chemistry; D051580:Neuronal Calcium-Sensor Proteins / Q000737:chemistry* / Q000378:metabolism; D009479:Neuropeptides / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017448:Receptors, Dopamine D2 / Q000737:chemistry* / Q000378:metabolism; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides; D010455:Peptides; D017448:Receptors, Dopamine D2; C101464:frequenin calcium sensor proteins; D008274:Magnesium; D002118:Calcium