Interaction between the D2 Dopamine Receptor and Neuronal Calcium Sensor-1 Analyzed by Fluorescence Anisotropy

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• 作者：Matthew P. Woll ; Dan A. De Cotiis ; Maria C. Bewley ; Diana M. Tacelosky ; Robert Levenson ; John M. Flanagan
• 刊名：Biochemistry
• 出版年：2011
• 出版时间：October 18, 2011
• 年：2011
• 卷：50
• 期：41
• 页码：8780-8791
• 全文大小：449K
• 年卷期：v.50,no.41(October 18, 2011)
• ISSN：1520-4995

文摘

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) Ca²⁺/Mg²⁺ FA titration experiments revealed that NCS-1 EF-hands 2鈥? (EF2鈥?) contributed to binding with the D2R peptide. EF2 appears to have the highest affinity for Ca²⁺, 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鈥揇2R 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.