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 is 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 is 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 HEK293 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 2-4 contributed to binding with the D2R peptide. EF-2 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 co-factor 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.

Single-Molecule Folding Mechanisms of the apo- and Mg2+-Bound States of Human Neuronal Calcium Sensor-1

“…Upon calcium activation, the N-domain-D2R interaction may become tighter and the conformational changes of the C-domain, mostly through binding of calcium into EF4, enable further coupling with the N-domain, which may fully expose the second binding site for GRK2. This would be consistent with evidence which suggest that occupancy of calcium in EF2 is sufficient for the NCS-1-D2R interaction (54). In this respect, an important next step to understand these processes in detail will be to directly observe and characterize the activation of NCS-1 from a predominantly Mg 2þ -bound state to the fully calcium-bound state.…”

“…Presumably, D2R and GRK2 are bound to separate NCS-1 domains. No detailed structural information on the interactions is available but biochemical and structural models have suggested different scenarios regarding the stoichiometry and domain involvement in binding (53,54). Our results indicate that the extension and folding rate of the N-domain is similar in its Mg 2þ -bound and Ca 2þ -bound states.…”

A non-cleavable hexahistidine affinity tag at the carboxyl-terminus of the HIV-1 Pr55Gag polyprotein alters nucleic acid binding properties

“…The observed anisotropies as a function of protein concentration were fit to a variety of models using an in-house non-linear least squares fitting MATLAB (Mathworks) script that minimized the difference in chi-square (χ 2 ) between the observed ( A obs ) and calculated ( A calc ) anisotropy values [10]: $${\chi }^2=\sum _{i=1}^N\left\{\frac{1}{{\sigma }^2}{\left[A_{\mathrm{italicobs},i}-A_{\mathrm{italiccalc},i}\right]}^2\right\}$$…”

Single-Molecule Folding Mechanism of an EF-Hand Neuronal Calcium Sensor

“…The EF sites were individually disabled by introducing the D73A/E84Q (EF2), D109A/E120Q (EF3), or D157A/E168Q (EF4) mutations (De Cotiis et al, 2008;Woll et al, 2011;Aravind et al, 2008;Muralidhar et al, 2005;Jeromin et al, 2004). The E. coli strain BL21(DE3) was used to express unmyristoylated human NCS1 and variants and was grown at 37 C in Luria-Bertani medium.…”