Characterisation of the Interaction of the C-Terminus of the Dopamine D2 Receptor with Neuronal Calcium Sensor-1

Abstract

NCS-1 is a member of the neuronal calcium sensor (NCS) family of EF-hand Ca2+ binding proteins which has been implicated in several physiological functions including regulation of neurotransmitter release, membrane traffic, voltage gated Ca2+ channels, neuronal development, synaptic plasticity, and learning. NCS-1 binds to the dopamine D2 receptor, potentially affecting its internalisation and controlling dopamine D2 receptor surface expression. The D2 receptor binds NCS-1via a short 16-residue cytoplasmic C-terminal tail. We have used NMR and fluorescence spectroscopy to characterise the interactions between the NCS-1/Ca2+ and D2 peptide. The data show that NCS-1 binds D2 peptide with a Kd of ∼14.3 µM and stoichiometry of peptide binding to NCS-1 of 2∶1. NMR chemical shift mapping confirms that D2 peptide binds to the large, solvent-exposed hydrophobic groove, on one face of the NCS-1 molecule, with residues affected by the presence of the peptide spanning both the N and C-terminal portions of the protein. The NMR and mutagenesis data further show that movement of the C-terminal helix 11 of NCS-1 to fully expose the hydrophobic groove is important for D2 peptide binding. Molecular docking using restraints derived from the NMR chemical shift data, together with the experimentally-derived stoichiometry, produced a model of the complex between NCS-1 and the dopamine receptor, in which two molecules of the receptor are able to simultaneously bind to the NCS-1 monomer.

The guanine-exchange factor Ric8a binds the calcium sensor NCS-1 to regulate synapse number and probability of release

“…In the presence of Ric8, the target will displace the H10 helix and proper insertion will occur (supplementary material Movie 2). A similar mechanism has been proposed for yeast and human Frq and KChIP1 interacting with their corresponding targets, PI4K3b (Ames et al, 2000;Strahl et al, 2007), dopamine D2/D3 receptors (Lian et al, 2011) and the Kv4.3 channel (Pioletti et al, 2006;Wang et al, 2007). It is plausible that the transient occlusion of the crevice by H10 will serve as a mechanism to quantitatively regulate the probability of binding to Ric8a, as suggested for human Frq (Heidarsson et al, 2012) and similar to the chain-and-ball model of voltagedependent K + channels for its open-closed status (Fan et al, 2012).…”

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

“…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.…”

The Structure of Neuronal Calcium Sensor-1 in Solution Revealed by Molecular Dynamics Simulations

“…In this sense, we propose that a mutation that consists in replacing Arg102 with an uncharged residue could affect the mobility of H9 and L3, compromising the docking process of L3 into the HC. In fact, it is known that the substitution of Arg102 with glutamine affects the structural and dynamical features of the C-terminal tail, providing a molecular counterpart to the hypothesis that the Arg102Gln mutation is implicated in the autism disease [10], [19], [29].…”

“…NCS proteins bind target ligands trough a conserved hydrophobic binding pocket, which can differ in shape and size to regulate the target specificity [1], [3], [17], [18], [29]. However, to guarantee the target specificity, each member of the NCS family seems to use multiple regulatory mechanisms.…”