NCS-1 protein regulates TRPA1 channel through the PI3K pathway in breast cancer and neuronal cells.

The physical and functional interaction between transient receptor potential channel ankyrin 1 (TRPA1) and neuronal calcium sensor 1 (NCS-1) was assessed. NCS-1 is a calcium (Ca2+) sensor found in many tissues, primarily neurons, and TRPA1 is a Ca2+ channel involved not only in thermal and pain sensation but also in conditions such as cancer and chemotherapy-induced peripheral neuropathy, in which NCS-1 is also a regulatory component.We explored the interactions between these two proteins by employing western blot, qRT-PCR, co-immunoprecipitation, Ca2+ transient monitoring with Fura-2 spectrophotometry, and electrophysiology assays in breast cancer cells (MDA-MB-231) with different levels of NCS-1 expression and neuroblastoma cells (SH-SY5Y).Our findings showed that the expression of TRPA1 was directly correlated with NCS-1 levels at both the protein and mRNA levels. Additionally, we found a physical and functional association between these two proteins. Physically, the NCS-1 and TRPA1 co-immunoprecipitate. Functionally, NCS-1 enhanced TRPA1-dependent Ca2+ influx, current density, open probability, and conductance, where the functional effects depended on PI3K. Conclusion: NCS-1 appears to act not only as a Ca2+ sensor but also modulates TRPA1 protein expression and channel function in a direct fashion through the PI3K pathway. These results contribute to understanding how Ca2+ homeostasis is regulated and provides a mechanism underlying conditions where Ca2+ dynamics are compromised, including breast cancer. With a cellular pathway identified, targeted treatments can be developed for breast cancer and neuropathy, among other related diseases.

[Neuronal Calcium Sensor-1: A Zinc/Redox-Dependent Protein of Nervous System Signaling Pathways].

Intracellular calcium signaling is involved in regulating the key functional mechanisms of the nervous system. The control of neuronal excitability and plasticity by calcium ions underlies the mechanisms of higher nervous activity, and the mechanisms of this control are of particular interest to researchers. A family of highly specialized neuronal proteins described in recent decades can translate the information contained in calcium signals into the regulation of channels, enzymes, receptors, and transcription factors. Neuronal calcium sensor-1 (NCS-1) is the most common member of the family, which is intensely expressed in central nervous system (CNS) cells; and controls several vital processes, such as neuronal growth and survival, reception, neurotransmission, and synaptic plasticity. In addition to calcium ions, NCS-1 can bind the so-called mobile, or signaling intracellular zinc, an increased concentration of which is a characteristic feature of cells in oxidative stress. Zinc coordination under these conditions stimulates NCS-1 oxidation to form a disulfide dimer (dNCS-1) with altered functional properties. A combined effect of mobile zinc and an increased redox potential of the medium can thus induce aberrant NCS-1 activity, including signals that promote survival of neuronal cells or induce their apoptosis and, consequently, the development of neurodegenerative processes. The review details the localization, expression regulation, structure, and molecular properties of NCS-1 and considers the current data on its signaling activity in health and disease, including zinc-dependent redox regulation cascades.

Neuronal Calcium Sensor-1 Protects Cortical Neurons from Hyperexcitation and Ca2+ Overload during Ischemia by Protecting the Population of GABAergic Neurons.

A defection of blood circulation in the brain leads to ischemia, damage, and the death of nerve cells. It is known that individual populations of GABAergic neurons are the least resistant to the damaging factors of ischemia and therefore they die first of all, which leads to impaired inhibition in neuronal networks. To date, the neuroprotective properties of a number of calcium-binding proteins (calbindin, calretinin, and parvalbumin), which are markers of GABAergic neurons, are known. Neuronal calcium sensor-1 (NCS-1) is a signaling protein that is expressed in all types of neurons and is involved in the regulation of neurotransmission. The role of NCS-1 in the protection of neurons and especially their individual populations from ischemia and hyperexcitation has not been practically studied. In this work, using the methods of fluorescence microscopy, vitality tests, immunocytochemistry, and PCR analysis, the molecular mechanisms of the protective action of NCS-1 in ischemia/reoxygenation and hyperammonemia were established. Since NCS-1 is most expressed in GABAergic neurons, the knockdown of this protein with siRNA led to the most pronounced consequences in GABAergic neurons. The knockdown of NCS-1 (NCS-1-KD) suppressed the basic expression of protective proteins without significantly reducing cell viability. However, ischemia-like conditions (oxygen-glucose deprivation, OGD) and subsequent 24-h reoxygenation led to a more massive activation of apoptosis and necrosis in neurons with NCS-1-KD, compared to control cells. The mass death of NCS-1-KD cells during OGD and hyperammonemia has been associated with the induction of a more pronounced network hyperexcitation symptom, especially in the population of GABAergic neurons, leading to a global increase in cytosolic calcium ([Ca2+]i). The symptom of hyperexcitation of neurons with NCS-1-KD correlated with a decrease in the level of expression of the calcium-binding protein-parvalbumin. This was accompanied by an increase in the expression of excitatory ionotropic glutamate receptors, N-methyl-D-aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (NMDAR and AMPAR) against the background of suppression of the expression of glutamate decarboxylase (synthesis of γ-aminobutyric acid).

Nanomolar affinity of EF-hands in neuronal calcium sensor 1 for bivalent cations Pb2+, Mn2+, and Hg2.

Abiogenic metals Pb and Hg are highly toxic since chronic and/or acute exposure often leads to severe neuropathologies. Mn2+ is an essential metal ion but in excess can impair neuronal function. In this study, we address in vitro the interactions between neuronal calcium sensor 1 (NCS1) and divalent cations. Results showed that non-physiological ions (Pb2+ and Mn2+) bind to EF-hands in NCS1 with nanomolar affinity and lower equilibrium dissociation constant than the physiological Ca2+ ion. (Kd, Pb2+ = 7.0 ± 1.0 nM; Kd, Mn2+ = 34.0 ± 6.0 nM; K). Native ultra-high resolution mass spectrometry (FT-ICR MS) and trapped ion mobility spectrometry-mass spectrometry (nESI-TIMS-MS) studies provided the NCS1-metal complex compositions-up to four Ca2+ or Mn2+ ions and three Pb2+ ions (M⋅Pb1-3Ca1-3, M⋅Mn1-4Ca1-2, and M⋅Ca1-4) were observed in complex-and similarity across the mobility profiles suggests that the overall native structure is preserved regardless of the number and type of cations. However, the non-physiological metal ions (Pb2+, Mn2+, and Hg2+) binding to NCS1 leads to more efficient quenching of Trp emission and a decrease in W30 and W103 solvent exposure compared to the apo and Ca2+ bound form, although the secondary structural rearrangement and exposure of hydrophobic sites are analogous to those for Ca2+ bound protein. Only Pb2+ and Hg2+ binding to EF-hands leads to the NCS1 dimerization whereas Mn2+ bound NCS1 remains in the monomeric form, suggesting that other factors in addition to metal ion coordination, are required for protein dimerization.

Boosting ER-mitochondria calcium transfer to treat Wolfram syndrome.

Wolfram syndrome is a rare genetic disorder characterized by endocrine dysfunction and progressive neurodegeneration. By targeting intracellular calcium dysregulations, a sigma-1 receptor agonist rescued neurological deficits in preclinical models of Wolfram syndrome.

Disulfide Dimerization of Neuronal Calcium Sensor-1: Implications for Zinc and Redox Signaling.

Neuronal calcium sensor-1 (NCS-1) is a four-EF-hand ubiquitous signaling protein modulating neuronal function and survival, which participates in neurodegeneration and carcinogenesis. NCS-1 recognizes specific sites on cellular membranes and regulates numerous targets, including G-protein coupled receptors and their kinases (GRKs). Here, with the use of cellular models and various biophysical and computational techniques, we demonstrate that NCS-1 is a redox-sensitive protein, which responds to oxidizing conditions by the formation of disulfide dimer (dNCS-1), involving its single, highly conservative cysteine C38. The dimer content is unaffected by the elevation of intracellular calcium levels but increases to 10-30% at high free zinc concentrations (characteristic of oxidative stress), which is accompanied by accumulation of the protein in punctual clusters in the perinuclear area. The formation of dNCS-1 represents a specific Zn2+-promoted process, requiring proper folding of the protein and occurring at redox potential values approaching apoptotic levels. The dimer binds Ca2+ only in one EF-hand per monomer, thereby representing a unique state, with decreased α-helicity and thermal stability, increased surface hydrophobicity, and markedly improved inhibitory activity against GRK1 due to 20-fold higher affinity towards the enzyme. Furthermore, dNCS-1 can coordinate zinc and, according to molecular modeling, has an asymmetrical structure and increased conformational flexibility of the subunits, which may underlie their enhanced target-binding properties. In HEK293 cells, dNCS-1 can be reduced by the thioredoxin system, otherwise accumulating as protein aggregates, which are degraded by the proteasome. Interestingly, NCS-1 silencing diminishes the susceptibility of Y79 cancer cells to oxidative stress-induced apoptosis, suggesting that NCS-1 may mediate redox-regulated pathways governing cell death/survival in response to oxidative conditions.

A Novel Approach to Bacterial Expression and Purification of Myristoylated Forms of Neuronal Calcium Sensor Proteins.

N-terminal myristoylation is a common co-and post-translational modification of numerous eukaryotic and viral proteins, which affects their interaction with lipids and partner proteins, thereby modulating various cellular processes. Among those are neuronal calcium sensor (NCS) proteins, mediating transduction of calcium signals in a wide range of regulatory cascades, including reception, neurotransmission, neuronal growth and survival. The details of NCSs functioning are of special interest due to their involvement in the progression of ophthalmological and neurodegenerative diseases and their role in cancer. The well-established procedures for preparation of native-like myristoylated forms of recombinant NCSs via their bacterial co-expression with N-myristoyl transferase from Saccharomyces cerevisiae often yield a mixture of the myristoylated and non-myristoylated forms. Here, we report a novel approach to preparation of several NCSs, including recoverin, GCAP1, GCAP2, neurocalcin δ and NCS-1, ensuring their nearly complete N-myristoylation. The optimized bacterial expression and myristoylation of the NCSs is followed by a set of procedures for separation of their myristoylated and non-myristoylated forms using a combination of hydrophobic interaction chromatography steps. We demonstrate that the refolded and further purified myristoylated NCS-1 maintains its Са2+-binding ability and stability of tertiary structure. The developed approach is generally suited for preparation of other myristoylated proteins.

Membrane Binding of Neuronal Calcium Sensor-1: Highly Specific Interaction with Phosphatidylinositol-3-Phosphate.

Neuronal calcium sensors are a family of N-terminally myristoylated membrane-binding proteins possessing a different intracellular localization and thereby targeting unique signaling partner(s). Apart from the myristoyl group, the membrane attachment of these proteins may be modulated by their N-terminal positively charged residues responsible for specific recognition of the membrane components. Here, we examined the interaction of neuronal calcium sensor-1 (NCS-1) with natural membranes of different lipid composition as well as individual phospholipids in form of multilamellar liposomes or immobilized monolayers and characterized the role of myristoyl group and N-terminal lysine residues in membrane binding and phospholipid preference of the protein. NCS-1 binds to photoreceptor and hippocampal membranes in a Ca2+-independent manner and the binding is attenuated in the absence of myristoyl group. Meanwhile, the interaction with photoreceptor membranes is less dependent on myristoylation and more sensitive to replacement of K3, K7, and/or K9 of NCS-1 by glutamic acid, reflecting affinity of the protein to negatively charged phospholipids. Consistently, among the major phospholipids, NCS-1 preferentially interacts with phosphatidylserine and phosphatidylinositol with micromolar affinity and the interaction with the former is inhibited upon mutating of N-terminal lysines of the protein. Remarkably, NCS-1 demonstrates pronounced specific binding to phosphoinositides with high preference for phosphatidylinositol-3-phosphate. The binding does not depend on myristoylation and, unexpectedly, is not sensitive to the charge inversion mutations. Instead, phosphatidylinositol-3-phosphate can be recognized by a specific site located in the N-terminal region of the protein. These data provide important novel insights into the general mechanism of membrane binding of NCS-1 and its targeting to specific phospholipids ensuring involvement of the protein in phosphoinositide-regulated signaling pathways.

Mice lacking neuronal calcium sensor-1 show social and cognitive deficits.

Neuronal calcium sensor-1 or Frequenin is a calcium sensor widely expressed in the nervous system, with roles in neurotransmission, neurite outgrowth, synaptic plasticity, learning, and motivated behaviours. Neuronal calcium sensor-1 has been implicated in neuropsychiatric disorders including autism spectrum disorder, schizophrenia, and bipolar disorder. However, the role of neuronal calcium sensor-1 in behavioural phenotypes and brain changes relevant to autism spectrum disorder have not been evaluated. We show that neuronal calcium sensor-1 deletion in the mouse leads to a mild deficit in social approach and impaired displaced object recognition without affecting social interactions, behavioural flexibility, spatial reference memory, anxiety-like behaviour, or sensorimotor gating. Morphologically, neuronal calcium sensor-1 deletion leads to increased dendritic arbour complexity in the frontal cortex. At the level of hippocampal synaptic plasticity, neuronal calcium sensor-1 deletion leads to a reduction in long-term potentiation in the dentate gyrus, but not area Cornu Ammonis 1. Metabotropic glutamate receptor-induced long-term depression was unaffected in both dentate and Cornu Ammonis 1. These studies identify roles for neuronal calcium sensor-1 in specific subregions of the brain including a phenotype relevant to neuropsychiatric disorders.

NCS-1 Deficiency Is Associated With Obesity and Diabetes Type 2 in Mice.

Neuronal calcium sensor-1 (NCS-1) knockout (KO) in mice (NCS-1-/- mice) evokes behavioral phenotypes ranging from learning deficits to avolition and depressive-like behaviors. Here, we showed that with the onset of adulthood NCS-1-/- mice gain considerable weight. Adult NCS-1-/- mice are obese, especially when fed a high-fat diet (HFD), are hyperglycemic and hyperinsulinemic and thus develop a diabetes type 2 phenotype. In comparison to wild type (WT) NCS-1-/- mice display a significant increase in adipose tissue mass. NCS-1-/- adipocytes produce insufficient serum concentrations of resistin and adiponectin. In contrast to WT littermates, adipocytes of NCS-1-/- mice are incapable of up-regulating insulin receptor (IR) concentration in response to HFD. Thus, HFD-fed NCS-1-/- mice exhibit in comparison to WT littermates a significantly reduced IR expression, which may explain the pronounced insulin resistance observed especially with HFD-fed NCS-1-/- mice. We observed a direct correlation between NCS-1 and IR concentrations in the adipocyte membrane and that NCS-1 can be co-immunoprecipitated with IR indicating a direct interplay between NCS-1 and IR. We propose that NCS-1 plays an important role in adipocyte function and that NCS-1 deficiency gives rise to obesity and diabetes type 2 in adult mice. Given the association of altered NCS-1 expression with behaviorial abnormalities, NCS-1-/- mice may offer an interesting perspective for studying in a mouse model a potential genetic link between some psychiatric disorders and the risk of being obese.

Emerging Roles of Neuronal Ca2+ Sensor-1 in Cardiac and Neuronal Tissues: A Mini Review.

The EF-hand calcium (Ca2+)-binding protein, neuronal Ca2+ sensor-1 (NCS-1/frequenin), is predominantly expressed in neuronal tissues and plays a crucial role in neuronal functions, including synaptic transmission and plasticity. NCS-1 has diverse functional roles, as elucidated in the past 15 years, which include the regulation of phosphatidylinositol 4-kinase IIIß (PI-4K-ß) and several ion channels such as voltage-gated K+ and Ca2+ channels, the D2 dopamine receptors, and inositol 1,4,5-trisphosphate receptors (InsP3Rs). Functional analyses demonstrated that NCS-1 enhances exocytosis and neuronal survival after injury, as well as promotes learning and memory in mice. NCS-1 is also expressed in the heart including the Purkinje fibers (PFs) of the conduction system. NCS-1 interacts with KV4 K+ channels together with dipeptidyl peptidase-like protein-6 (DPP-6), and this macromolecule then composes the transient outward current in PFs and contributes to the repolarization of PF action potential, thus being responsible for idiopathic arrhythmia. Moreover, NCS-1 expression was reported to be significantly high at the immature stage and at hypertrophy in adults. That report demonstrated that NCS-1 positively regulates cardiac contraction in immature hearts by increasing intracellular Ca2+ signals through interaction with InsP3Rs. With the related signals, NCS-1 activates nuclear Ca2+ signals, which would be a mechanism underlying hormone-induced cardiac hypertrophy. Furthermore, NCS-1 contributes to stress tolerance in cardiomyocytes by activating mitochondrial detoxification pathways, with a key role in Ca2+-dependent pathways. In this review, we will discuss recent findings supporting the functional significance of NCS-1 in the brain and heart and will address possible underlying molecular mechanisms.

Photoreceptor calcium sensor proteins in detergent-resistant membrane rafts are regulated via binding to caveolin-1.

Rod cell membranes contain cholesterol-rich detergent-resistant membrane (DRM) rafts, which accumulate visual cascade proteins as well as proteins involved in regulation of phototransduction such as rhodopsin kinase and guanylate cyclases. Caveolin-1 is the major integral component of DRMs, possessing scaffolding and regulatory activities towards various signaling proteins. In this study, photoreceptor Ca2+-binding proteins recoverin, NCS1, GCAP1, and GCAP2, belonging to neuronal calcium sensor (NCS) family, were recognized as novel caveolin-1 interacting partners. All four NCS proteins co-fractionate with caveolin-1 in DRMs, isolated from illuminated bovine rod outer segments. According to pull-down assay, surface plasmon resonance spectroscopy and isothermal titration calorimetry data, they are capable of high-affinity binding to either N-terminal fragment of caveolin-1 (1-101), or its short scaffolding domain (81-101) via a novel structural site. In recoverin this site is localized in C-terminal domain in proximity to the third EF-hand motif and composed of aromatic amino acids conserved among NCS proteins. Remarkably, the binding of NCS proteins to caveolin-1 occurs only in the absence of calcium, which is in agreement with higher accessibility of the caveolin-1 binding site in their Ca2+-free forms. Consistently, the presence of caveolin-1 produces no effect on regulatory activity of Ca2+-saturated recoverin or NCS1 towards rhodopsin kinase, but upregulates GCAP2, which potentiates guanylate cyclase activity being in Ca2+-free conformation. In addition, the interaction with caveolin-1 decreases cooperativity and augments affinity of Ca2 + binding to recoverin apparently by facilitating exposure of its myristoyl group. We suggest that at low calcium NCS proteins are compartmentalized in photoreceptor rafts via binding to caveolin-1, which may enhance their activity or ensure their faster responses on Ca2+-signals thereby maintaining efficient phototransduction recovery and light adaptation.

Functional Status of Neuronal Calcium Sensor-1 Is Modulated by Zinc Binding.

Neuronal calcium sensor-1 (NCS-1) protein is abundantly expressed in the central nervous system and retinal neurons, where it regulates many vital processes such as synaptic transmission. It coordinates three calcium ions by EF-hands 2-4, thereby transducing Ca2+ signals to a wide range of protein targets, including G protein-coupled receptors and their kinases. Here, we demonstrate that NCS-1 also has Zn2+-binding sites, which affect its structural and functional properties upon filling. Fluorescence and circular dichroism experiments reveal the impact of Zn2+ binding on NCS-1 secondary and tertiary structure. According to atomic absorption spectroscopy and isothermal titration calorimetry studies, apo-NCS-1 has two high-affinity (4 × 106 M-1) and one low-affinity (2 × 105 M-1) Zn2+-binding sites, whereas Mg2+-loaded and Ca2+-loaded forms (which dominate under physiological conditions) bind two zinc ions with submicromolar affinity. Metal competition analysis and circular dichroism studies suggest that Zn2+-binding sites of apo- and Mg2+-loaded NCS-1 overlap with functional EF-hands of the protein. Consistently, high Zn2+ concentrations displace Mg2+ from the EF-hands and decrease the stoichiometry of Ca2+ binding. Meanwhile, one of the EF-hands of Zn2+-saturated NCS-1 exhibits a 14-fold higher calcium affinity, which increases the overall calcium sensitivity of the protein. Based on QM/MM molecular dynamics simulations, Zn2+ binding to Ca2+-loaded NCS-1 could occur at EF-hands 2 and 4. The high-affinity zinc binding increases the thermal stability of Ca2+-free NCS-1 and favours the interaction of its Ca2+-loaded form with target proteins, such as dopamine receptor D2R and GRK1. In contrast, low-affinity zinc binding promotes NCS-1 aggregation accompanied by the formation of twisted rope-like structures. Altogether, our findings suggest a complex interplay between magnesium, calcium and zinc binding to NCS-1, leading to the appearance of multiple conformations of the protein, in turn modulating its functional status.

Identification of critical amino acid residues and functional conservation of the Neurospora crassa and Rattus norvegicus orthologues of neuronal calcium sensor-1.

Neuronal calcium sensor-1 (NCS-1) is a member of neuronal calcium sensor family of proteins consisting of an amino terminal myristoylation domain and four conserved calcium (Ca2+) binding EF-hand domains. We performed site-directed mutational analysis of three key amino acid residues that are glycine in the conserved site for the N-terminal myristoylation, a conserved glutamic acid residue responsible for Ca2+ binding in the third EF-hand (EF3), and an unusual non-conserved amino acid arginine at position 175 in the Neurospora crassa NCS-1. The N. crassa strains possessing the ncs-1 mutant allele of these three amino acid residues showed impairment in functions ranging from growth, Ca2+ stress tolerance, and ultraviolet survival. In addition, heterologous expression of the NCS-1 from Rattus norvegicus in N. crassa confirmed its interspecies functional conservation. Moreover, functions of glutamic acid at position 120, the first Ca2+ binding residue among all the EF-hands of the R. norvegicus NCS-1 was found conserved. Thus, we identified three critical amino acid residues of N. crassa NCS-1, and demonstrated its functional conservation across species using the orthologue from R. norvegicus.

Neuronal calcium sensor-1 deletion in the mouse decreases motivation and dopamine release in the nucleus accumbens.

Calcium sensors detect intracellular calcium changes and interact with downstream targets to regulate many functions. Neuronal Calcium Sensor-1 (NCS-1) or Frequenin is widely expressed in the nervous system, and involved in neurotransmission, synaptic plasticity and learning. NCS-1 interacts with and regulates dopamine D2 receptor (D2R) internalization and is implicated in disorders like schizophrenia and substance abuse. However, the role of NCS-1 in behaviors dependent on dopamine signaling in the striatum, where D2R is most highly expressed, is unknown. We show that Ncs-1 deletion in the mouse decreases willingness to work for food. Moreover, Ncs-1 knockout mice have significantly lower activity-dependent dopamine release in the nucleus accumbens core in acute slice recordings. In contrast, food preference, responding for conditioned reinforcement, ability to represent changes in reward value, and locomotor response to amphetamine are not impaired. These studies identify novel roles for NCS-1 in regulating activity-dependent striatal dopamine release and aspects of motivated behavior.

Membrane binding of Neuronal Calcium Sensor-1 (NCS1).

Neuronal Calcium Sensor-1 (NCS1) belongs to the family of Neuronal Calcium Sensor (NCS) proteins. NCS1 is composed of four EF-hand motifs and an N-terminal myristoylation. However, the presence of a calcium-myristoyl switch in NCS1 and its role in the membrane binding are controversial. The model of Langmuir lipid monolayers is thus used to mimic the cell membrane in order to characterize the membrane interactions of NCS1. Two binding parameters are calculated from monolayer measurements: the maximum insertion pressure, up to which protein binding is energetically favorable, and the synergy, reporting attractive or repulsive interactions with the lipid monolayers. Binding membrane measurements performed in the presence of myristoylated NCS1 reveal better binding interactions for phospholipids composed of phosphoethanolamine polar head groups and unsaturated fatty acyl chains. In the absence of calcium, the membrane binding measurements are drastically modified and suggest that the protein is more strongly bound to the membrane. Indeed, the binding of calcium by three EF-hand motifs of NCS1 leads to a conformation change. NCS1 arrangement at the membrane could thus be reshuffled for better interactions with its substrates. The N-terminal peptide of NCS1 is composed of two amphiphilic helices involved in the membrane interactions of NCS1. Moreover, the presence of the myristoyl group has a weak influence on the membrane binding of NCS1 suggesting the absence of a calcium-myristoyl switch mechanism in this protein. The myristoylation could thus have a structural role required in the folding/unfolding of NCS1 which is essential to its multiple biological functions.

Chronic social defeat stress increases dopamine D2 receptor dimerization in the prefrontal cortex of adult mice.

The present study aimed to examine the effects of chronic social defeat stress on the dopamine receptors and proteins involved in post-endocytic trafficking pathways. Adult mice were divided into susceptible and unsusceptible groups after 10 days of social defeat stress. Western blot analysis was used to measure the protein expression levels of dopamine D2 receptors (D2Rs), a short (D2S) and a long form (D2L) and, D2R monomers and dimers, dopamine D1 receptors (D1Rs), neuronal calcium sensor-1 (NCS-1) and G protein-coupled receptor-associated sorting protein-1 (GASP-1), and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure the mRNA expression levels of D2S, D2L, D2R monomers and dimers, and D1Rs in different brain areas. We observed increased expression of D2S, D2L and D2Rs dimers in the prefrontal cortex (PFC) of susceptible and/or unsusceptible mice compared with controls. The only significant findings with regard to mRNA expression levels were lower expression of D2S mRNA in the amygdala (AMYG) of susceptible and unsusceptible mice compared with controls. The present study demonstrated that chronic social defeat stress induced increased expression of D2S, D2L, and D2R dimers in the PFC of susceptible and/or unsusceptible mice.

Neuronal Calcium Sensor-1 Binds the D2 Dopamine Receptor and G-protein-coupled Receptor Kinase 1 (GRK1) Peptides Using Different Modes of Interactions.

Neuronal calcium sensor-1 (NCS-1) is the primordial member of the neuronal calcium sensor family of EF-hand Ca(2+)-binding proteins. It interacts with both the G-protein-coupled receptor (GPCR) dopamine D2 receptor (D2R), regulating its internalization and surface expression, and the cognate kinases GRK1 and GRK2. Determination of the crystal structures of Ca(2+)/NCS-1 alone and in complex with peptides derived from D2R and GRK1 reveals that the differential recognition is facilitated by the conformational flexibility of the C-lobe-binding site. We find that two copies of the D2R peptide bind within the hydrophobic crevice on Ca(2+)/NCS-1, but only one copy of the GRK1 peptide binds. The different binding modes are made possible by the C-lobe-binding site of NCS-1, which adopts alternative conformations in each complex. C-terminal residues Ser-178-Val-190 act in concert with the flexible EF3/EF4 loop region to effectively form different peptide-binding sites. In the Ca(2+)/NCS-1·D2R peptide complex, the C-terminal region adopts a 310 helix-turn-310 helix, whereas in the GRK1 peptide complex it forms an α-helix. Removal of Ser-178-Val-190 generated a C-terminal truncation mutant that formed a dimer, indicating that the NCS-1 C-terminal region prevents NCS-1 oligomerization. We propose that the flexible nature of the C-terminal region is essential to allow it to modulate its protein-binding sites and adapt its conformation to accommodate both ligands. This appears to be driven by the variability of the conformation of the C-lobe-binding site, which has ramifications for the target specificity and diversity of NCS-1.