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Previous Article | Next Article
The Journal of Neuroscience, October 1, 2002, 22(19):8476-8486
Interaction with Neuronal Calcium Sensor NCS-1 Mediates Desensitization of the D2 Dopamine Receptor
Nadine Kabbani1, Laszlo Negyessy3, Ridwan Lin2, Patricia Goldman-Rakic3, and Robert Levenson1 1 Department of Pharmacology and 2 Neuroscience Graduate Program, Penn State College of Medicine, Hershey, Pennsylvania 17033, and 3 Department of Neurobiology, Yale University School of Medicine, New Haven, Connecticut 06510
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
Dopaminergic transmission within limbic regions of the brain is highly dependent on the regulation of D2 receptor activity. Here we show that the neuronal calcium sensor-1 (NCS-1) can mediate desensitization of D2 dopamine receptors. Analysis of D2 receptors expressed in human embryonic kidney 293 cells indicates that NCS-1 attenuates agonist-induced receptor internalization via a mechanism that involves a reduction in D2 receptor phosphorylation. This effect of NCS-1 was accompanied by an increase in D2 receptor-mediated cAMP inhibition after dopamine stimulation. The ability of NCS-1 to modulate D2 receptor signaling was abolished after a single amino acid mutation in NCS-1 that has been shown to impair the calcium-binding properties of NCS-1. Coimmunoprecipitation experiments from striatal neurons reveal that NCS-1 is found in association with both the D2 receptor and G-protein-coupled receptor kinase 2, a regulator of D2 receptor desensitization. Colocalization of NCS-1 and D2 receptors was examined in both primate and rodent brain. In striatum, NCS-1 and D2 receptors were found to colocalize within sites of synaptic transmission and in close proximity to intracellular calcium stores. NCS-1-D2 receptor interaction may serve to couple dopamine and calcium signaling pathways, thereby providing a critical component in the regulation of dopaminergic signaling in normal and diseased brain.
Key words: dopamine receptor; neuronal calcium sensor-1; GRK; GPCR desensitization; frequenin; calcium signaling; yeast two-hybrid
INTRODUCTION
Dopaminergic neurotransmission is mediated via the distinct signaling properties of D1-like (D1 and D5) and D2-like (D2, D3, and D4) dopamine receptor subfamilies (Civelli et al., 1993
; Missale et al., 1998
Dopamine receptors belong to the superfamily of G-protein-coupled receptors (GPCRs) (Civelli et al., 1993
To identify proteins involved in mediating D2 receptor signaling, we used the C terminus of the D2 receptor as bait to screen a human brain cDNA library. One clone isolated from this screen corresponds to neuronal calcium sensor-1 (NCS-1). NCS-1 is the mammalian ortholog of frequenin, a calcium-binding protein implicated in mediating several aspects of neurotransmission, including ion channel regulation (Weiss et al., 2000
MATERIALS AND METHODS
DNA constructs and protein interaction assays. All constructs were generated by subcloning PCR amplification products into appropriate vectors, and each construct was verified by automated DNA sequencing. cDNA fragments encoding the C terminus of the D1 (residues 365-446), D2 (residues 428-443), D3 (residues 385-400), D4 (residues 370-387), and D5 (residues 360-477) receptors were ligated into the yeast GAL4 DNA-binding domain expression vector pAS2-1 (Clontech, Palo Alto, CA). For the D2 receptor screen, the D2-pAS2-1 bait plasmid and the human brain cDNA library in the GAL4 activation domain vector pACT2 (Clontech) were simultaneously cotransformed into the yeast strain MaV103 as previously described (Lin et al., 2001
/Trp
-galactosidase activity via the nitrocellulose filter lift method (Lin et al., 2001
To identify the sites of interaction between D2 or D3 receptors and NCS-1, truncated or full-length receptor constructs (in pAS2-1) were assayed for interaction against truncated or full-length NCS-1 constructs (in pACT2) using the yeast two-hybrid method. Bait and prey plasmids were simultaneously transformed into the yeast strain MaV103 and interactions were assayed as described above.
For expression studies, full-length NCS-1 cDNA was subcloned into the mammalian expression vectors pEGFP-N [C-terminal enhanced green fluorescent protein (EGFP) tag; Clontech] or pCB6 (Brewer and Roth, 1991
Cell culture and transfection. Human embryonic kidney (HEK) 293 cells were maintained in DMEM supplemented with 10% fetal bovine serum. HEK 293 cells stably expressing FLAG-tagged D2L receptors (293-D2 cells) were provided by Dr. Mark von Zastrow (University of California, San Francisco). 293-D2 cells were maintained in DMEM supplemented with 10% fetal bovine serum and 300 µg/ml geneticin/G418 (Invitrogen, Grand Island, NY). Cells were transfected using the Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA) under conditions described by the manufacturer. Striatal cell cultures were prepared from embryonic day 18 (E18) rat embryos as previously described (Lin et al., 2001
Glutathione S-transferase pull-down and coimmunoprecipitation. Fusion protein glutathione S-transferase (GST)-NCS-1 (amino acids 1-190) was constructed in the expression vector pGEX-4T-1 (Amersham Biosciences, Piscataway, NJ). GST-NCS-1 fusion protein was induced in Escherichia coli strain BL21 (DE3) then purified with glutathione-sepharose (Amersham) according to manufacturer's instructions. GST-pull-down assays were performed as previously described (Lin et al., 2001
Immunoprecipitations were performed from crude membranes or total cell lysates as previously described by Karpa et al. (2000)
Immunofluorescence. HEK 293 cells were transiently transfected with plasmids encoding either FLAG-tagged D2 or FLAG-tagged D3 receptors. Detection of dopamine receptors was done by immunostaining with anti-FLAG M2 monoclonal (1:1000 dilution of the monoclonal antibody; Sigma). Immunofluorescence was visualized by confocal laser microscopy using a Zeiss LSM 210 confocal microscope. For immunostaining of striatal cultures, cells were fixed and permeabilized as previously described (Lin et al., 2001
Immunoelectron microscopy. Animals used for immunohistochemistry were housed and treated according to institutional guidelines. Three adult Wistar rats and two adult rhesus monkeys (Macaca mulatta) of both sexes were perfused, and brain tissue was prepared as described (Mrzljak et al., 1998
Cleavable biotin and cAMP assays. Receptor internalization and cell surface labeling assays were performed using the cleavable biotin method as described by Vickery and von Zastrow (1999)
cAMP assays were performed on cell lysates prepared from 293-D2 cells transiently transfected with either wild-type NCS-1 or the E120Q NCS-1 mutant. In some experiments, cells were subjected to a 12 hr preincubation with 100 µg/ml pertussis toxin (Calbiochem) before initiation of the cAMP assay. Measurement of total intracellular cAMP was performed using a cAMP enzyme immunoassay kit (Amersham) according to instructions provided by the manufacturer. Statistical analysis was performed using the Statistica software package.
Whole-cell phosphorylation assays. Whole-cell phosphorylation assays were performed essentially as described by Tiberi et al. (1996)
RESULTS
Interaction of NCS-1 with dopamine receptors
To identify dopamine receptor interacting proteins, we used the C terminus of the D2 dopamine receptor (amino acid residues 428-443) to screen an adult human brain cDNA library. In control experiments the bait did not cause autologous activation of the reporter gene, as indicated by lack of
To examine the specificity of the D2 receptor-NCS-1 interaction, we used the yeast two-hybrid system to test the interaction of NCS-1 with additional dopamine receptor family members. Bait constructs encoding the C-terminal segments of the D1, D2, D3, D4, and D5 receptors were tested for interaction with NCS-1. Using this assay, we found that NCS-1 interacts with the D2, D3, and D5 receptors, but not with the D1 or D4 receptor subtypes. These results indicate that NCS-1 can associate with some but not all members of the D1-like and D2-like subfamilies in the yeast system.
To confirm interaction between NCS-1 and the D2 receptor, we tested the ability of D2 receptors to associate with a GST fusion protein containing the complete NCS-1 ORF. Crude membranes were prepared from 293-D2 cells and tested for the ability to associate with the GST-NCS-1 fusion protein. As shown in Figure 1A, a Western blot containing crude membranes prepared from 293-D2 cells produced a 44 kDa band immunoreactive with the anti-FLAG M2 monoclonal antibody. This band corresponds in size to FLAG-tagged D2 receptors detected in unadsorbed cell lysates. Immunoreactive D2 receptors were not visualized when membranes were adsorbed onto GST alone or when membranes were prepared from nontransfected HEK 293 cells.
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Figure 1. Interaction of D2 dopamine receptor with NCS-1. A, GST-NCS-1 fusion protein was used to pull-down the D2 receptor from crude membrane preparations of 293-D2 cells. FLAG-tagged D2 receptors were detected in unabsorbed cell lysates and pull-down lanes using FLAG-specific M2 antibodies. Coimmunoprecipitation of NCS-1 with D2 receptors (B) or D3 receptors (C). An anti-GFP antibody was used to immunoprecipitate GFP-NCS-1 from crude membrane preparations of 293-D2 cells transiently transfected with GFP-NCS-1 (B), or HEK 293 cells transiently transfected with both GFP-NCS-1 and FLAG-tagged D3 receptors (C). Blots were probed for either D2 or D3 receptors using anti-FLAG M2 antibodies.
Interaction between dopamine receptors and NCS-1 was also verified by coimmunoprecipitation experiments. To demonstrate interaction, we tested the ability of an anti-green fluorescent protein (GFP) antibody to coimmunoprecipitate NCS-1 and D2 receptors from 293-D2 cells transiently expressing GFP-tagged NCS-1. As shown in Figure 1B, anti-GFP antibody was capable of coimmunoprecipitating D2 receptors. We also tested the ability of an anti-GFP antibody to coimmunoprecipitate NCS-1 and D3 receptors from HEK 293 cells transiently transfected with GFP-tagged NCS-1 plus FLAG-tagged D3 receptors. As shown in Figure 1C, anti-GFP antibody was capable of coimmunoprecipitating D3 receptors in these cells. In these experiments, FLAG-tagged D2 and D3 receptors were not coimmunoprecipitated from cells transfected with GFP alone or when the GFP antibody was omitted from the immunoprecipitation reaction. Taken together, these studies provide strong evidence for an association between NCS-1 and D2-like dopamine receptors in transfected mammalian cells.
Mapping protein interaction domains
We performed deletion mapping studies to localize sites within NCS-1 that contribute to NCS-1-D2 receptor interaction. A series of truncation fragments of NCS-1 were tested for their interaction with the C terminus of the D2 receptor (amino acids 428-443) in the yeast two-hybrid assay. As shown in Figure 2A, a minimal fragment containing the N-terminal 71 residues of NCS-1 tested positive in the
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Figure 2. D2 receptor and NCS-1 interaction domains. A, Schematic representation of constructs encoding truncations of NCS-1. Constructs were tested for interaction with the C-terminal tail of the D2 receptor in the two-hybrid assay. Interaction is indicated by the presence or absence of
Interaction of NCS-1 and D2 receptors in cultures of rat striatum
To further characterize NCS-1-D2 receptor interaction, we examined the expression of NCS-1 and D2 receptors in cultures of rat striatum. In double-labeling experiments, coexpression of NCS-1 and the neuronal marker MAP2 was detected within somatic and dendritic neuronal regions (Fig. 3A-C). NCS-1 did not colocalize with the astrocytic marker glial fibrillary acidic protein (GFAP) (data not shown). Double labeling with anti-D2 and anti-NCS-1 antibodies indicates colocalization of the proteins within somata and processes of striatal neurons (Fig. 3D-I). These observations suggest that NCS-1 colocalizes with D2 receptors in striatal neurons. Coimmunoprecipitation experiments were performed to test whether NCS-1 and D2 receptors can associate in striatal cultures. D2 receptors were immunoprecipitated from striatal membrane preparations and immunocomplexes probed for the presence of NCS-1. Western blot analysis using anti-NCS-1 antibodies showed the presence of an ~22 kDa band whose mobility is virtually coincident with that of endogenous NCS-1 expressed in HEK 293 cells (Fig. 3J). This band was not detected in mock immunoprecipitations in which the anti-D2 receptor antibody was omitted or in immunoprecipitations using a nonspecific antibody. These results indicate that NCS-1 and D2 receptors can associate in striatal neurons.
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Figure 3. Expression of NCS-1 and D2 receptors in primary striatal neurons from rat brain. Detection of anti-MAP2 (A) and anti-NCS-1 (B) in double labeling within neurons. Merged image (C) shows MAP2 and NCS-1 coexpression within neuronal perikarya and dendritic process of virtually all striatal neurons. Detection of D2 receptors (D) and NCS-1 (E) in double-labeled cultures. Merged image (F) shows D2 receptor and NCS-1 coexpression within the cell body and processes of neurons. High magnification of a representative neuron (indicated by arrowheads in D-F) showing expression of D2 receptors (G), NCS-1 (H), and colocalization of the two proteins (I). Numerous D2-positive, NCS-1-negative cells display an apparent astrocytic morphology (D-F). J, Interaction of D2 receptors and NCS-1 in striatal cultures Anti-D2 receptor antibody was used to immunoprecipitate D2 receptors from crude membrane preparations of striatal cells. Immunocomplexes were then probed with anti-NCS-1 antibodies. The position of NCS-1 endogenously expressed in HEK 293 cell membranes is shown (HEK 293 lane).
NCS-1 and D2 receptors colocalize at sites of synaptic activity
Double immunoelectron microscopic analysis of rat and monkey striatum was used to gain insight into the subcellular distribution of NCS-1 and D2 dopamine receptors. NCS-1 immunoreactivity was localized in dendrites, spines, and occasionally in axonal boutons in the neuropil of the rat and monkey striatum. In double-labeling experiments, dendrites and spines were the most frequently observed structures exhibiting both NCS-1 and D2 receptor immunoreactivity (Fig. 4a-g). In monkey striatum, D2 and NCS-1 immunoreactivity was found in very close proximity, and sometimes overlapped, within dendrites (Fig. 4a-c). A similar overlapping expression of NCS-1 and D2 receptors was also detected within the spines of both monkey (Fig. 4d,e) and rat (Fig. 4f,g) striatum. Spines coexpressing NCS-1 and D2 receptors usually formed asymmetric synaptic contacts with axon terminals containing round synaptic vesicles (Fig. 4d-g). Within double-labeled structures, immunometal particles representing D2 receptor immunoreactivity were often associated with the cytoplasmic surface of the plasma membrane and were found peri- and/or extrasynaptically (Fig. 4a-g). Morphological characterization of the double labeling indicates that D2-NCS-1 complexes are situated in close proximity to intracellular calcium stores. This is the case in spines where double labeling is often associated with the spine apparatus (Fig. 4f,g) and in dendrites where immunolabeling associated with the plasma membrane was often found in close proximity to mitochondria (Fig. 4a-c). Given that the method used does not allow preservation of the fine membranous structures of the endoplasmic reticulum, it is highly likely that the immunolabeling pattern observed in dendrites represents sites where intracellular calcium stores approach the neuronal plasma membrane (Berridge, 1998
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Figure 4. NCS-1 and D2 receptors colocalize within dendritic shafts and spines. Representative images of immunolabeling of NCS-1 (DAB reaction product) and D2 (immunometal particles) receptors within dendritic shafts (a-c) and spines (d-g) of neurons within monkey (a-e) and rat (f, g) striatum. Silver grains (representing D2 receptors) were detected extrasynaptically (a-d, f, g) and perisynaptically (e). Within dendritic spines, the spine apparatus appears to be immunoreactive for NCS-1 (d-g). D2 receptor immunoreactivity is associated with the membrane of the NCS-1-immunoreactive spine apparatus (f, g). Note the close spatial contiguity of DAB deposits (arrows) and silver particles in the dendrite as well as within spines (b, d, g). An immunonegative spine is present in the top right corner of a-c. Arrowheads point to the postsynaptic density of asymmetric synaptic contacts (d, e). Scale bar, 200 nm.
NCS-1-D2 receptor interaction attenuates ligand-induced D2 receptor internalization
To determine the functional significance of NCS-1-D2 receptor interaction, we examined the effect of overexpressing NCS-1 on D2 receptor signaling. HEK 293 cells were found to endogenously express NCS-1. A 10-fold increase in NCS-1 expression was observed after transient transfection of HEK 293 cells with an EGFP-tagged NCS-1 construct (data not shown). Members of the NCS family are predicted to play a role in regulating desensitization of GPCRs (Burgoyne and Weiss, 2001
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Figure 5. NCS-1-D2 interaction attenuates D2 receptor internalization. 293-D2 cells were transiently transfected with expression plasmids encoding either EGFP-NCS-1 or EGFP alone. A cleavable biotinylation assay was used to examine the effect of NCS-1 on D2 receptor internalization. A, Anti-FLAG M2 antibody was used to immunoprecipitate D2 receptors from lysates of cells treated with 10 µM dopamine for 20 min, 10 µM dopamine for 40 min, 10 µM dopamine for 20 min in the presence of 1 µM haloperidol, or untreated controls. B, Anti-FLAG antibody was used to immunoprecipitate D2 receptors from crude membranes prepared from biotinylated cells. The ratio of D2 receptor expressed at the cell surface was normalized to total D2 receptor to obtain the relative value of plasma membrane (pm) expression. C, Anti-FLAG M2 antibody was used to immunoprecipitate D2 receptors from total lysates of cells treated with 10 µM dopamine for 20 min. The asterisks refer to Student's t tests performed to indicate statistical significance between groups; p 0.05.
NCS-1 overexpression attenuates GRK-mediated internalization of D2 and D3 receptors
We also examined the effect of NCS-1 overexpression on GRK-mediated internalization of activated D2 and D3 dopamine receptors. In these experiments, FLAG-tagged D2 or D3 receptors were transiently transfected into HEK 293 cells, and receptor internalization was monitored via confocal microscopy and receptor biotinylation assays. In HEK 293 cells, D2 and D3 receptors were found predominantly at the plasma membrane in the absence of dopamine stimulation (Fig. 6A,D). After dopamine treatment both the D2 and D3 receptor subtypes exhibited a time-dependent translocation from the plasma membrane to cytosolic compartments (Fig. 6A-F). Consistent with previous reports (Kim et al., 2001
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Figure 6. Effect of NCS-1 on D2-D3 receptor internalization. Confocal images of HEK 293 cells transiently transfected with FLAG-tagged D2 (A-C) or FLAG-tagged D3 (D-F) receptors. Representative images of cells stained with anti-FLAG M2 antibody. A, D, No dopamine; 10 µM dopamine treatment for 25 (B, E); or 60 min (C, F). A cleavable biotinylation assay was used to examine the effect of NCS-1 on D2 receptor internalization in the presence of GRK2 overexpression (G), and D3 receptor internalization in the presence of GRK3 overexpression (H).
NCS-1-D2 receptor interaction attenuates receptor phosphorylation and enhances D2 receptor signaling
To determine the effect of NCS-1 on GRK-mediated phosphorylation of the D2 receptor, we measured D2 receptor phosphorylation in 293-D2 cells. In these cells, D2 receptor phosphorylation increased by more than twofold after 10 min of dopamine treatment (Fig. 7B). As shown in Figure 7A, dopamine-induced D2 receptor phosphorylation in 293-D2 cells was enhanced by the overexpression of GRK2, GRK3, and GRK5. Previous reports have shown GRK2 to be a potent regulator of D2 receptor phosphorylation (Ito et al., 1999
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Figure 7. NCS-1 overexpression attenuates D2 receptor phosphorylation and increases D2 receptor signaling. D2 receptor phosphorylation was assayed by immunoprecipitation with an anti-FLAG M2 after a 10 min stimulation with 10 µM dopamine. A, 293-D2 cells were transiently transfected with GRK2, GRK3, or GRK5 cDNA. B, 293-D2 cells were transiently transfected with GRK2, GRK2 plus EGFP-NCS-1, or EGFP-NCS-1 alone. C, D, 293-D2 cells were transiently transfected with EGFP-NCS-1, EGFP-E120Q, or EGFP alone. Total cAMP levels were measured in cells treated for 5 min with 100 µM forskolin followed by a 10 min exposure to 10 µM dopamine. C, Transfected 293-D2 cells were incubated with 100 µg/ml pertussis toxin for 12 hr before the assay. Error bars indicate SEM. Statistical analysis was performed using a one-way ANOVA; n = 9; *p < 0.05.
In most mammalian cell lines, activation of D2 receptors leads to a decrease in intracellular cAMP levels via coupling of these receptors to inhibitory subsets of G-proteins (Missale et al., 1998
We investigated the effects of calcium binding to NCS-1 on D2 receptor signaling by expressing an NCS-1 mutant (E120Q) in 293-D2 cells. The E120Q mutation occurs in the third EF calcium-binding hand of NCS-1 and prevents the protein from undergoing calcium-dependent conformational changes (Weiss et al., 2000
D2 receptors, NCS-1, and GRK2 form a signaling complex
Members of the neuronal calcium sensor family have been reported to directly interact with GRKs and regulate signaling through GPCRs (Sallese et al., 2000
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Figure 8. Interaction of NCS-1 with D2 receptors and GRK2. A, Anti-NCS-1 antibody was used to immunoprecipitate endogenous NCS-1 from crude membrane fractions of 293-D2 cells. Immunocomplexes were probed using anti-GRK2 antibodies. B, The blot in A was stripped and reprobed with anti-D2 receptor antibodies. C, Anti-NCS-1 antibody was used to immunoprecipitate endogenous NCS-1 from crude membrane fractions of 293-D2 cells that were treated with forskolin, pretreated with H-89, or exposed to H-89 alone. The blot was probed with anti-GRK2 antibodies. D, GST-NCS-1 fusion protein was used to pull-down endogenous GRK2 from lysates prepared from 293-D2 cells. Pull-downs were performed in the presence of either 1 mM Ca2+ or 10 mM EDTA. The blot was probed using an anti-GRK2 antibody. E, The blot in D was stripped and reprobed using anti-D2 receptor antibodies. F, GST-NCS-1 fusion protein was used to pull-down endogenous GRK2 from HEK 293 cells lysate. Pull-downs were performed in the presence of 100 nM, 1 µM, 10 µM calcium, or in the presence of 10 mM EDTA. The blot was probed using anti-GRK2 antibodies.
It is well documented that stimulation of cAMP signaling pathways is associated with enhanced PKA activity (Pawson and Scott, 1997
Association between NCS-1-D2 receptor and NCS-1-GRK2 was also examined under differing calcium conditions. Total cell lysate prepared from 293-D2 cells was tested for the ability to associate with GST-NCS-1 fusion protein in the presence or absence of calcium. As shown in Figure 8D, an immunoreactive band corresponding to endogenous GRK2 was detected when calcium was present in the pull-down. When the same blot was stripped and reprobed with anti-D2 receptor antibodies, a 44 kDa band immunoreactive with the anti-D2 receptor antibody was detected either when calcium was present or chelated in the pull-down assay (Fig. 8E). These results indicate that association between NCS-1 and the D2 receptor is calcium-independent, whereas, association between NCS-1 and GRK2 is calcium-dependent.
Interaction between NCS-1 and GRK2 was also tested in the absence of D2 receptor expression. In these experiments, GST-NCS-1 fusion proteins were tested for their ability to associate with GRK2 (endogenously expressed in HEK 293 cells) at various calcium concentrations. As shown in Figure 8F, an immunoreactive band corresponding to native GRK2 was detected at calcium concentrations in the pull-down reaction ranging from 100 nM-10 µM. Consistent with earlier findings, GRK2 failed to associate with NCS-1 in the presence of the calcium chelator EDTA. These results indicate that NCS-1-GRK2 interaction can occur in the absence of the D2 receptor, and that association between these two proteins may ensue over a broad range of intracellular calcium concentrations.
We used triple label immunofluorescence to determine whether NCS-1, D2 receptors, and GRK2 colocalize within neurons. In striatal cultures, many neurons coexpress all three proteins (Fig. 9A-C). Overlapping staining of NCS-1, D2 receptors, and GRK2 was detected predominantly in cell bodies and dendritic processes (Fig. 9D). NCS-1 was immunoprecipitated from lysates of primary striatal neurons and immunocomplexes probed for the presence of GRK2. As demonstrated in Figure 9E, an immunoreactive band corresponding to GRK2 was detected in the NCS-1 immunoprecipitate, whereas no band was detected when lysates were immunoprecipitated with an irrelevant antibody. Interaction between these proteins is therefore likely to play an important role in modulating signaling through the D2 dopamine receptor.
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Figure 9. Coexpression of D2 receptors, NCS-1, and GRK2 in primary striatal cultures. Epifluorescent detection of NCS-1 (A), D2 receptor (B), and GRK2 (C) triple labeling within striatal neurons. The merged image (D) shows coexpression of the three proteins within striatal neurons. E, Interaction of NCS-1 and GRK2 in striatal cultures. Anti-NCS-1 antibody was used to immunoprecipitate endogenous NCS-1 from lysates of primary striatal cultures. Immunocomplexes were probed with anti-GRK2 antibody. GRK2 was detected in lysates from striatal cell cultures as well as HEK 293 cells. GRK2 immunoreactivity was not detected when striatal lysates were immunoprecipitated with a nonspecific ( ) antibody.
DISCUSSION
We have demonstrated that D2 dopamine receptors can functionally interact with NCS-1, a member of the neuronal calcium sensor family of EF hand calcium-binding proteins (Burgoyne and Weiss, 2001
NCS-1 belongs to the recoverin subfamily of EF hand calcium sensor proteins (Burgoyne and Weiss, 2001
We found that the level of the NCS-1-GRK2-D2 receptor complex was significantly increased after treatment of cells with the adenylyl cyclase activator forskolin, raising the possibility that formation of the NCS-1-GRK2-D2 receptor complex may also be mediated via PKA activation. Our data indicate that treatment of HEK 293 cells (stably expressing the D2 receptor) with the PKA-inhibitor H-89 significantly attenuated the interaction between NCS-1 and GRK2. This result suggests that PKA activation is likely to participate in the formation of the NCS-1-GRK2-D2 receptor complex. This is consistent with the observation that both PKA- and GRK-mediated phosphorylation events play a critical role in the desensitization of dopamine receptors (Jiang and Sibley, 1999
The fact that NCS-1 can regulate desensitization of D2 dopamine receptors supports the hypothesis that NCS proteins are likely direct regulators of GPCR signaling (Iacovelli et al., 1999
In directed yeast two hybrid screens, we also detected interaction between NCS-1 and D3 (D2-like) and D5 (D1-like) dopamine receptors. It will clearly be of interest to determine the physiological significance of NCS-1 interaction with each of the different dopamine receptor subtypes. Truncation mapping identified a 9-amino acid-long segment located in the C terminus of the D2 receptor that is responsible for binding NCS-1. The identical sequence is present in the C terminus of the D3 receptor, suggesting that this segment also represents the site of D3-NCS-1 interaction. BLAST analysis failed to identify a region of sequence homology, either in the C terminus or any other segment, within the D5 dopamine receptor. Because biochemical experiments support the validity of the D5 receptor-NCS-1 interaction (Kabbani, unpublished observations), it seems likely that alternative sequence motifs may be capable of contributing to the interaction between NCS-1 and different dopamine receptor subtypes. The interaction of NCS-1 with the D5 receptor raises another issue of interest. D1-like and D2-like dopamine receptors are believed to signal via distinct second messenger pathways (Missale et al., 1998
Previous studies have shown that NCS-1 functions to regulate calcium-dependent neurotransmitter release (McFerran et al., 1998
FOOTNOTES Received Feb. 6, 2002; revised July 22, 2002; accepted July 24, 2002.
This work was supported by National Institute of Mental Health Grant P50-MH44866. We thank Dr. Allan Levey (Emory University) for D2 dopamine receptor antibody and Dr. J. L. Benovic (Thomas Jefferson University) for GRK cDNA. We are grateful to Klara Szigeti for excellent assistance with electron microscopy. We thank Drs. Blaise Peterson, Clare Bergson, and Bernhard Luscher for helpful comments on this manuscript.
Correspondence should be addressed to Robert Levenson, Penn State College of Medicine, Department of Pharmacology, Hershey, PA 17033. E-mail: rlevenson{at}hmc.psu.edu.
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