SEARCH

SEARCH BY CITATION

Keywords:

  • cyclic AMP;
  • D1 dopamine receptor;
  • desensitization;
  • G protein coupling;
  • phosphorylation;
  • protein kinase C

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

J. Neurochem. (2010) 115, 1655–1667.

Abstract

The D1 dopamine receptor (D1DAR) is robustly phosphorylated by multiple protein kinases, yet the phosphorylation sites and functional consequences of these modifications are not fully understood. Here, we report that the D1DAR is phosphorylated by protein kinase C (PKC) in the absence of agonist stimulation. Phosphorylation of the D1DAR by PKC is constitutive in nature, can be induced by phorbol ester treatment or through activation of Gq-mediated signal transduction pathways, and is abolished by PKC inhibitors. We demonstrate that most, but not all, isoforms of PKC are capable of phosphorylating the receptor. To directly assess the functional role of PKC phosphorylation of the D1DAR, a site-directed mutagenesis approach was used to identify the PKC sites within the receptor. Five serine residues were found to mediate the PKC phosphorylation. Replacement of these residues had no effect on D1DAR expression or agonist-induced desensitization; however, G protein coupling and cAMP accumulation were significantly enhanced in PKC-null D1DAR. Thus, constitutive or heterologous PKC phosphorylation of the D1DAR dampens dopamine activation of the receptor, most likely occurring in a context-specific manner, mediated by the repertoire of PKC isozymes within the cell.

Abbreviations used:
AC

adenylyl cyclase

DA

dopamine

DAG

diacylglycerol

DAR

DA receptor

DMEM

Dulbecco’s modified essential medium

EBSS

Earle’s balanced salt solution

GPCR

G protein-coupled receptor

GRK

G protein-coupled receptor kinase

ICL3

third intracellular loop

m1AchR

m1 muscarinic acetylcholine receptor

PDBu

phorbol 12,13-dibutyrate

PDK-1

phosphoinositide-dependent kinase-1

PKA

cAMP-dependent protein kinase

PKC

protein kinase C

PMA

phorbol 12-myristate,13-acetate

TE

Tris–EDTA

WT

wild-type

Dopaminergic signaling is involved in a variety of biological mechanisms. Dopamine (DA) acts as a neurotransmitter in the CNS where it participates in such processes as long term memory persistence (Rossato et al. 2009) and synaptic plasticity (Reynolds and Wickens 2000); however, aberrant dopaminergic signaling contributes to schizophrenia, movement disorders, and Parkinson’s disease. DA can also act in a paracrine fashion by regulating sodium excretion within the kidney (Yu et al. 2006). Dopaminergic signaling is mediated by five G protein-coupled receptors (GPCRs) that belong to the Class A family (Gainetdinov et al. 2004). DA receptors (DARs) are further grouped into two subclasses based upon common structural, pharmacological, and physiological properties (Neve et al. 2004). The D1-like receptor subclass is composed of the D1 and D5 DARs that couple to Gs/Golf leading to an increase in cAMP accumulation upon agonist activation. The D2-like receptor subclass is composed of the D2, D3, and D4 DARs that couple to Gi/Go resulting in attenuation of cAMP accumulation and modulation of K+ and Ca2+ channel conductances.

G protein-coupled receptors comprise the largest family of genes within the mammalian genome and mediate a variety of signaling pathways (Fredriksson and Schioth 2005). Most of these receptors exhibit some degree of phosphorylation which is proposed to mediate receptor desensitization—the process of diminishing receptor response under continual agonist stimulation (Krupnick and Benovic 1998; Ferguson 2001). Homologous forms of desensitization are believed to involve G protein-coupled receptor kinase (GRK, EC 2.7.11.16) phosphorylation resulting in uncoupling of receptor from G protein and enhanced arrestin binding with a concomitant abrogation of second messenger production followed by receptor internalization (Krupnick and Benovic 1998; Ferguson 2001). Heterologous desensitization of GPCRs appears to result from receptor phosphorylation by a kinase that is activated by a separate receptor system and is generally mediated by second messenger-activated kinases such as cAMP-dependent protein kinase (PKA, EC 2.7.11.11) and/or protein kinase C (PKC, EC 2.7.11.13) (Krupnick and Benovic 1998; Ferguson 2001).

The D1DAR exhibits both basal and robust agonist-induced phosphorylation mediated by multiple classes of protein kinases. The majority of the agonist-induced phosphorylation of the D1DAR is mediated by GRK isoforms GRK2, GRK3, and GRK5 (Tiberi et al. 1996; Rankin et al. 2006), while agonist-promoted PKA phosphorylation at T268 within the third intracellular loop (ICL3) of the D1DAR regulates the onset of receptor desensitization (Jiang and Sibley 1999) and receptor trafficking (Mason et al. 2002). In contrast, little is known about PKC-mediated phosphorylation of the D1DAR, although cells treated with PKC inhibitors display a reduction in basal and phorbol 12-myristate,13-acetate (PMA)-induced receptor phosphorylation (Gardner et al. 2001; Rex et al. 2008), indicating that PKC does phosphorylate D1DAR; however, the PKC sites within the receptor and the physiological significance of this modification remain unknown.

The PKC family of kinases is comprised of ten isozymes divided into three subgroups based on sequence homology and cofactor activation requirements. These isozymes display specific subcellular localization patterns and activities (Gould and Newton 2008; Reyland 2009; Newton 2010). The conventional subgroup is comprised of PKC α, βI, βII, and γ, and requires both Ca2+ and diacylglycerol (DAG) for kinase activation. The novel PKCs (δ, ε, η, and θ) require DAG, but not Ca2+ for activation, while the atypical PKCs (ζ and λ) require neither DAG nor Ca2+ for activation, but rather rely on protein-protein interactions and phosphoinositide-dependent kinase-1 (PDK-1) phosphorylation for activation. PKCμ/PKD1 and PKCν/PKD3 were originally categorized as the fourth (PDK) subgroup within the PKC family; however, these kinases have been re-categorized as a novel subgroup within the CamK family (Manning et al. 2002b).

Our laboratory has previously reported that PKCs phosphorylate the D2DAR and that this phosphorylation results in functional desensitization and receptor internalization (Namkung and Sibley 2004). We have also shown that ethanol-dependent regulation of the D1DAR is mediated through modification of PKC activities, resulting in decreased D1DAR phosphorylation (Rex et al. 2008). We now report that D1DAR is phosphorylated by multiple PKC isozymes in the absence of agonist stimulation. This constitutive PKC phosphorylation of the D1DAR dampens DA activation of the receptor thus attenuating D1DAR-mediated signaling pathways.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Materials

HEK293Tsa201 (HEK293T) cells (Heinzel et al. 1988) were a gift from Dr. Vanitha Ramakrishnan. [3H]SCH-23390 (86.00 Ci/mmol), [32P]orthophosphate (carrier-free, 10 mCi/mL), [3H]cAMP (28.1 Ci/mmol), and [35S]GTPγS (1250 Ci/mmol) were obtained from Perkin Elmer Life Sciences (Waltham, MA, USA). DA, 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one (Ro-20-1724), sodium metabisulfite, (+)-butaclamol, anti-FLAG M2 affinity gel, PMA, phorbol 12,13-dibutyrate (PDBu), 4α-phorbol-12,13-didecanoate (4αPDD), Gö6983 and Gö6976 were purchased from Sigma (St. Louis, MO, USA). Cell culture media and reagents were purchased from Invitrogen (Carlsbad, CA, USA). Calcium phosphate transfection kits were obtained from Clontech (Mountain View, CA, USA). MiniCompleteTM protease inhibitor cocktail was purchased from Roche Applied Science (Indianapolis, IN, USA). QuikChange Multi Site-Directed Mutagenesis kit was purchased from Stratagene (La Jolla, CA, USA). Mutagenesis primers were synthesized by Eurofins MWG Operon (Huntsville, AL, USA). NuPage gels and buffers were purchased from Invitrogen. The m1 muscarinic acetylcholine receptor (m1AchR) expression construct was a gift from Dr. Jurgen Wess. Expression constructs for human PKCε, mouse PKCλ, human PKCμ, rat PKCζ, PKCδ, and human PDK-1 were gifts from Dr. Alex Toker. Expression constructs for rat PKCβI and rat PKCγ were gifts from Dr. Alexandra Newton. The human PKCν/PKD3 expression construct was purchased from Open Biosystems (Huntsville, AL, USA) and the mouse PKCη (Mischak et al. 1993) construct was purchased from Addgene (Cambridge, MA, USA). The PKCα construct was a gift from Mohammed Akbar.

Cell culture and transfection

HEK293T cells were cultured in Dulbecco’s modified essential medium (DMEM) supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 50 units/mL penicillin, 50 μg/mL streptomycin, and 10 μg/mL gentamycin. Cells were grown at 37°C in 5% CO2 and 90% humidity. An amino terminal FLAG epitope-tagged construct for the rat D1DAR (Monsma et al. 1990) was created from pSFβ2, an expression construct containing a FLAG-tagged β2-adrenergic receptor (Guan et al. 1992) to create the wild-type (WT) pSFD1 (D1DAR) as previously reported (Gardner et al. 2001). All site-directed mutagenesis was performed using the QuikChange Multi Site-Directed Mutagenesis kit. For phosphorylation mutants, serine residues were mutated to alanine residues and threonine residues were mutated to valine residues. All mutant FLAG-D1 constructs were verified by DNA sequence analysis prior to use. HEK293T cells were transfected using the calcium phosphate precipitation method for each experiment as follows: 5 million cells were seeded in 150 mm culture dishes, then transfected 24 h later with 20–30 μg of DNA (for [35S]GTPγS binding assays) or 3.5 million cells were seeded in 100 mm culture dishes, then transfected 24 h later with 10–20 μg DNA (for radioligand binding, in situ phosphorylation, and cAMP accumulation assays). For those experiments where PKC was cotransfected with D1DAR constructs, the ratio of PKC to receptor DNA was 1 : 5. After 24 h, the transfected cells were divided and reseeded as described below for each assay for experiments performed the following day.

In situ phosphorylation assays

These assays were performed as previously described (Gardner et al. 2001; Rankin et al. 2006). Briefly, 1 day prior to the experiment, transfected HEK293T cells were seeded at 1.5 × 106 cells per well of a 6-well poly-d-lysine coated Biocoat plate (BD Biosciences, San Jose, CA, USA) and 2 × 106 cells of the same transfection were seeded in a 100 mm culture dish and cultured overnight. Cells in the 6-well plates were washed with Earle’s Balanced Salt Solution (EBSS) and incubated for 1 h in phosphate-free DMEM. Media was then removed and replaced with 1 mL of fresh phosphate-free DMEM containing 106 μCi of [32P]orthophosphoric acid and returned to the incubator for 45 min. Cells were then challenged with 10 μM DA as noted for the time period listed then placed on ice. Cells were washed twice with cold EBSS and solubilized for 1 h at 4°C in 1 mL of solubilization buffer (50 mM HEPES, 1 mM EDTA, 10% glycerol, 1% Triton X-100, pH 7.4, 50 mM NaF, 40 mM sodium pyrophosphate, and 150 mM NaCl) supplemented with MiniCompleteTM protease inhibitor cocktail. The samples were cleared by centrifugation and the protein concentration was determined using the Pierce BCA Protein Assay kit (Pierce, Rockford, IL, USA). The specific activity of D1DAR expression in each transfection group was determined by radioligand binding assays performed using cells in the 100 mm culture dishes described above. After quantifying the receptors in each transfection group, equal amounts of receptor protein were transferred to fresh tubes containing 50 μL of equilibrated anti-FLAG M2-affinity gel and incubated overnight with mixing at 4°C. The samples were washed three times with the following solutions in the order of 0.5 M NaCl solubilization buffer, 0.15 M NaCl solubilization buffer, and TE (Tris–EDTA, pH 7.4) at 4°C. Proteins were eluted from the affinity gel by addition of 35 μL of 2× lithium dodecyl sulfate sample buffer containing reducing agent (Invitrogen) and incubated at 37°C for 1 h. Proteins were resolved on 4–12% NuPage Bis–Tris gradient gels run in 3-(N-Morpholino)propanesulfonic acid sodium dodecyl sulfate running buffer, dried, and subjected to autoradiography. Receptor phosphorylation was quantitated by scanning the autoradiographs and measuring the band intensity using the software Labworks 4.0 (UVP, Inc., Upland, CA, USA).

Radioligand binding assays

HEK293T cells were harvested by incubation with 5 mM EDTA in EBSS lacking CaCl2 and MgSO4 and collected by centrifugation at 300 g for 10 min. The cells were resuspended in lysis buffer (5 mM Tris, pH 7.4 and 5 mM MgCl2) at 4°C and were disrupted using a dounce homogenizer followed by centrifugation at 34 000 g for 15 min. The resulting membrane pellet was resuspended in binding buffer (50 mM Tris, pH 7.4) and 100 μL of the membrane suspension was added to assay tubes containing [3H]SCH-23390 in a final volume of 1 mL and a portion of the membrane suspension was quantitated using the BCA protein assay. (+)-Butaclamol was added at a final concentration of 3 μM to determine non-specific binding. The assay tubes were incubated at 25°C for 1.5 h and the reaction was terminated by rapid filtration through GF/C filters pre-treated with 0.6% polyethyleneimine. Radioactivity bound to filters was quantitated in a Beckman LS 6500 scintillation counter (Beckman Coulter, Brea, CA, USA).

cAMP accumulation assays

Eighteen hours post-transfection, the HEK293T cells were reseeded into 24-well poly-d-lysine-coated, Biocoat plates (BD Biosciences) at a density of 80 000 cells/well and allowed to incubate for 24 h. Cells were pre-treated as described in DMEM buffer lacking serum or supplements for the time indicated. Duplicate wells were exposed to 250 μL DA dilutions (in 20 mM HEPES-buffered DMEM with 200 μM sodium metabisulfite, and 30 μM RO-20-1724) ranging from 1 × 10−10 M to 1 × 10−4 M with two wells receiving no DA. The plates were then incubated at 37°C for 20 min. The reaction was terminated by discarding the medium and adding 200 μL of 3% perchloric acid to each well for 30 min then neutralized with 80 μL of 15% KHCO3. cAMP accumulation was measured using the competitive binding of cAMP to PKA as described by Watts and Neve (Watts and Neve 1996) with slight modifications. Briefly, 50 μL of neutralized reaction lysate was added to Neptune 1.1 mL minitubes (VWR, West Chester, PA, USA) containing 200 μL of PKA lysate in TE, pH 7.4 buffer and 50 μL of [3H]cAMP, 0.182 μCi/mL of TE, pH 7.4 and incubated overnight at 4°C. Removal of unbound isotope was accomplished by adding 250 μL of charcoal solution (20 mg/mL carbon and 10 mg/mL bovine serum albumin in water) and centrifugation at 1000 g for 30 min. Supernatants were collected and quantitated in a Beckman LS 6500 scintillation counter (Beckman Coulter). The quantity of cAMP produced in each sample was calculated from a standard curve ranging from 0.4 to 100 pmol unlabeled cAMP/assay.

[35S]GTPγS binding

The [35S]GTPγS binding assays were carried out as described by Gardner et al. (1996) (Gardner et al. 1996) with modifications. Briefly, 5 million HEK293T cells were cultured in 100 mm culture dishes for 24 h prior to transfection. 24 h post-transfection, cells were transferred to 150 mm poly-d-lysine-coated Biocoat plates (BD Biosciences) for experiments the next day. Cells were pre-incubated with the indicated chemical in DMEM buffer (non-supplemented) for the time indicated. Cells were placed on ice and rinsed with EBSS twice. 8 mL membrane preparation buffer (50 mM Tris, pH 7.4, 10 mM sodium pyrophosphate, 10 mM NaF, 5 mM EDTA) was added to each dish and the cells were harvested and transferred to a dounce homogenizer followed by centrifugation at 34 000 g for 30 min. The supernatant was discarded and 10 mL of HEPES buffer (20 mM HEPES, pH 7.4, 6 mM MgCl2, and 100 mM NaCl) was added to each membrane pellet and centrifuged again at 34 000 g for 30 min. The supernatant was discarded and 4 mL HEPES buffer was added and resuspended in a Dounce homogenizer. The membrane suspension (30–55 μg of protein) was incubated in HEPES buffer with 0.1 mM dithiothreitol, 0.2 mM sodium metabisulfite, 5 μM GDP, and 100 pM [35S]GTPγS and various DA concentrations at 30°C for 30 min in a final reaction volume of 1 mL. Basal binding was determined in the absence of agonist and non-specific binding was determined with 10 μM unlabeled GTPγS. The reaction was terminated by rapid filtration through GF/C filters with three washes of 4 mL ice-cold membrane preparation buffer. Radioactivity bound to filters was quantitated in a Beckman LS 6500 scintillation counter (Beckman Coulter). Free [35S]GTPγS (0.1 fmol) was counted to calculate fmol [35S]GTPγS/cpm. The protein concentration in each membrane suspension was determined using the BCA protein assay. DA-stimulated [35S]GTPγS binding was calculated by subtracting basal binding and normalizing with the quantity of membrane protein present in each reaction.

Data analysis

All phosphorylation assays were performed at least three times. Figures depict representative autoradiography obtained for each experimental condition. Relative intensities of the phosphorylated bands were determined by scanning the autoradiographs and analyzing the bands using LabWorks 4.0 (UVP, Inc.). Binding assays and cAMP experiments were performed three to four times. Radioligand binding parameters, KD and Bmax, as well as EC50 values for DA-stimulated cAMP production were calculated using GraphPad Prism 5.02 curve-fitting program (GraphPad Software Inc., La Jolla, CA, USA).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

PKC phosphorylates the D1DAR to modulate receptor signaling

Phosphorylation of the D1DAR by PKC was examined by expressing the FLAG-tagged receptor in HEK293T cells and performing in situ phosphorylation assays. We previously determined that HEK293T cells express members of all four classes of PKCs: PKCα, PKCβI, PKCδ, PKCε, PKCζ, PKCμ, and PKCν, making them an appropriate model system for such studies (Rex et al. 2008). Whole cells expressing the D1DAR were treated with the active phorbol esters PDBu and PMA (for direct activation of all classes of PKCs except the atypical PKCs) or the inactive phorbol ester 4α-phorbol-12,13-didecanoate prior to solubilization, immunoprecipitation, and autoradiography. Only the cells treated with active phorbol esters produced a robust increase in phosphorylation above that of basal phosphorylation (Fig. 1a, quantitated in Figure S1). The phorbol ester-promoted phosphorylation was transient in nature, exhibiting maximal effects within 15–30 min. of stimulation and returning to basal levels within an hour (Fig 1b). Both basal and phorbol ester-stimulated phosphorylation were significantly reduced in the presence of the PKC inhibitors Gö6976 (inhibitor of PKC α, β, and γ) and Gö6983 (inhibitor of PKC α, β, γ, δ, and ζ) (Fig. 1c). Importantly, the observation that PKC inhibitor treatment nearly abolishes basal phosphorylation of the D1DAR indicates that the receptor is phosphorylated constitutively by PKC(s).

image

Figure 1.  PKC phosphorylation of the D1DAR. In situ phosphorylation assays performed on HEK293T cells expressing the rat D1DAR as described in Materials and Methods. (a) Cells were treated for 30 min with 1 μM concentrations of PDBu (lane 2), PMA (lane 3), or 4αPDD (lane 4) prior to cell lysis. Lane 1 represents cells exposed to media alone. 1.2 pmol of D1DAR were loaded into each lane. (b) Time course for PDBu-induced phosphorylation of the D1DAR. Cells were exposed to 1 μM PDBu for the times indicated prior to cell lysis and immunoprecipitation. 1.5 pmol of D1DAR were loaded into each lane. (c) (+) lanes indicate that the cells were exposed to the indicated chemicals, while (−) lanes indicate an absence of the indicated chemicals in the media. Cells were pre-treated for 45 min with 10 μM Gö6976 or Gö6983 PKC inhibitors prior to stimulation with 10 μM PDBu for 30 min. 1.4 pmol of D1DAR were loaded into each lane. This experiment was performed three times with similar results.

Download figure to PowerPoint

As phorbol esters directly activate PKCs, we wished to investigate if the D1DAR could be phosphorylated in a heterologous fashion through activation of a Gq-linked GPCR. The m1AchR was utilized for this purpose based upon previously published studies (Namkung and Sibley 2004). When the m1AchR is stimulated with the agonist carbachol, the second messengers DAG and inositol triphosphate are produced resulting in the activation of PKC. The m1AchR was over-expressed with the D1DAR in HEK293T cells followed by carbachol stimulation and in situ phosphorylation analysis (Fig. 2). Carbachol stimulation resulted in a significant increase in D1DAR phosphorylation only in cells over-expressing the m1AchR, supporting the idea that the D1DAR can be phosphorylated by PKC, and that this phosphorylation can occur constitutively as well as in a heterologous fashion.

image

Figure 2.  Activation of Gq-linked muscarinic receptors can lead to phosphorylation of the D1DAR. In situ phosphorylation assay performed on HEK293T cells cotransfected with the rat D1DAR and the mouse m1 mAchR (or empty vector) as described in Materials and Methods. (a) (+) lanes correspond to cells treated for 30 min with the 100 μM carbachol prior to cell lysis and immunoprecipitation. 1.2 pmol of D1DAR were loaded into each lane. This experiment was performed three times with similar results. (b) The mean ± SEM of the normalized data collected from three individual experiments are reported in the histogram (anova followed by Bonferroni pair-wise comparisons, where *p < 0.5).

Download figure to PowerPoint

Having confirmed that the D1DAR can be phosphorylated by PKC, we were interested in identifying which PKC isozymes might mediate this phosphorylation. To address this question, in situ phosphorylation assays were performed on HEK293T cells with over-expression of various PKC isozymes. The rationale was that cotransfection of cells with the D1DAR and a specific PKC isozyme would result in an increase in D1DAR phosphorylation if that PKC isozyme is capable of mediating D1DAR phosphorylation. Activation of PKC isozymes was accomplished with the use of phorbol ester treatment except for the atypical PKCs. For the atypical PKCζ and PKCλ experiments, PDK-1 was additionally cotransfected. For these PKCs, PDK-1 phosphorylates residues within the activation loop located in the highly conserved catalytic kinase domain, and this phosphorylation event converts the PKC to an activated conformation (Le Good et al. 1998). We obtained expression constructs for most of the PKC isozymes identified to date and performed in situ phosphorylation assays as described. Figure 3(a) depicts a representative autoradiograph of an in situ phosphorylation assay performed with wild-type D1DAR and the over-expression of PKCβI. Figure 3(b) depicts the quantitation of the various in situ phosphorylation assays performed using the indicated PKC isoforms over-expressed with wild-type D1DAR. (Representative autoradiographs for wild-type D1DAR and the over-expression of various PKC isoforms are presented in Figure S2). To summarize our results, the PKC isozymes that were found to phosphorylate the D1DAR included α (alpha) (Rex et al. 2008), βI (betaI), γ (gamma), δ (delta), and ε (epsilon). The PKC isozymes that were incapable of increasing D1DAR phosphorylation were η(eta), λ (lambda), ζ (zeta), μ (mu), and ν (nu). Lack of D1DAR phosphorylation by these co-expressed PKC isozymes was not because of lack of PKC expression, as the presence of auto-phosphorylated PKC isozymes was observed on the autoradiographs (see Figure S2).

image

Figure 3.  Identification of individual PKC isozymes that can mediate D1DAR phosphorylation. (a) In situ phosphorylation assay performed on HEK293T cells expressing the D1DAR and empty vector (V) or PKCβI as described in Materials and Methods. (+) lanes correspond to cells treated for 30 min with the indicated PKC activator PDBu and/or the PKC inhibitors Gö6976 and Gö6983. The amount of D1DAR resolved for each lane is 0.5 pmol. Representative autoradiographs for D1DAR + PKCγ, δ, ε, μ, λ, and ζ co-expression experiments are presented in Figure S2. (b) Summary histogram of D1DAR + PKC isoform co-expression analysis. Co-expression of PKC βΙ, δ, ε, and γ (solid black bars indicated with an asterisk *) produce statistically greater phosphorylation of the D1DAR upon phorbol ester (PE) stimulation as compared to D1DAR + Vector stimulated with PE (checkered bar, second from left). The mean ± SEM of the normalized data collected from three to nine individual in situ phosphorylation assays performed as described in Fig. 3(a) are reported in the histogram (anova followed by Bonferroni pair-wise comparisons, where *p < 0.05).

Download figure to PowerPoint

Protein kinase C isozymes βII (betaII), θ (theta), and υ (upsilon) were not tested. Thus, it appears that many but not all PKC isozymes are capable of mediating D1DAR phosphorylation.

To explore the functional effects that PKC phosphorylation of the D1DAR has on receptor signaling, we examined DA-stimulated cAMP accumulation in cells that were pre-treated with PKC activators or inhibitors that were previously found to modulate receptor phosphorylation. Surprisingly, either activation (with PMA) or inhibition (with Gö6983) of PKC resulted in a potentiation of the maximal amount of D1DAR-stimulated cAMP accumulation without a change in the EC50 for DA (Fig. 4). Given that both of these agents had opposite effects on D1DAR phosphorylation (Fig. 1), these results suggest that additional mechanisms must be involved in modulating the cAMP response. Indeed PKC is well known to phosphorylate specific adenylyl cyclase (AC) isoforms leading to increased hormone-stimulation of AC activity (Sibley et al. 1986; Yoshimasa et al. 1987; Sunahara and Taussig 2002). Thus, in order to delineate the functional effects of PKC phosphorylation on the receptor alone, we decided that the most direct approach would be to identify all of the PKC phosphorylation sites within the D1DAR and eliminate them using site-directed mutagenesis.

image

Figure 4.  Effect of PKC activation or inhibition on cAMP accumulation in cells expressing the D1DAR. Measurement of cAMP produced by HEK293T cells expressing the D1DAR receptor when exposed to the indicated stimuli as described in Materials and Methods. (a) Cells were pre-treated for 30 min with 1 μM PMA or 1 h with 3 μM Gö6983 prior to stimulation with the indicated concentrations of DA. EC50 values [mean (95% C.I.)] for each WT D1DAR sample are as follows: control = [7.6 × 10−8 M (4.6 × 10−8 to 1.3 × 10−7)], PMA = [1.4 × 10−7 M (9.2 × 10−8 to 2.2 × 10−7)], and Gö6983 = [1.1 × 10−7 M (7.6 × 10−8 to 1.6 × 10−7)]. (b) Summary histogram of Emax cAMP (pmol/well) from cells treated as described above. Individual experimental data were divided by the Emax obtained for WT control (untreated) group to take into account varying receptor expression levels between experiments. The mean ± SEM of the normalized data collected from six individual experiments are reported in the histogram (anova followed by Bonferroni pair-wise comparisons, where *p < 0.5 and **p < 0.01).

Download figure to PowerPoint

Identification of the PKC phosphorylation sites on the D1DAR

Our goal was to generate a D1DAR construct devoid of PKC phosphorylation sites thus providing a D1DAR mutant incapable of direct regulation by PKC phosphorylation. To this end, site-directed mutagenesis was used to initially create a series of carboxyl tail truncation mutants by inserting STOP codons into amino acid positions 347, 369, 394, and 404, as well as creating several site-specific mutants by replacing serine or threonine residues with alanine or valine residues, respectively (Fig. 5). Both WT and the D1DAR truncation mutants were expressed in HEK293T cells, stimulated with DA or PDBu, and then subjected to in situ phosphorylation analyses. Truncation of the carboxyl terminus downstream of amino acid 394 resulted in a dramatic reduction of both basal and PDBu-stimulated phosphorylation, indicating that PKC phosphorylation sites exist in the D1DAR carboxyl tail downstream of amino acid 394 (Fig. 6a).

image

Figure 5.  Diagram of the rat D1DAR illustrating the constructs used in this study. All intracellular serine and threonine residues are indicated by shading. Truncation mutants are indicated in the figure at the point of STOP codon insertion and include T404, T394, T369, and T347. Site-specific mutants were created using site-directed mutagenesis to replace serine residues with alanine and threonine residues with valine and include ‘3rd TOTAL’ where all serine and threonine residues are mutated in the third intracellular loop, ‘Tail TOTAL’ where all serine and threonine residues are mutated in the carboxyl tail, ‘All TOTAL’ which is a combination of 3rd TOTAL and Tail TOTAL. The ‘PKC-null’ mutant lacks the five serine residues indicated by black circles (S259, S397, S398, S417, and S421).

Download figure to PowerPoint

image

Figure 6.  Identification of PKC phosphorylation sites in the D1DAR. In situ phosphorylation assays performed on HEK293T cells expressing the indicated D1DAR constructs as described in Fig. 5 and Materials and Methods. (a and b) WT, truncation, and site-specific mutants of the D1DAR were stimulated with either DA or PDBu for 15 min prior to lysis, immunoprecipitation, resolution by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and autoradiography. In the figure labels, the pan mutants All TOTAL, 3rd TOTAL, and Tail TOTAL are abbreviated to ‘TOT.’ (c) Reverse (Rev) constructs were created by site-directed mutagenesis using the Tail TOTAL as the template and reintroducing the indicated WT serine or threonine residues (numbering scheme for carboxyl serines and threonines is listed in Fig. 5). Cells expressing the indicated reverse constructs were treated as described in (a). (d) Cells expressing the D1DAR PKC-null construct were treated with control media or media supplemented with the PKC inhibitor Gö6983 for 45 min prior to stimulation with control, DA-, or PDBu-supplemented media for 15 min and processed as described in panels (a)–(c). These experiments were performed three times with similar results.

Download figure to PowerPoint

Mutation of all of the serine and threonine residues within the ICL3 led to a decrease in basal, agonist-induced, and phorbol ester-induced phosphorylation (Fig. 6b, compare lanes 1–3 to lanes 10–12), suggesting that there is at least one PKC phosphorylation site within ICL3. Interestingly, complete truncation (Fig. 6a, lanes 13–15) or mutation of all serine and threonine residues in the carboxyl tail (Fig. 6b, lanes 7–9) results in a completely phosphorylation-null receptor even though the eight serine/threonine residues in ICL3 remain intact, indicating that phosphorylation must occur initially in the carboxyl tail prior to phosphorylation of ICL3 sites. This hierarchical nature and mechanism of D1DAR phosphorylation is currently undergoing further investigation (Rankin and Sibley, in preparation). Mutation of serine or threonine residues within the first or second intracellular loop did not affect PKC phosphorylation of the D1DAR (data not shown) indicating that these regions of the receptor do not harbor phosphorylation sites.

Because the D1DAR is heavily phosphorylated, even in the basal state, we found it generally difficult to detect the loss of a single phosphorylation site using our in situ phosphorylation assay. Thus, we used a reverse-mutational approach where all possible phosphorylation sites in the receptor region were mutated to reduce the background level of phosphorylation, and then single potential phosphorylation sites were mutated back to WT serines or threonines to see if there was a gain in phosphorylation. This ‘add back’ approach was initially performed on the carboxyl tail region of the receptor. The D1DAR Tail TOTAL construct (Fig. 5) is a phosphorylation-null receptor (Fig. 6) with a WT ICL3 region and a mutated carboxyl tail region where all twenty serine and threonine residues were replaced with alanine and valine residues, respectively. Individually and in small clusters, alanine residues were returned to serine residues, and valine residues were reverted to threonine residues then subjected to in situ phosphorylation analyses. Ninety-six individual mutants were screened for phosphorylation by stimulating cells with control media or with media supplemented with phorbol ester or DA. D1DAR mutants that displayed predominantly DA-mediated phosphorylation were identified as receptors containing GRK sites (Fig. 6c, left panel), while receptor constructs that displayed predominantly phorbol-ester induced phosphorylation were identified as receptors containing PKC phosphorylation sites (Fig. 6c, middle and right panels). This methodology identified four carboxyl tail serine residues that are phosphorylated upon phorbol ester treatment: S397, S398, S417, and S421. The GRK sites phosphorylated in response to agonist treatment will be reported separately (Rankin and Sibley, in preparation).

Once all the PKC phosphorylation sites were identified in the carboxyl tail, these sites were removed by site-directed mutagenesis in the WT D1DAR construct, and this new construct was used as a template to remove each of the eight individual ICL3 serine and threonine residues followed by screening for phosphorylation as described above. Only residue S259 in ICL3 consistently produced an abolition of phorbol ester-induced phosphorylation, and was thus included as a fifth PKC residue in the D1DAR PKC-null construct (Fig. 5). The D1DAR construct with the five serines, S397, S398, S417, S421, and S259, simultaneously mutated displays significantly reduced basal phosphorylation as compared to the WT D1DAR (compare Fig. 6a, lane 1 to Fig. 6d, lane 2), is resistant to phosphorylation upon phorbol ester stimulation, and is unresponsive to PKC inhibitor treatment (Fig. 6d). Hereafter, we refer to this construct as D1DAR PKC-null.

Effects of eliminating PKC-mediated phosphorylation of the D1DAR

Removal of PKC phosphorylation sites within the D1DAR has no effect on receptor expression compared to the WT receptor (Figure S3); however, maximal DA-stimulated cAMP accumulation is significantly enhanced in cells expressing the PKC-null D1DAR as compared to cells expressing the WT D1DAR at similar levels (Fig. 7a–c, control samples). Treatment of cells with phorbol ester (PMA) to activate PKC still results in potentiation of maximal cAMP accumulation for both WT and PKC-null D1DAR expressing cells; however, treatment with a PKC inhibitor (Gö6983) no longer displays an enhancement of cAMP accumulation in cells expressing the PKC-null D1DAR (Fig. 7), supporting the successful removal of PKC phosphorylation sites within the D1DAR. These results further indicate that PKC-inhibitor enhancement of D1DAR-stimulation of cAMP accumulation is mediated by reduction of basal receptor phosphorylation. In contrast, the PKC activator enhancement of D1-DAR stimulation of cAMP accumulation appears not to be mediated by PKC phosphorylation of the receptor, but rather by phosphorylation of non-receptor components. Notably, PKC modulation of the D1DAR does not interfere with agonist-mediated regulation of the receptor as removal of PKC phosphorylation sites has no effect on DA-induced desensitization (Figure S4).

image

Figure 7.  Effect of PKC activation and inhibition on WT vs. PKC-null D1DAR cAMP accumulation. cAMP accumulation assays were performed on cells expressing the WT or PKC-null D1DAR. Cells were pre-treated with 1 μM PMA for 30 min or 3 μM Gö6983 PKC inhibitor for 1 h prior to stimulation with the indicated concentrations of DA for 20 min followed by cell lysis and cAMP quantitation. Representative cAMP accumulation assays performed on cells expressing similar levels (∼ 4.5 pmol/mg protein) of the (a) WT D1DAR, control Emax = 21.9 pmol cAMP/well; EC50 [mean (95% C.I.)] = [2.64 × 10−8 M (1.6 × 10−8 to 4.5 × 10−8)] or (b) PKC null, control Emax = 33.4 pmol cAMP/well; EC50 [mean (95% C.I.)] = [2.57 × 10−8 M (1.2 × 10−8 to 5.4 × 10−8)]. (c) Summary histogram of Emax cAMP (pmol/well) from cells treated as described above where PE represents ‘phorbol ester.’ Each individual experiment was performed on cell samples expressing the same amount of WT or PKC-null receptor. Individual experimental data were divided by the Emax obtained for WT control (untreated) group to take into account varying receptor expression levels between experiments. The mean ± SEM of the normalized data collected from five individual experiments are reported in the histogram (anova followed by Bonferroni pair-wise comparisons, where *p < 0.5 and **p < 0.01; note that the WT D1DAR data is reiterated from Fig. 4b for comparative purposes).

Download figure to PowerPoint

To further determine, if the observed enhancement in cAMP accumulation upon PMA treatment is receptor mediated or because of downstream effectors, [35S]GTPγS binding assays were performed to determine if the increased second messenger production was because of more effective/increased G protein coupling to the receptor. Notably, as observed with the cAMP accumulation assays, removal of PKC phosphorylation sites within the D1DAR results in an increase of maximal G protein coupling in cells expressing similar levels of WT or PKC-null receptor without a change in DA potency (Fig. 8). These results confirm that PKC phosphorylation of the D1DAR reduces the ability of the receptor to activate G proteins and produce a signaling response.

image

Figure 8.  Removal of PKC phosphorylation sites within the D1DAR increases G protein-receptor coupling. [35S]GTPγS binding assay performed on cells expressing either the WT or PKC-null D1DAR and Gαs in a ratio of 5 : 1. (a) Representative binding experiment performed on cell membranes expressing 3.9 pmol/mg protein of either WT D1DAR (Emax = 23.7 fmol/mg protein, [35S]GTPγS bound; EC50 [mean (95% C.I.)] = [1.61 × 10−7 M (6.9 × 10−8 to 3.8 × 10−7)] or PKC-null D1DAR (Emax = 39.6 fmol/mg protein, [35S]GTPγS bound; EC50 [mean (95% C.I.)] = [1.37 × 10−7 M (4.3 × 10−8 to 4.4 × 10−7)] stimulated with the indicated concentrations of DA. (b) Summary histogram of Bmax (fmol/mg protein, [35S]GTPγS bound) from cells treated as described in (a). Each individual experiment was performed on cell samples expressing similar amounts of WT or PKC-null receptor. Data were divided by WT control Bmax for each individual experiment to take into account varying expression levels between experiments. The mean ± SEM of the normalized data collected from four individual experiments are reported in the histogram, where *p < 0.05 paired Student’s t-test.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The idea that phosphorylation of GPCRs represents a universal mechanism whereby agonist-bound, phosphorylated receptor becomes a substrate for arrestin binding, inducing receptor desensitization and internalization is no longer broadly applicable given the recent accumulation of evidence for phosphorylation-independent arrestin recruitment, desensitization, and internalization (Chen et al. 2004; Jala et al. 2005; Richardson et al. 2003; Namkung et al. 2009b,a). Additionally, evidence is mounting that GPCRs can be differentially regulated not only by a particular kinase but also by the location within the receptor and the time-frame in which the phosphorylation occurs (Busillo et al. 2010).

The D1DAR is the most widely expressed dopamine receptor in the CNS where it functions in a variety of neuronal processes, yet it is also expressed in peripheral tissue including the parathyroid gland (Sunahara et al. 1990), seminal vesicles (Hyun et al. 2002), heart, and kidney (Ozono et al. 1997). Given the diversity of tissue types in which the receptor is expressed, tissue-specific D1DAR functional regulation mechanisms must exist to refine tissue-specific function. Phosphorylation represents the most prevalent means of post-translationally modifying a protein to effect functional change (Manning et al. 2002a), so an attractive model for tissue-specific receptor regulation can be achieved through tissue-specific kinase expression, activation and/or subcellular localization (Tobin et al. 2008). To this end, we have begun identifying which kinases phosphorylate the D1DAR, where this phosphorylation occurs within the receptor, and the functional consequence(s) of this modification.

The major finding of our current study is that the D1DAR is phosphorylated by PKC either constitutively or heterologously, and that this negatively regulates receptor-G protein coupling and downstream signaling. Through the use of PKC inhibitors and mutagenesis (see below) we found that the vast majority of D1DAR phosphorylation in the basal state is mediated by PKC. The small amount of remaining basal phosphorylation is because of GRK(s) and an unidentified protein kinase (Rankin and Sibley, unpublished observations). The exact mechanism of basal phosphorylation of the D1DAR by PKC is unclear, but may be because of constitutively active PKC isozymes within the cell or through the scaffolding activities of PKC binding proteins. We have recently reported that RanBP9 and RanBP10 can bind to the D1DAR and to PKCδ or PKCγ, providing a mechanism by which these PKC isozymes can be directed to the receptor to increase D1DAR basal phosphorylation (Rex et al. 2010). We also found that PKC phosphorylation of the D1DAR could be increased through either direct PKC activation with phorbol esters or through activation of a Gq-linked muscarinic receptor leading to PKC activation. The latter mechanism may be involved in heterologous forms of GPCR desensitization. PKC activation appears not to be involved in homologous forms of agonist-induced receptor phosphorylation as we previously found that PKC inhibitors do not block this response (Gardner et al. 2001). Agonist-stimulated receptor phosphorylation appears to be predominantly mediated by GRKs (Rankin and Sibley, in preparation).

It appears as if multiple PKC isozymes are capable of phosphorylating the D1DAR. In our over-expression studies, we found that PKCs α, βI, γ, δ, and ε all increased D1DAR phosphorylation whereas PKCs η, λ, ζ, μ, and ν were ineffective. We previously found that HEK293T cells express PKCs α, βI, δ, ε, ζ, μ, and ν (Rex et al. 2008). From these comparative analyses, it would appear as if PKCs α, βI, δ, and ε may be involved in D1DAR phosphorylation, at least within the HEK293T cells. A distinct possibility, however, is that different PKC isozymes may play different roles in basal versus cell-stimulated phosphorylation and/or that distinct PKCs phosphorylate different sites on the receptor. All of these possibilities will require further investigation.

In order to specifically delineate the role of receptor phosphorylation per se, we found it necessary to identify all of the potential PKC phosphorylation sites on the receptor. This was a complicated analysis because of the fact that the D1DAR is a robustly phosphorylated receptor with 32 potential serine/threonine phospho-acceptor sites located within the intracellular domains of the receptor. We determined that the carboxyl tail and the ICL3 comprise the two domains that are phosphorylated in the D1DAR and these alone contain 28 potential sites of serine/threonine phosphorylation. A further complication in this analysis is that phosphorylation of the D1DAR appears to occur in a hierarchical fashion in that sites in the carboxyl terminus must be phosphorylated prior to phosphorylation of sites in the ICL3. We first obtained evidence for this in examining DA-stimulated (i.e., GRK-mediated) D1DAR phosphorylation (Kim et al., 2004). While this mechanism also appears to apply to PKC-mediated receptor phosphorylation, we are currently exploring this phenomenon more fully using GRK-mediated phosphorylation as a read-out (Rankin and Sibley, in preparation).

Given the above, we undertook a combination of mutational analyses, which involved ‘loss of function’ mutations, in which serine/threonine residues were mutated to alanine/valine residues and a loss of phosphorylation was examined, as well as ‘gain of function’ mutations in which all (or many) of the serine/threonine residues were first mutated to alanine/valine residues to create a phosphorylation-null or phosphorylation-reduced receptor and then specific alanine/valine residues were mutated back to serine/threonine residues and a gain of phosphorylation was detected. Examination of over 100 mutated receptor constructs (for PKC alone) resulted in the identification of five serine residues—four in the carboxyl terminus and one in the ICL3 that appeared to account for all of the receptor phosphorylation by PKC in both the basal and stimulated states. When all five of these serine residues were simultaneously mutated, basal phosphorylation of the receptor was dramatically reduced and there was no effect of PKC activators or inhibitors on receptor phosphorylation. Interestingly, the four serine residues in the carboxyl terminus are in relative close proximity to each other (Fig. 5) suggesting that this region may comprise a potential binding site for PKC(s) within the carboxyl terminus.

Importantly, PKC- and GRK-mediated phosphorylation of the D1DAR appears to occur independently from one another. This is suggested by the fact that PKC inhibitor pre-treatment did not affect DA-induced receptor phosphorylation (Gardner et al. 2001) and the PKC-null receptor construct desensitized normally in response to agonist (this study). The role of PKC phosphorylation in regulating D1DAR function proved to be rather complicated as we found that both intracellular activators and inhibitors of PKCs led to enhanced DA-stimulated cAMP accumulation. However, it has been previously shown that PKC phosphorylation of specific AC isoforms will increase hormone-stimulated cAMP accumulation (Sibley et al. 1986; Yoshimasa et al. 1987; Sunahara and Taussig 2002) whereas PKC phosphorylation of GPCRs generally leads to desensitization or reduced receptor activity (Sibley et al. 1984; Bouvier 1990). In order to differentiate between these two possibilities we created a PKC-null mutant as described above. In comparison to the WT D1DAR, the PKC-null construct was refractory to the cAMP enhancing effects of PKC inhibitors on cAMP accumulation, whereas the mutant construct responded fully (enhancement of cAMP accumulation) to PKC activators when compared to the WT receptor. These results would seem to confirm that the enhanced cAMP response subsequent to PKC activation is non-receptor mediated whereas the enhanced response to PKC inhibition is mediated by a reduction in the basal or constitutive phosphorylation of the D1DAR. These results were confirmed using [35S]GTPγS binding assays to directly measure D1DAR activation of Gs. In these experiments, the PKC-null receptor construct was significantly more effective in stimulating [35S]GTPγS binding than the WT receptor, indicating that PKC phosphorylation diminishes the ability of the receptor to activate Gs. Importantly, in this model, one must invoke the hypothesis that under basal conditions, PKC phosphorylation of the receptor, rather than non-receptor components, exhibits the predominant functional effect otherwise PKC inhibitor treatment would be expected to decrease rather than increase DA-stimulated cAMP accumulation. In contrast, when PKC is maximally activated such as when the cells are stimulated with phorbol esters, non-receptor components provide the predominant effect of enhancing cAMP accumulation.

In summary, we have shown that the D1DAR is phosphorylated either constitutively or heterologously by PKC and that this negatively regulates receptor-G protein coupling and downstream signaling. We have also previously shown that the D1DAR can be constitutively phosphorylated and desensitized by GRK4 (Rankin et al. 2006). Taken together, these results demonstrate the importance of constitutive mechanisms of GPCR regulation in general and of the D1DAR in particular, and provide a means of modulating the signaling efficacy of the D1DAR in a context-specific manner.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

This research was supported in part by the Intramural Research Program of NINDS and NIMH, NIH. M.L.R. was supported by an Intramural NINDS Competitive Fellowship. We would like to thank the NINDS DNA Sequencing Facility for generating all sequencing data used in this study. We would also like to thank Dr. Varnitha Ramakrishana for the HEK293Tsa201 cells.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
  • Bouvier M. (1990) Cross-talk between second messengers. Ann. N Y Acad. Sci. 594, 120129.
  • Busillo J. M., Armando S., Sengupta R., Meucci O., Bouvier M. and Benovic J. L. (2010) Site-specific phosphorylation of CXCR4 is dynamically regulated by multiple kinases and results in differential modulation of CXCR4 signaling. J. Biol. Chem. 285, 78057817.
  • Chen C. H., Paing M. M. and Trejo J. (2004) Termination of protease-activated receptor-1 signaling by beta-arrestins is independent of receptor phosphorylation. J. Biol. Chem. 279, 1002010031.
  • Ferguson S. S. (2001) Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol. Rev. 53, 124.
  • Fredriksson R. and Schioth H. B. (2005) The repertoire of G-protein-coupled receptors in fully sequenced genomes. Mol. Pharmacol. 67, 14141425.
  • Gainetdinov R. R., Premont R. T., Bohn L. M., Lefkowitz R. J. and Caron M. G. (2004) Desensitization of G protein-coupled receptors and neuronal functions. Annu. Rev. Neurosci. 27, 107144.
  • Gardner B., Hall D. A. and Strange P. G. (1996) Pharmacological analysis of dopamine stimulation of [35S]-GTP gamma S binding via human D2short and D2long dopamine receptors expressed in recombinant cells. Br. J. Pharmacol. 118, 15441550.
  • Gardner B., Liu Z. F., Jiang D. and Sibley D. R. (2001) The role of phosphorylation/dephosphorylation in agonist-induced desensitization of D1 dopamine receptor function: evidence for a novel pathway for receptor dephosphorylation. Mol. Pharmacol. 59, 310321.
  • Gould C. M. and Newton A. C. (2008) The life and death of protein kinase C. Curr. Drug Targets 9, 614625.
  • Guan X. M., Kobilka T. S. and Kobilka B. K. (1992) Enhancement of membrane insertion and function in a type IIIb membrane protein following introduction of a cleavable signal peptide. J. Biol. Chem. 267, 2199521998.
  • Heinzel S. S., Krysan P. J., Calos M. P. and DuBridge R. B. (1988) Use of simian virus 40 replication to amplify Epstein-Barr virus shuttle vectors in human cells. J. Virol. 62, 37383746.
  • Hyun J. S., Baig M. R., Yang D. Y., Leungwattanakij S., Kim K. D., Abdel-Mageed A. B., Bivalacqua T. J. and Hellstrom W. J. (2002) Localization of peripheral dopamine D1 and D2 receptors in rat and human seminal vesicles. J. Androl. 23, 114120.
  • Jala V. R., Shao W. H. and Haribabu B. (2005) Phosphorylation-independent beta-arrestin translocation and internalization of leukotriene B4 receptors. J. Biol. Chem. 280, 48804887.
  • Jiang D. and Sibley D. R. (1999) Regulation of D(1) dopamine receptors with mutations of protein kinase phosphorylation sites: attenuation of the rate of agonist-induced desensitization. Mol. Pharmacol. 56, 675683.
  • Kim O. J., Gardner B. R., Williams D. B., Marinec P. S., Cabrera D. M., Peters J. D., Mak C. C., Kim K. M. and Sibley D. R. (2004) The role of phosphorylation in D1 dopamine receptor desensitization: evidence for a novel mechanism of arrestin association. J. Biol. Chem. 279, 79998010.
  • Krupnick J. G. and Benovic J. L. (1998) The role of receptor kinases and arrestins in G protein-coupled receptor regulation. Annu. Rev. Pharmacol. Toxicol. 38, 289319.
  • Le Good J. A., Ziegler W. H., Parekh D. B., Alessi D. R., Cohen P. and Parker P. J. (1998) Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1. Science 281, 20422045.
  • Manning G., Plowman G. D., Hunter T. and Sudarsanam S. (2002a) Evolution of protein kinase signaling from yeast to man. Trends Biochem. Sci. 27, 514520.
  • Manning G., Whyte D. B., Martinez R., Hunter T. and Sudarsanam S. (2002b) The protein kinase complement of the human genome. Science 298, 19121934.
  • Mason J. N., Kozell L. B. and Neve K. A. (2002) Regulation of dopamine D(1) receptor trafficking by protein kinase A-dependent phosphorylation. Mol. Pharmacol. 61, 806816.
  • Mischak H., Pierce J. H., Goodnight J., Kazanietz M. G., Blumberg P. M. and Mushinski J. F. (1993) Phorbol ester-induced myeloid differentiation is mediated by protein kinase C-alpha and -delta and not by protein kinase C-beta II, -epsilon, -zeta, and -eta. J. Biol. Chem. 268, 2011020115.
  • Monsma Jr F. J., Mahan L. C., McVittie L. D., Gerfen C. R. and Sibley D. R. (1990) Molecular cloning and expression of a D1 dopamine receptor linked to adenylyl cyclase activation. Proc. Natl Acad. Sci. USA 87, 67236727.
  • Namkung Y. and Sibley D. R. (2004) Protein kinase C mediates phosphorylation, desensitization, and trafficking of the D2 dopamine receptor. J. Biol. Chem. 279, 4953349541.
  • Namkung Y., Dipace C., Javitch J. A. and Sibley D. R. (2009a) G protein-coupled receptor kinase-mediated phosphorylation regulates post-endocytic trafficking of the D2 dopamine receptor. J. Biol. Chem. 284, 1503815051.
  • Namkung Y., Dipace C., Urizar E., Javitch J. A. and Sibley D. R. (2009b) G protein-coupled receptor kinase-2 constitutively regulates D2 dopamine receptor expression and signaling independently of receptor phosphorylation. J. Biol. Chem. 284, 3410334115.
  • Neve K. A., Seamans J. K. and Trantham-Davidson H. (2004) Dopamine receptor signaling. J. Recept. Signal Transduct. Res. 24, 165205.
  • Newton A. C. (2010) Protein kinase C: poised to signal. Am. J. Physiol. Endocrinol. Metab. 298, E395E402.
  • Ozono R., O’Connell D. P., Wang Z. Q., Moore A. F., Sanada H., Felder R. A. and Carey R. M. (1997) Localization of the dopamine D1 receptor protein in the human heart and kidney. Hypertension 30, 725729.
  • Rankin M. L., Marinec P. S., Cabrera D. M., Wang Z., Jose P. A. and Sibley D. R. (2006) The D1 dopamine receptor is constitutively phosphorylated by G protein-coupled receptor kinase 4. Mol. Pharmacol. 69, 759769.
  • Rex E. B., Rankin M. L., Ariano M. A. and Sibley D. R. (2008) Ethanol regulation of D(1) dopamine receptor signaling is mediated by protein kinase C in an isozyme-specific manner. Neuropsychopharmacology 33, 29002911.
  • Rex E., Rankin M. L., Yang Y., Lu Q., Gerfen C. R., Jose P. A. and Sibley D. R. (2010) Identification of RanBP 9/10 as Interacting Partners for Protein Kinase C{gamma}/{delta} and the D1 Dopamine Receptor: regulation of PKC-mediated receptor phosphorylation. Mol. Pharmacol. 78, 6980.
  • Reyland M. E. (2009) Protein kinase C isoforms: multi-functional regulators of cell life and death. Front. Biosci. 14, 23862399.
  • Reynolds J. N. and Wickens J. R. (2000) Substantia nigra dopamine regulates synaptic plasticity and membrane potential fluctuations in the rat neostriatum, in vivo. Neuroscience 99, 199203.
  • Richardson M. D., Balius A. M., Yamaguchi K., Freilich E. R., Barak L. S. and Kwatra M. M. (2003) Human substance P receptor lacking the C-terminal domain remains competent to desensitize and internalize. J. Neurochem. 84, 854863.
  • Rossato J. I., Bevilaqua L. R., Izquierdo I., Medina J. H. and Cammarota M. (2009) Dopamine controls persistence of long-term memory storage. Science 325, 10171020.
  • Sibley D. R., Nambi P., Peters J. R. and Lefkowitz R. J. (1984) Phorbol diesters promote beta-adrenergic receptor phosphorylation and adenylate cyclase desensitization in duck erythrocytes. Biochem. Biophys. Res. Commun. 121, 973979.
  • Sibley D. R., Jeffs R. A., Daniel K., Nambi P. and Lefkowitz R. J. (1986) Phorbol diester treatment promotes enhanced adenylate cyclase activity in frog erythrocytes. Arch. Biochem. Biophys. 244, 373381.
  • Sunahara R. K. and Taussig R. (2002) Isoforms of mammalian adenylyl cyclase: multiplicities of signaling. Mol. Interv. 2, 168184.
  • Sunahara R. K., Niznik H. B., Weiner D. M. et al. (1990) Human dopamine D1 receptor encoded by an intronless gene on chromosome 5. Nature 347, 8083.
  • Tiberi M., Nash S. R., Bertrand L., Lefkowitz R. J. and Caron M. G. (1996) Differential regulation of dopamine D1A receptor responsiveness by various G protein-coupled receptor kinases. J. Biol. Chem. 271, 37713778.
  • Tobin A. B., Butcher A. J. and Kong K. C. (2008) Location, location, location...site-specific GPCR phosphorylation offers a mechanism for cell-type-specific signalling. Trends Pharmacol. Sci. 29, 413420.
  • Watts V. J. and Neve K. A. (1996) Sensitization of endogenous and recombinant adenylate cyclase by activation of D2 dopamine receptors. Mol. Pharmacol. 50, 966976.
  • Yoshimasa T., Sibley D. R., Bouvier M., Lefkowitz R. J. and Caron M. G. (1987) Cross-talk between cellular signalling pathways suggested by phorbol-ester-induced adenylate cyclase phosphorylation. Nature 327, 6770.
  • Yu P., Asico L. D., Luo Y., Andrews P., Eisner G. M., Hopfer U., Felder R. A. and Jose P. A. (2006) D1 dopamine receptor hyperphosphorylation in renal proximal tubules in hypertension. Kidney Int. 70, 10721079.

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Figure S1. Quantitation of PKC phosphorylation of the D1DAR. The mean ± SEM of the normalized data collected from seven individual in situ phosphorylation assays performed as described in Fig. 1(c) are reported in the histogram (paired Student’s t-test, where *< 0.05, **< 0.01, and ***< 0.001).

Figure S2. Identification of individual PKC isozymes that can mediate D1DAR phosphorylation. In situ phosphorylation assays performed on HEK293T cells expressing the D1DAR and empty vector (V) or the indicated PKC isozyme as described in Materials and Methods. (+) lanes correspond to cells treated for 30 min with the indicated PKC activator PDBu and/or the PKC inhibitors Gö6976 and Gö6983. Activation of the atypical PKCλ and PKCζ was accomplished by cotransfection of the upstream activator kinase PDK-1. Here, cells were exposed to [32P]orthophosphoric acid for 45 min prior to cell lysis. The amount of D1DAR resolved for each experiment is as follows: 0.5 pmole/lane for PKCβI and PKCδ, 1.4 pmole/lane for PKC and PKCμ, and 1.5 pmole/lane for PKCλ and PKCζ, and 1 pmole/lane for PKCγ. High molecular weight bands present in the PKC and PKCγ autoradiographs represent co-immunoprecipitated FLAG-tagged PKC expression constructs. While PKCμ is not FLAG-tagged, it efficiently co-immunoprecitated with the D1DAR. These experiments were performed at least three times with similar results.

Figure S3. Removal of PKC phosphorylation sites within the D1DAR has no effect on receptor expression. Radioligand binding assay performed on cells expressing the WT or PKC-null D1DAR. In this representative experiment, binding parameters were Bmax = 7.6 pmole/mg protein, KD = 0.20 for WT receptor and Bmax = 7.3 pmole/mg protein, KD = 0.17 for PKC-null D1DAR. This experiment was performed four times with similar results.

Figure S4. Removal of PKC phosphorylation sites within the D1DAR has no effect on DA-induced receptor desensitization. cAMP accumulation assays performed on cells expressing (a) WT or (b) PKC-null D1DAR. Cells were pre-treated for 1 h with 10 μM DA, washed, then challenged with various concentrations of DA followed by cell lysis and cAMP quantitation as described in Materials and Methods. This experiment was performed three times with similar results; the extent of DA-induced desensitization was not statistically different between the two groups (mean ± SEM %desensitization for WT = 42.5 ± 4.9 and for PKC-null = 37.0 ± 2.8), p = 0.26, paired Student’s t-test.

As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

FilenameFormatSizeDescription
JNC_7074_sm_FigS1.eps1229KSupporting info item
JNC_7074_sm_FigS2.eps3381KSupporting info item
JNC_7074_sm_FigS3.eps1058KSupporting info item
JNC_7074_sm_FigS4.eps1237KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.