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Molecular Endocrinology, doi:10.1210/me.2005-0084
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Molecular Endocrinology 20 (8): 1772-1785
Copyright © 2006 by The Endocrine Society

Ghrelin Amplifies Dopamine Signaling by Cross Talk Involving Formation of Growth Hormone Secretagogue Receptor/Dopamine Receptor Subtype 1 Heterodimers

Hong Jiang, Lorena Betancourt and Roy G. Smith

Huffington Center of Aging (H.J., L.B., R.G.S.), and Departments of Molecular and Cellular Biology (H.J., L.B., R.J.S.) and Medicine (R.G.S.), Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: Roy G. Smith, Huffington Center of Aging, Baylor College of Medicine, One Baylor Plaza, M320, Houston, Texas 77030. E-mail: rsmith{at}bcm.tmc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Our objective is to determine the neuromodulatory role of ghrelin in the brain. To identify neurons that express the ghrelin receptor [GH secretagogue receptor (GHS-R)], we generated GHS-R-IRES-tauGFP mice by gene targeting. Neurons expressing the GHS-R exhibit green fluorescence and are clearly evident in the hypothalamus, hippocampus, cortex, and midbrain. Using immunohistochemistry in combination with green fluorescent protein fluorescence, we identified neurons that coexpress the dopamine receptor subtype 1 (D1R) and GHS-R. The potential physiological relevance of coexpression of these two receptors and the direct effect of ghrelin on dopamine signaling was investigated in vitro. Activation of GHS-R by ghrelin amplifies dopamine/D1R-induced cAMP accumulation. Intriguingly, amplification involves a switch in G protein coupling of the GHS-R from G{alpha}11/q to G{alpha}i/o by a mechanism consistent with agonist-dependent formation of GHS-R/D1R heterodimers. Most importantly, these results indicate that ghrelin has the potential to amplify dopamine signaling selectively in neurons that coexpress D1R and GHS-R.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IN 1996, WE REPORTED the expression cloning of an orphan G protein-coupled receptor (GPCR) for the synthetic GH secretagogues (GHS), MK-0677 (1). MK-0677 was developed to restore the amplitude of episodic release of GH in the elderly primarily by activating GHRH neurons in the arcuate nucleus and amplifying GHRH signaling in pituitary somatotrophs (2). Subsequently, by screening tissue extracts in cell lines engineered to stably express the GHS receptor (GHS-R), ghrelin was identified by Kojima and co-workers (3, 4) as an endogenous agonist of GHS-R that stimulated hypothalamic centers involved in regulating GH release and appetite. These properties were later shown to be mediated by the GHS-R, because ghsr–/– mice were refractory to ghrelin treatment (5).

Cross talk between different GPCRs is well known and often results in amplification of cellular responses (6, 7). By investigating the mechanism through which MK-0677 rejuvenated the GH/IGF-I axis, we determined in primary cultures of rat pituitary cells that somatotrophs endogenously coexpress the GHS-R and GHRH receptor (GHRH-R). We showed that MK-0677 amplified GH release by augmenting GHRH-induced cAMP accumulation (6, 8). In addition to rejuvenating the GH axis in elderly persons and in rodents (6), administration of a ghrelin mimetic improved immune function and body composition (9, 10). Because ghrelin receptor (GHS-R) mRNA was also localized in areas of the brain that regulate mood, memory, and learning (11), we speculated that central nervous system function in the elderly might also benefit from administration of ghrelin or a ghrelin mimetic.

Attenuation of dopamine signaling is associated with aging (12). Dopamine receptor subtype 1 (D1R), like the GHRH-R, transduces its signal through cAMP; therefore, we reasoned that ghrelin might also rescue the dopamine aging phenotype if the GHS-R was produced in D1R-expressing neurons. To address this possibility, it was first necessary to determine whether GHS-R and D1R are expressed in the same neurons. Although we had developed antibodies that could detect specific GHS-R expression in stably transfected cell lines by Western blots (13), under rigorously controlled conditions we failed to unequivocally demonstrate specific GHS-R localization in brain sections by immunohistochemistry. We now report the generation of Ghsr-IRES-tauGFP mice by gene targeting and in these mice GHS-R-expressing neurons are clearly identified by green fluorescent protein (GFP) fluorescence. Using D1R-immunofluorescence in brain sections from these mice, we show that GHS-R and D1R are coexpressed in CA1, CA2, CA3, and dentate gyrus of the hippocampal structures as well as in midbrain, substantia nigra, and ventral tegmental area.

The demonstration that the ghrelin receptor (GHS-R) and D1R are coexpressed in certain neurons implicates ghrelin as a neuromodulator of dopamine signaling. Indeed, neuromodulation of dopamine signaling by ghrelin in vivo was suggested by the observation that ghrelin administration to rats dose-dependently augments cocaine-induced hyperactivity (14). Having shown that Ghsr and D1R are coexpressed in a population of neurons, it became important to determine whether ghrelin had the potential to modulate dopamine signaling. Although, ideally, the direct effects of ghrelin on these neurons should be studied in vivo, unequivocally distinguishing direct from indirect actions of ghrelin on dopamine signaling is confounded by the observation in Ghsr-IRES-tauGFP mice that not all populations of neurons coexpress both receptors, but express Ghsr and D1R independently.

Possible interactions between the GHS-R and D1R signaling pathways had not been previously been investigated; therefore, we wanted to ascertain whether ghrelin was capable of directly modifying dopamine action in cells that express both receptors and, if so, by what mechanism. To test the potential functional significance of GHS-R and D1R coexpression, we exploited cell models. We found that ghrelin potentiates dopamine-induced cAMP accumulation, and that potentiation is dependent upon expression of the GHS-R. Remarkably, the mechanism involves alteration of GHS-R G protein coupling, which is associated with formation of GHS-R/D1R heterodimers.

The demonstration of augmentation of dopamine-induced cAMP accumulation by activation of the ghrelin receptor may have profound implications for age-related memory loss. Indeed, extensive studies by Kandel and co-workers (15) show that learning and memory are improved by increasing cAMP accumulation in neurons. The studies described herein provide a novel approach of selectively increasing cAMP levels in specific neurons.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of Neurons Coexpressing the Ghrelin Receptor (GHS-R) and D1R
By using the targeting vector shown in Fig. 1Go, we established ES cell clones with LNL-IRES-tauGFP (16) integrated into the Ghsr-locus. After selection of appropriately targeted clones, neo was excised by Cre recombination. The ES cells were injected into mouse blastocysts to produce germline chimeras and subsequently a colony of Ghsr-IRES-tauGFP mice. Translation and processing of the bicistronic mRNA produced from the derivatized Ghsr-locus produces Ghsr and tauGFP, allowing identification of Ghsr-expressing neurons. Examination of serial brain sections from Ghsr-IRES-tauGFP mice showed GFP fluorescence in CA1, CA2, CA3, and dentate gyrus of the hippocampus, in neurons of the substantia nigra, ventral tegmental area, arcuate nucleus, medial septal nucleus, amygdala, lateral entorhinal and auditory/somatosensory area of the cortex, olfactory tubercule, and medial preoptic area (data not shown). Indeed, localization of Ghsr neurons in these areas is consistent with previous results, where we localized expression of Ghsr mRNA by in situ hybridization (11). Figure 1CGo illustrates GFP expression in neurons of a Ghsr-IRES-tauGFP mouse.


Figure 1
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  		Fig. 1. Generation of Ghsr-IRES-tauGFP Knock-In Mice

A, Schematic representative of the endogenous Ghsr locus, targeting vector, and targeted allele. a, Wild-type Ghsr locus: black lines represent noncoding, and red boxes represent coding regions; restriction sites are indicated for BamHI; also shown are ATG start and TGA stop codons. b, Ghsr-IRES-tauGFP-LNL targeting vector: the white box labeled "i" represents the IRES sequence; the blue box represents the coding sequence of tauGFP; the gray box labeled "neo" represents the neo-selectable marker LNL flanked by loxP sites (indicated by black triangles). c, Ghsr locus after homologous recombination with the Ghsr-IRES-tauGFP-LNL targeting vector: the probe used to detect homologous recombination in Southern blots is represented as a black box on the right. d, Ghsr locus after Cre-mediated excision of the neo cassette: the PCR probe used to genotype mice is represented as {rightleftarrows}. B, a, Southern blot analysis of DNA from Ghsr-IRES-tauGFP-LNL targeted ES cells (T) and wild type ES cells (WT). Genomic DNAs were digested with BamHI and the 5.8- and 13.5-kb bands represent targeted and endogenous alleles, respectively. b, PCR genotyping of DNA from Ghsr-IRES-tauGFP homozygous (G) and wild-type mice (WT). M represents the DNA kilobase markers. C, GFP-labeled neurons in the brain of Ghsr-IRES-tauGFP knock-in mouse (high magnification).

 
To investigate whether Ghsr and D1R were expressed in the same neurons, we immunostained brain sections from homozygous Ghsr-IRES-tauGFP mice with mouse D1R antibody and a second antibody conjugated with Alexa 594. We found that DR1 is coexpressed with Ghsr in neurons localized in cortex, hippocampal structures, substantia nigra, midbrain, and ventral tegmental areas. As examples, we show DR1 and Ghsr (GFP) coexpression in neurons localized in ventral tegmental area and substantia nigra (Fig. 2Go, A and B).


Figure 2
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  		Fig. 2. Identification of Neurons Coexpressing Ghsr and D1R in Mouse Brain

Serial sections of brains from Ghsr-IRES-tauGFP knock-in homozygous mice were stained with mouse D1R antibody. Ghsr-expressing neurons are localized by GFP imaged in green in a and d. D1R expression neurons are localized by Alexa 594 imaged in red fluorescence in b and e. Coexpression of Ghsr and D1R can be seen by the yellow-orange color in the merged images in c and f. A, Neurons are localized in ventral tegmental areas. B, Neurons are localized in substantia nigra.

 
Functional Interaction of GHS-R and D1R in HEK293 Cells
Dopamine activation of D1R increases intracellular cAMP accumulation. To investigate the potential for receptor cross talk, we treated HEK293 cells that express human GHS-R and D1R with ghrelin and dopamine. HEK293 cells were selected because they have been used extensively for establishing mechanisms of G protein interactions and regulation of adenylyl cyclase (AC) isozyme activity. Treatment of D1R/GHS-R-expressing cells with dopamine showed a dose-dependent increase in cAMP accumulation. Remarkably, treatment with dopamine and ghrelin together produced a marked increase (3- to 4-fold) in cAMP accumulation compared with cells treated with dopamine alone (Fig. 3AGo). The enhanced D1R signaling required both receptors and both agonists, because it was not observed when ghrelin was added to cells expressing D1R alone (Fig. 3BGo). Furthermore, the increase in cAMP accumulation was a result of synergy, because in the absence of dopamine, ghrelin activation of GHS-R in the D1R/GHS-R-expressing cells did not increase cAMP accumulation (Fig. 3CGo). The observation that ghrelin alone had no effect on cAMP levels is consistent with previous characterization of GHS-R signaling through the phospholipase C pathway, which occurs in primary cultures of rat pituitary cells, as well as in HEK293 cells (1, 6). Together, these results demonstrate that ghrelin-mediated amplification of dopamine-induced cAMP accumulation is a consequence of a functional interaction between GHS-R and D1R.


Figure 3
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  		Fig. 3. Coactivation of GHS-R and D1R Results in Amplification of Dopamine-Induced cAMP Accumulation in HEK293 Cells

A, Synergistic effects of ghrelin on dopamine-induced cAMP accumulation in HEK293 cells expressing GHS-R and D1R. The cells were incubated with various concentrations of dopamine without or with 100 nM ghrelin for 15–20 min. The cAMP levels were determined as described in Materials and Methods. B, Ghrelin has no effect on dopamine-induced cAMP accumulation in HEK293 cells expressing dopamine D1R alone. The cells were incubated with various concentrations of dopamine without or with 100 nM ghrelin for 15–20 min. C, Effects of various concentrations of ghrelin on cAMP accumulation in HEK293 cells expressing GHS-R and D1R. The cells were incubated with ghrelin alone for 15–20 min. Intracellular basal (nonstimulated) cAMP level was used for comparison. The data represents the mean ± SEM of three independent experiments performed in triplicate for each concentration point.

 
We next investigated whether cross talk between D1R and GHS-R modifies GHS-R signaling. As reported previously, GHS-R activation transduces its signal through phospholipase C and increases intracellular Ca2+ (6). We routinely measure changes in intracellular Ca2+ in response to ghrelin and ghrelin mimetics by Ca2+-induced aequorin bioluminescence in HEK293 cells stably expressing aequorin (HEK293-AEQ17) (13). HEK293-AEQ17 cells were transfected with D1R and GHS-R expression plasmids, and the effects of dopamine alone, ghrelin alone, and ghrelin plus dopamine on Ca2+-induced aequorin bioluminescence were compared. Ghrelin, but not dopamine, induced aequorin bioluminescence and the combination of ghrelin and dopamine failed to increase aequorin bioluminescence beyond that induced by ghrelin (data not shown).

Ghrelin Enhances Dopamine-Induced cAMP Accumulation Independent of Protein Kinase C (PKC) Signaling
The potential for ghrelin and ghrelin mimetics to amplify a GPCR-induced cAMP increase was first observed in pituitary somatotrophs that express GHS-R and GHRH-R endogenously (8). Treating somatotrophs with GHRH increased cAMP accumulation; coadministration of GHRH with ghrelin mimetics potentiated GHRH-induced cAMP accumulation and GH release through a PKC-mediated pathway (8, 17). To determine whether these physiologically relevant results might extrapolate to HEK293 cells, we compared the effects of GHRH, ghrelin, and GHRH plus ghrelin in HEK293 cells transiently expressing the GHS-R and GHRH-R. In agreement with our results in somatotrophs, ghrelin amplified GHRH-induced cAMP accumulation (data not shown). Furthermore, as observed in primary pituitary cells, the effects of ghrelin were partially mimicked by the PKC activator Phorbol 12-myristate 13-acetate (PMA) and inhibited by a selective PKC inhibitor bisindolylmaleimide I (Bis) (data not shown).

Accordingly, we investigated whether PKC signaling was involved in amplification of dopamine-induced cAMP accumulation. When D1R/GHS-R-expressing HEK293 cells were treated with dopamine and PMA, the cAMP level was not changed compared with treatment with dopamine alone (Fig. 4AGo). Furthermore, when D1R/GHS-R cells were pretreated with Bis, ghrelin still caused amplification of dopamine-induced cAMP accumulation (Fig. 4BGo), indicating that PKC signaling is not involved in the synergistic effect of ghrelin on D1R signaling.


Figure 4
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  		Fig. 4. Amplification of D1R Signaling by Ghrelin in HEK293 Cells Is Not Mediated by PKC but by a PTX-Inhibitable Pathway

A, HEK293 cells expressing D1R/GHS-R were incubated with or without the PKC activator, PMA (phorbol 12-myristate 13-acetate), and dopamine for 15 min. The cAMP levels were determined as described in Materials and Methods. Intracellular basal (nonstimulated) cAMP of HEK293 cells expressing D1R/GHS-R was standardized as unity. The data represent the mean ± SEM of three independent experiments performed in triplicate for each concentration point. B, HEK293 cells expressing D1R/GHS-R were preincubated with Bis, a PKC inhibitor for 30 min, and then stimulated with both dopamine and ghrelin for 15–20 min. Intracellular basal (nonstimulated) cAMP of HEK293 cells expressing D1R/GHS-R was standardized as unity. The data represent the mean ± SEM of three independent experiments performed in triplicate for each concentration point. C, HEK293 cells expressing D1R/GHS-R were preincubated overnight with PTX, and then stimulated with both dopamine and ghrelin for 15–20 min. The cAMP levels were determined as described in Materials and Methods. PTX treatment has no significant effects on basal (nonstimulated) cAMP level and dopamine-induced cAMP level, but significantly reduced ghrelin amplification of dopamine-induced cAMP accumulation. *, P < 0.05 compared with no treatment with PTX. Intracellular basal (nonstimulated) cAMP of HEK293 cells expressing D1R/GHS-R was standardized as unity. The data represent the mean ± SEM of three independent experiments performed in triplicate for each concentration point.

 
Coupling with G{alpha}i/o Protein Is Involved in Amplification of D1R-Induced cAMP Accumulation by Ghrelin
Ghrelin binds to the GHS-R which couples to phospholipase C via G{alpha}11, and transduces its signal via inositol 1,4,5-triphosphate, diacylglycerol, and PKC (6). However, the results shown in Fig. 4Go, A and B, indicate that PKC signaling is not involved in the mechanism of amplification of dopamine/D1R signaling. To investigate whether ghrelin and dopamine in combination transduced their signal by a mechanism involving coupling to G{alpha}i/o, we asked whether pertussis toxin (PTX) would attenuate ghrelin amplification of dopamine signaling. As shown in Fig. 4CGo, PTX inhibits the synergistic effect of ghrelin on dopamine-induced cAMP accumulation. These results indicate that a PTX-sensitive G{alpha}i/o protein is involved in the amplification of cAMP signaling. In parallel, we investigated the effects of PTX on ghrelin amplification of GHRH-R signaling. PTX failed to block ghrelin amplification of GHRH-induced cAMP accumulation (data not shown).

D1R and GHS-R Form Heterodimers in Living Cells
We next addressed how coactivation of D1R and GHS-R causes switching of GHS-R G protein coupling from G{alpha}11/q to G{alpha}i/o, and we investigated the possibility of GHS-R and D1R forming heterodimers. First, we investigated the subcellular distribution of D1R and GHS-R in cells by fluorescence deconvolution microscopy in a neuronal cell line. SK-N-SH cells were transfected with D1R and GHS-R-GFP expression plasmids. The cells were immunostained with antihuman D1R antibody and Texas Red-conjugated second antibody. As shown in Fig. 5AGo, in the absence of agonists, both D1R (red fluorescence) and GHS-R-GFP (green fluorescence) are prominently colocalized on the plasma membrane. When cells were treated with dopamine and ghrelin, redistribution and coaggregation were observed (Fig. 5BGo). Agonist-dependent aggregation and colocalization indicated that D1R and GHS-R could cooperate and associate via the same trafficking pathway.


Figure 5
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  		Fig. 5. Distribution of D1R and GHS-R in SK-N-SH Cells

SK-N-SH cells cotransfected with D1R and GHS-R-GFP were incubated for 15–20 min with medium in the absence (A) or presence (B) of 10 µM dopamine and 100 nM ghrelin, and processed for immunostaining using Texas Red-conjugated rabbit anti-D1R antibody. Deconvolution microscope was employed to visualize GFP (green images) and Texas Red (red images). GFP and Texas Red images were merged using deconvolution microscope softWoRx software to reveal D1R/GHS-R-GFP colocalization (yellow images).

 
To determine whether D1R and GHS-R were physically associated as a heteromeric complex, we used a D1R antibody to immunoprecipitate D1R from cell lysates of HEK293 cells expressing D1R and Xpress-tagged GHS-R. Western blot analysis using anti-Xpress showed that the D1R antibody coimmunoprecipitated the Xpress-GHS-R 48-kDa protein (Fig. 6AGo, lanes 4 and 5). This band was not detected when an irrelevant rabbit IgG was used (Fig. 6AGo, lanes 2 and 3) or when extracts from cells expressing D1R alone were analyzed (Fig. 6AGo, lane 1). Further confirmation of heterodimerization was obtained when anti-Xpress was used to perform immunoprecipitation followed by Western analysis with anti-D1R. In this case, an anti-D1R reactive band of 46 kDa was observed in D1R/GHS-R cells treated with ghrelin and dopamine (Fig. 6BGo, lane 3), but not in extracts from GHS-R cells (Fig. 6BGo, lane 1). Western analyses also indicated the formation of GHS-R and D1R immunoactive molecular aggregates (~100 kDa) that represented approximately 80% of the immunoprecipitated protein. These immunoactive aggregates were only observed in anti-D1R and anti-Xpress immunoprecipitates and not when an irrelevant IgG was used. This indicates that the majority of the GHS-R/D1R heterodimers immunoprecipitate as oligomers that are resistant to dissociation (data not shown). Although these results are consistent with a physical association between GHS-R and D1R in HEK293 cells, they are inconclusive because of the tendency for highly hydrophobic GPCRs to aggregate during cell lysis.


Figure 6
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  		Fig. 6. Coimmunoprecipitation of D1R and GHS-R in HEK293 Cells

A, HEK293 cells were transfected with either D1R alone (lane 1), or with D1R and Xpress-GHS-R (lanes 2–5); before lysis, the cells were treated without (lanes 1, 2, and 5) or with 10 µM dopamine and 100 nM ghrelin (lanes 3 and 4) for 15–20 min at 37 C. Cell lysates were centrifuged at 12,000 x g for 10 min, supernatants were withdrawn, and aliquots (500 µg protein) were incubated with protein A-agarose and immunoprecipitated with anti-D1R antibody (lanes 1, 4, and 5) or with an irrelevant rabbit IgG (lanes 2 and 3). Samples were subjected to SDS-PAGE and immunoblotted with anti-Xpress. B, HEK293 cells were transfected with either Xpress-GHS-R alone (lane 1), or with D1R and Xpress-GHS-R (lanes 2 and 3). The cell lysates were centrifuged at 12,000 x g for 10 min, supernatants were withdrawn, and aliquots (500 µg protein) were incubated with protein G-agarose and immunoprecipitated with anti-Xpress antibody and immunoblotted with anti-D1R.

 
To seek more conclusive evidence for GHS-R/D1R heterodimerization, we investigated the association of D1R with GHS-R using bioluminescence resonance energy transfer (BRET). This proximity assay, based on nonradiative transfer between a bioluminescent donor Renilla luciferase (Rluc) and a fluorescent acceptor (GFP), allows real-time monitoring of protein-protein interactions in living cells (18). Most importantly, the BRET assay is far more sensitive than immunoprecipitation. It allows real-time measurements in cells at physiologically relevant GPCR concentrations and ample sensitivity to conduct GPCR/GPCR dose titrations.

For the BRET assay, GHS-R and D1R were tagged at their carboxyl terminus with Rluc or GFP, so that each receptor could be expressed in HEK293 cells with either GFP or Rluc tags. BRET was measured in HEK293 cells cotransfected with D1R-Rluc and GHS-R-GFP expression plasmids. As shown in Fig. 7AGo, a significant increase in the BRET ratios was detected when the D1R-Rluc/GHS-R-GFP-expressing cells were incubated with dopamine and ghrelin (P < 0.05), suggesting that the two agonists promoted heterodimerization of GHS-R and D1R.


Figure 7
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  		Fig. 7. Agonist-Dependent Heterodimerization of D1R and GHS-R and Constitutive GHS-R Homodimerization in Living Cells Detected by BRET

A, BRET ratio was measured in HEK293 cells cotransfected with D1R-Rluc (0.1 µg) and GHS-R-GFP in different amount (0, 0.1, 0.4, 0.8 µg), and the total amount of DNA transfected in each well was completed to 1 µg with empty vector. The cells were incubated in the presence or absence of dopamine (10 µM) and ghrelin (100 nM) for 15–20 min before addition of Rluc substrate (DeepBlue C). The data represent the means ± SD of three independent experiments each performed in triplicate. *, P < 0.05 compared with in the absence of both ligands. B, BRET ratio was measured in HEK293 cells that were cotransfected with 0.1 µg GHS-R-Rluc or 0.1 µg D1R-Rluc and different amounts ranging from 0.1–1.8 µg of GHS-R-GFP in the absence of agonists. The total fluorescence and luminescence were used as relative measures of total expression of the acceptor and donor proteins, respectively. Saturation curve was obtained by plotting BRET ratio as a function of the [acceptor]/[donor] ratio. The figure represents one of three experiments that gave similar results. C, BRET ratio was measured in HEK293 cells cotransfected with GHS-R-Rluc (0.1 µg) and GHRH-R-GFP in different amount (0.1, 0.4, and 0.8 µg), and the total amount of DNA transfected in each well was completed to 1 µg with empty vector. The cells were incubated in the presence or absence of 100 nM ghrelin and different concentrations of GHRH (1, 10, and 100 nM). The data represent the means ± SD of three independent experiments, each performed in triplicate.

 
Besides being consistent with agonist-induced promotion of heterodimer formation, the increased BRET ratio shown in Fig. 7AGo could also be explained by a change in conformation of preexisting heterodimers of GHS-R and D1R resulting in closer proximity or more favorable orientation of donor and acceptor (19). To investigate this possibility, we conducted BRET titration assays. As shown in Fig. 7BGo, significant concentration-dependent increases in the BRET ratio were observed for the GHS-R-GFP/GHS-R-Rluc pair but not for GHS-R-GFP/D1R-Rluc pair. Importantly, the observation that the BRET signal increases as a hyperbolic function of GHS-R-GFP/GHS-R-Rluc ratio is consistent with a specific protein-protein interaction. Random molecular collisions would be expected to cause the BRET ratio to increase linearly over a wide range of GFP/Rluc concentrations (20, 21, 22). Hence, in the absence of ghrelin and dopamine, the GHS-R forms constitutive homodimers; ghrelin and dopamine are required to induce formation of GHS-R/D1R heterodimers.

To determine whether heterodimer formation was a common mechanism leading to amplification of cAMP accumulation, we investigated the GHRH-R as a potential GPCR partner for heterodimerization with the GHS-R. Like D1R, the GHRH-R is G{alpha}s coupled and when coexpressed with GHS-R in either HEK293 cells or endogenously in somatotrophs, GHRH-induced cAMP accumulation is enhanced by ghrelin treatment. BRET studies using GHS-R-Rluc and GHRH-R-GFP showed in contrast to GHS-R-Rluc and D1R-GFP the GHS-R and GHRH-R pair did not form heterodimers either in the presence or absence of ghrelin and GHRH (Fig. 7CGo). Hence, formation of GHS-R heterodimers is dependent upon the GPCR partner, and amplification of cAMP accumulation by ghrelin is not required for GHS-R/GHRH-R dimer formation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
To investigate the function of ghrelin in the brain, we generated Ghsr-IRES-tauGFP mice. These mice allow us to clearly identify Ghsr (ghrelin receptor)-expressing neurons and identify which neurotransmitter receptors are coexpressed. In this study, we focused on the dopamine pathway, because it had been shown that ghrelin dose-dependently amplified cocaine-induced hyperactivity in rats. By immunostaining brain sections from Ghsr-IRES-tauGFP mice to detect D1R, we identified neurons that expressed both Ghsr and D1R. This led us to ask whether activation of Ghsr by ghrelin might augment D1R signaling and potentially explain ghrelin modulation of dopamine action.

The presence of Ghsr and D1R in the same neurons suggested the possibility of cross talk between ghrelin and dopamine signaling pathways. Under physiological conditions, cells are stimulated by individual agonists or several agonists simultaneously according to the temporal pattern of endogenous release and half-life of each agonist. Thus, stimulation of one receptor leads to activation of a signaling pathway that has the potential to interact with pathways activated by other receptors. This process ensures exchange of information between individual signaling pathways and provides a molecular basis for their cooperation (23, 24, 25, 26). Because it is not possible to separate direct from indirect effects of ghrelin on dopamine pathways in vivo, we addressed the potential for cross talk in GHS-R- and D1R-expressing neurons by using a model system that allowed us to determine whether ghrelin and the GHS-R could directly modulate D1R signaling.

We selected HEK293 cells as a model to study the consequences of activating GHS-R and D1R in the same cell. To first determine whether results from the HEK293 cell system might reflect what occurs in native cells, we investigated ghrelin amplification of GHRH signaling. Pituitary somatotrophs coexpress GHS-R and GHRH-R endogenously, and we had previously shown that ghrelin mimetics amplify GHRH-induced cAMP accumulation in primary cultures of rat somatotrophs by a mechanism involving GHS-R-mediated activation of PKC (17, 27). We investigated whether this same mechanism applied when GHS-R and GHRH-R were expressed in HEK293 cells. As in somatotrophs endogenously expressing GHS-R and GHRH-R, we found that, in HEK293 cells transfected with GHS-R and GHRH-R, ghrelin amplified GHRH-induced cAMP accumulation and that augmentation was inhibited by a selective PKC inhibitor (Bis). Therefore, at least in the case of the GHS-R and GHRH-R, experiments in HEK293 cells reflect what occurs in native cells.

When HEK293 cells transfected with GHS-R and D1R were treated with ghrelin and dopamine, ghrelin caused augmentation of dopamine-induced cAMP accumulation (Fig. 3Go). Intriguingly, whereas both GHRH and dopamine activate their respective receptors in HEK293 cells to result in increased cAMP accumulation, in each case coactivation of the GHS-R by ghrelin augments cAMP accumulation through distinct mechanisms. With amplification of GHRH-R signaling, the well-characterized GHS-R-G{alpha}11/q signal transduction pathway (phospholipase C, diacylglycerol, PKC) is apparently involved and the synergy is blocked by a specific PKC inhibitor (Bis), but not by PTX. In the case of ghrelin amplification of D1R-induced cAMP accumulation, the synergism is not inhibited by Bis, but the ghrelin effect is blocked by PTX.

Signal transduction involving activation of AC is complex because 10 different AC isozymes are known (28). In most cell types, including somatotrophs and HEK293 cells, ghrelin activation of the GHS-R alone does not involve AC and transduces its signal through G{alpha}11/q and phospholipase C. Until recently, the contributions of Gß{gamma} subunits to the modulation of AC activity had been largely unappreciated (28). G{alpha}s subunits were assumed to predominate in the regulation of AC; however, the ß{gamma} subunits are powerful modulators of AC activity that can be stimulatory as with AC2, AC4, and AC7, or inhibitory, as for AC1 and AC8. The ß{gamma} subunits only stimulate cyclase activity when G{alpha}s is coactivated and accordingly establish a synergistic relationship. Therefore, activation of hormone receptors coupled to G{alpha}i subunits liberate ß{gamma} subunits and potentiate AC activity that is stimulated by a distinct, G{alpha}s-activated, hormone receptor. Importantly, AC isoforms that undergo stimulation by Gß{gamma} (AC2, AC4, and AC7) are not directly regulated by {alpha} subunits of the Gi family (28).

When the D1R and GHS-R are costimulated by their respective agonists, cross talk between their signaling pathways alters the signal transduction of GHS-R. We propose that the GHS-R activates a G protein of the G{alpha}i family, causing dissociation of ß{gamma} subunits that amplify G{alpha}s-D1R-mediated cAMP accumulation via activation of AC2 (28). Ghrelin alone does not change cAMP levels in HEK293 cells coexpressing GHS-R and D1R, but when both receptors are stimulated by treating the cells with ghrelin and dopamine, ghrelin increases dopamine-induced cAMP accumulation. Although PTX does not inhibit dopamine-induced cAMP accumulation, in the presence of both dopamine and ghrelin the synergistic effect of ghrelin is inhibited by PTX, which is consistent with potentiation of an AC2 mediated pathway by ß{gamma} subunits derived from G{alpha}i (28). A similar pathway was proposed to explain coactivation of AC2 by cross talk between ß-adrenergic receptors and bradykinin B2 receptors (29). Amplification of GHRH-induced cAMP accumulation by ghrelin can also be explained by amplification of AC2 activity, but in this case we speculate that increased AC2 activity is caused by PKC-mediated direct phosphorylation of AC2 (28), which is inhibited by Bis and not by PTX.

To explain how G protein coupling of GHS-R is modified by coexpression and coactivation of the D1R on a molecular basis, we hypothesized that ghrelin and dopamine induce GHS-R/D1R heterodimer formation to facilitate access of the G{alpha}i/o protein complex to the GHS-R (Fig. 8Go). The concept of GPCRs existing as functional homo- and heterodimers is gaining acceptance because of evidence for their role in receptor trafficking, cellular signaling, and pharmacological function (30, 31, 32, 33, 34, 35). To form heterodimers, the proteins must colocalize in the same subcellular area. By using deconvolution fluorescence imaging, we showed colocalization of GHS-R and D1R in the SK-N-SH neuronal cell line (Fig. 5Go).


Figure 8
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  		Fig. 8. Hypothetical Model Illustrating a Molecular Basis for Cross Talk between GHS-R and D1R Signaling that Augments Dopamine-Induced cAMP Accumulation

Dopamine and ghrelin induce the formation of [GHS-R/D1R]2 heterooligomers that for clarity are shown as heterodimers. D1R couples through G{alpha}s which activates AC2 isozyme (AC2); by conformational change induced by heterodimer formation, GHS-R couples with G{alpha}i protein, releasing ß{gamma} subunits that associate with AC2, thereby amplifying AC2 activity. Ghr, Ghrelin; DA, dopamine.

 
Colocalization of two proteins does not necessarily mean they have physical interaction. However, immunoprecipitation of cell lysates from cells expressing GHS-R and D1R using an antibody for tagged GHS-R, followed by Western blots with a D1R antibody, was consistent with formation of heterodimers in the presence of dopamine and ghrelin (Fig. 6AGo). Identical results were obtained when the D1R antibody was used for immunoprecipitation and an antibody for tagged GHS-R used in Western blots (Fig. 6BGo). A weak association between D1R and GHS-R was detected in the absence of both agonists when a D1R antibody was used to perform immunoprecipitation (Fig. 6AGo), but this is likely explained by low-level artifactual aggregation of receptors. Indeed, although GPCRs were reported to form both homodimers and heterodimers (19, 30, 31, 32, 36, 37, 38), there has been reluctance to acknowledge the physiological relevance of GPCR dimerization based on potential artifacts formed during immunoprecipitation. For example, low sensitivity of this method demands that the GPCRs must be expressed at high nonphysiological levels, and their extreme hydrophobicity require they be extracted from cell membranes before immunoprecipitating; under these conditions, artifactual dimerization might be created by nonspecific aggregation.

To avoid artifacts, more sensitive methods for detecting dimerization were developed (18) (19, 36). We selected the highly sensitive BRET assay to investigate the possible formation of GHS-R/D1R heterodimers in living cells, because it has the added advantage of monitoring interactions of proteins in real time (39). We showed by BRET assays that in the absence of dopamine and ghrelin no significant energy transfer was detected in D1R-Rluc/GHS-R-GFP cells, whereas, in the presence of dopamine and ghrelin, there was a significant increase in the BRET ratio, indicating an increase in the number of heterodimers formed.

We tested whether our BRET results could be explained by a change in conformation of preexisting complexes. By generating GHS-R/GHS-R and GHS-R/D1R BRET dose titration curves using different ratios of GHS-R and D1R, we showed that the BRET signal increased as a hyperbolic function, which is consistent with a specific protein-protein interaction. In the absence of agonists, the GHS-R/D1R pair does not form heterodimers (Fig. 7BGo). Interestingly, the titration curves indicate that the GHS-R constitutively forms homodimers, which has also been observed for the D1R (40); therefore, what we call GHS-R/D1R heterodimers might be large hetero-oligomers and because it is not easy to distinguish dimers from larger oligomers, we use the term dimer as the simplest form of oligomer. Indeed, to accommodate two heterotrimeric G protein complexes in a transition state, we postulate formation of a [GHS-R/D1R]2 heterotetramer (41).

The BRET assay monitors dimer formation, and not necessarily the state of activity of the receptor complex. However, our BRET results are complemented by functional data showing that heterodimerization induced by a combination of dopamine and ghrelin is associated with enhanced cAMP accumulation, as well as modification of GHS-R signaling. These observations suggest that ghrelin and dopamine induce GHS-R and D1R receptor heterodimerization to form a G protein complex with increased signaling capacity. In this particular case, heterodimerization provides a mechanism through which ghrelin can amplify dopamine signaling. Heterodimerization is not always required for signal amplification because ghrelin amplifies GHRH-induced cAMP accumulation in HEK293 cells coexpressing GHS-R and GHRH-R without forming BRET-detectable GHS-R/GHRH-R heterodimers.

An alternative explanation for the agonist-induced increased BRET ratio is molecular crowding of GHS-R and D1R in internalization vesicles. GHS-R, D1R, and GHRH-R internalize after agonist treatment. However, the modifications in G protein coupling and signal transduction caused by coactivation of GHS-R and D1R would precede internalization. In the case of GHS-R and D1R, ghrelin potentiation of dopamine-induced cAMP accumulation is associated with an increased BRET ratio, whereas ghrelin potentiation of GHRH-induced cAMP accumulation is not accompanied by changes in the BRET ratio. Therefore, we consider molecular crowding of GHS-R and D1R in vesicles after internalization a less likely interpretation of ghrelin- and dopamine-induced increase in the BRET ratio, but our experiments do not rule out this possibility.

Our proposed model for explaining the synergistic effect of ghrelin on dopamine signaling is depicted by Fig. 8Go. The amplification of dopamine-induced cAMP accumulation by ghrelin is associated with formation of GHS-R and D1R heterodimers that we suggest oligomerize to produce a heterotetrameric functional unit that will more readily accommodate two heterotrimeric G protein complexes (G{alpha}s and G{alpha}i/o) (41). We speculate that weak latent coupling exists between GHS-R and G{alpha}i/o proteins and that a conformational change accompanying GHS-R/D1R heterodimer formation strengthens the interaction with G{alpha}i/o. Indeed, although D1R is coupled primarily to G{alpha}s, D1R has the potential to couple to PTX-sensitive G{alpha}i/o (42, 43), which may facilitate G{alpha}i/o interactions with GHS-R/D1R.

An in vivo link between ghrelin and dopamine was suggested by the observation in rats that ghrelin administration increased dopamine-related hyperactivity (14). We have identified for the first time populations of neurons in the ventral tegmental area, substantia nigra, hippocampal structures, and dentate gyrus that coexpress GHS-R and D1R. Unambiguous demonstration of the effects of ghrelin and dopamine agonists on these neurons in vivo is confounded by independent effects on individual neurons that express either D1R or GHS-R. Nevertheless, in this paper, we establish the principle that ghrelin amplifies dopamine-induced cAMP accumulation in cells coexpressing GHS-R and D1R. The potential physiological and clinical significance of these results are profound. Increases in cAMP are involved in the acute effects of dopamine, in dopamine control of gene expression and synaptic plasticity, and in the long-lasting changes induced by repeated administration of drugs of abuse. Regulation of cAMP signaling in specific neurons plays important roles in locomotion, mood, memory, and learning functions. Extensive studies by Kandel and co-workers (15) show that learning and memory are improved by augmentation of cAMP accumulation. Therefore, amplification of cAMP-activated signaling pathways through specific receptor interactions in the same cell is likely to play important physiological roles.

In summary, our results demonstrate that GHS-R and D1R are coexpressed in the same neurons and that ghrelin has the capacity to amplify dopamine signaling by a mechanism associated with GHS-R/D1R heterodimer formation. Because ghrelin production is cyclical, this pathway provides temporal control over the magnitude of dopamine signaling in neurons expressing both receptors. A decline in dopamine signaling occurs during aging that is not accompanied by reduced D1R expression in humans, but might be linked to the precipitous decline in expression of the dopamine transporter (44, 45). Therefore, amplifying D1R signaling in a neuronal specific way by administering ghrelin or a ghrelin mimetic could counteract age-related attenuation of dopamine function and benefit cognitive function in the elderly. Finally, our findings provide new information on the potentially broad neuromodulatory roles of ghrelin and GHS-R and invite speculation on their potential for a more profound role in modifying intracellular signaling of other neurotransmitter-activated GPCRs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Construction of the Targeting Vector and Gene Targeting
PCR-isolated mouse Ghsr genomic DNA fragments were inserted onto XhoI and SacI sites of plasmid IRES-tauGFP-LNL (a gift from Dr. Peter Mombaerts, The Rockefeller University, New York, NY) to make the targeting vector. The targeting vector Ghsr-IRES-tauGFP-LNL was linearized at the unique PvuI site. Electroporation and cell culture of AB2.2 was done by The Darwin Transgenic Mouse Core of Baylor College of Medicine. Genomic DNA from G418-resistant ES colonies was digested with BamHI and analyzed by Southern blot hybridization, yielding a 5.8-kb band with a 3' probe external to the targeting vector (Fig. 1BaGo). Then the neo-selectable marker was removed from the targeted ES cells by Cre recombination (Fig. 1Go).

Generation of Ghsr-IRES-tauGFP Knock-In Mice
The targeted ES cells were microinjected into blastocysts. Germline transmission and homozygous mice were obtained and confirmed by PCR genotyping (Fig. 1BbGo). PCR primers used for genotyping were as follows: 5'-CTGAAGGATGAGTTCCCGGGCCTGGACAAAGT-3' and 5'-GAGAGCTCGTGCTCCCAAGGCACCTATCACTG-3'. The targeted allele yields 6.5-kb DNA fragments, and the wild-type allele yields 3.6-kb DNA fragments as shown in Fig. 1BGo. The engineered mice are in a mixed (129 x C57BL/6) background.

Immunofluorescence Analysis of Mice Brain Sections
Brains were harvested from adult male Ghsr-IRES-tauGFP mice. Tissues were placed in 10% formalin solution, and then paraffin embedded and sectioned at 5 µm. After standard deparaffinization steps, sections were heat treated with 0.01 M citric acid for a total of 4 min, and rinsed in PBS. Sections were washed in 0.3% Triton X-100, before room temperature incubation for 1 h in 10% normal goat serum blocking solution and 0.3% Triton X-100 in PBS. Sections were incubated at 4 C in a humidity chamber for 72 h in a 1:200 dilution of mouse anti-D1R antibody (Chemicon, Temecula, CA) in the blocking solution. Sections were then washed in 0.3% Triton X-100 and incubated for 2 h at room temperature in a 1:300 dilution of goat antimouse Alexa 594 antibody (Molecular Probes, Eugene, OR). After a final PBS wash, sections were coverslipped with Vectashield (Vector Laboratories, Burlingame, CA). The sections were examined with a Zeiss (Oberkochen, Germany) Axioskop 2 fluorescence microscope.

Expression Constructs
The human and rodent GHS-R encodes two isoforms, GHS-R1a and GHS-R1b. GHS-R1a is identified as full-length functional form, whereas GHS-R1b is a truncated nonfunctional form. The constructs for in vitro assays are GHS-R1a or tagged GHS-R1a cDNAs. For simplicity, we refer to GHS-R1a as GHS-R.

GHS-R.
RT-PCR product of the human GHS-R was cloned into EcoRI/NotI sites of pcDNA3 (Invitrogen, San Diego, CA).

Xpress-GHS-R.
The human EcoRI/NotI GHS-R cDNA fragment was inserted in-frame into pcDNA3.1/HisC vector (Invitrogen), which carries the Xpress epitope.

GHS-R-GFP.
The GHS-R cDNA fragment was inserted in-frame into EcoRI/EcoRV sites of the pGFP-N3 vector (PerkinElmer, Wellesley, MA).

GHS-R-Rluc.
Human GHS-R cDNA fragment was inserted in-frame into EcoRI/EcoRV sites of the pRluc-N1vector (PerkinElmer).

D1R-Rluc.
The human D1R construct in pcDNA3 (ResGen) was cut with BstBI, blunted with Klenow enzyme, and cut again with SacI; the purified D1R fragment was ligated in-frame into SacI/EcoRV sites of pRluc-N3 vector (PerkinElmer).

Integrity of Tagged GHS-R1a and D1R Expression Constructs
The integrity of all constructs was confirmed by nucleotide sequencing. To test whether the tagged receptors displayed functional characteristics identical with those of wild-type receptors, GHS-R-GFP, GHS-R-Rluc, and Xpress-GHS-R were tested for activation by ghrelin in the aequorin luminescence-based Ca2+ assay (1). The results of these experiments showed that both modified receptors mimicked the function of wild-type GHS-R (data not shown). Activation of D1R by dopamine causes increased cAMP accumulation. When cAMP accumulation in response to dopamine was measured in wild-type D1R- or D1R-Rluc-expressing HEK293 cells, both wild-type and tagged D1Rs responded similarly.

Cell Culture and Transfection
HEK293 and SK-N-SH cells were maintained in DMEM supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin (Invitrogen). HEK293 cells with stably expressed AEQ-17 were maintained in high-glucose DMEM (Invitrogen) containing 10% FBS, 25 mM HEPES, 500 µg/ml G418, and 100 U/ml penicillin/streptomycin. Transient transfection was performed using FuGene 6 (Roche, Indianapolis, IN). Forty-eight hours posttransfection, all cells were incubated in serum-free medium for 1–2 h before treatment with various agents.

Measurement of cAMP Accumulation
Transfected HEK293 cells were treated without or with different agonists in serum-free medium in the presence of 30 µM phosphodiesterase inhibitor rolipram (Sigma, St. Louis, MO) for 15–20 min at 37 C, and then cells were washed twice with PBS and lysed with 0.1 M HCl. Intracellular cAMP levels were determined by enzyme immunoassay kit (Assay Designs, Ann Arbor, MI). All assay points were measured in triplicate, and production of cAMP was normalized to the amount of the sample protein; protein determination was done according to the Bradford method.

Aequorin Bioluminescence Assay
The assay was carried out essentially as described with modifications (1, 13). Forty-eight after transfection, HEK293-AEQ17 cells were charged for 2 h as described (1) before they were harvested and resuspended in modified Ham’s F12 containing 0.1% FBS and 25 mM HEPES at 5 x 105 cell/ml. Human ghrelin and dopamine were diluted with modified Hanks’ balanced salt solution (25 mM HEPES at pH 7.3) and distributed into 96-well plates. Assays were performed with Luminoskan Luminometer (Labsystems, Franklin, MA). The fractional luminescence values for each well were calculated by taking the ratio of the integrated response to the initial challenge to the total integrated luminescence as well as the Triton X-100 lysis response. Fractional luminescence data for each point represent the average of triplicate measurements, and statistical analyses were performed using Student’s t test.

Deconvolution Imaging
SK-N-SH cells were plated on culture chamber slides and transfected with GHS-R-GFP and D1R expression plasmids. Forty-eight hours later, cells were treated with vehicle or dopamine and ghrelin in serum-free medium for 15–20 min at 37 C; the cells were then washed, fixed, and immunostained with antihuman D1R antibody (Calbiochem, San Diego, CA; 1:4000) and Texas Red-conjugated second antibody (Molecular Probes; 1:5000). Deconvolution microscopic observations were made with Applied Precision DeltaVision deconvolution microscope. The images were optimized with softWoRx software.

Immunoprecipitation
HEK293 cells were seeded at a density of 800,000 cells per dish on 60-mm dishes and transfected with 2 µg D1R cDNA and/or 2 µg Xpress-GHS-R cDNA; the total amount of DNA transfected in each dish was completed to 4 µg with empty vector. Transfected and agonist-treated HEK293 cells were washed twice with PBS and incubated in lysis buffer (cooled to 4 C) containing 1x PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 0.1 mg/ml phenylmethylsulfonylfluoride, and 1x protease inhibitor cocktail (Roche) for 30 min on ice. The cells were then sonicated and incubated for a further 30–60 min on ice. Cell lysates were centrifuged at 12,000 x g for 10 min, supernatants were withdrawn, and aliquots (500 µg protein) were incubated with protein A-agarose (Invitrogen), or protein G-agarose (Invitrogen) for 2 h at 4 C with gentle rocking. The cleared supernatants were incubated with anti-D1R and protein A-agarose, or with anti-Xpress and protein G-agarose, or with a control rabbit IgG and protein A-agarose overnight at 4 C on a rocking platform. All immunoprecipitates were washed twice in wash buffer (1x PBS, 0.1% Nonidet P-40, 0.05% sodium deoxycholate, 0.01% sodium dodecyl sulfate, and 0.1 mg/ml phenylmethylsulfonylfluoride) and once in PBS. The immune complexes retained on beads were eluted with SDS-PAGE sample buffer and resolved by SDS-PAGE (10%) gels. The proteins were transferred to polyvinylidene difluoride membranes, and Western blotting was performed. The presence of Xpress-GHS-R was detected using anti-Xpress-HRP antibody (1:4000; Invitrogen). The presence of D1R was detected using anti-human D1R antibody (1:1000; Calbiochem) and peroxidase-conjugated antirabbit antibody (1:10,000; Amersham Biosciences, Piscataway, NJ).

BRET Assay
HEK293 cells were seeded at a density of 400,000 cells per well in six-well dishes and cotransfected with D1R-Rluc and GHS-R-GFP expression plasmids in different ratios; the total amount of DNA transfected in each well was adjusted to 1 µg with empty vector. Forty-eight hours posttransfection, HEK293 cells were detached using nonenzymatic cell dissociation solution (Sigma) and washed with D-PBS. Approximately 1.5–200,000 cells/well in D-PBS containing 2 µg/ml aprotinin (Roche) were distributed in a 96-well plate (white optiplate from PerkinElmer) and incubated in the presence or absence of dopamine (Sigma) and ghrelin (Phoenix, Mountain View, CA) for 10–15 min at 37 C. The coelenterazine derivative DeepBlue C (Packard Biosciences, Meriden, CT) was added at a final concentration of 5 µM and the signal was detected immediately using a Victor V microplate analyzer (PerkinElmer) with 410- and 515-nm emission filters. Background was taken as the area of this region of the spectrum without transfectants. Data are represented as a BRET ratio defined as follows: [(emission at 515 nm) – (background emission at 515 nm)]/[(emission at 410 nm) – (background emission at 410 nm)]. Statistical analyses were performed using Student’s test.

For BRET titration experiments, HEK293 cells were seeded at a density of 400,000 cells per well in six-well dishes and transfected with 0.1 µg GHS-R-Rluc or 0.1 µg D1R-Rluc and different amounts ranging from 0.1–1.8 µg GHS-R-GFP; the total amount of DNA transfected in each well was adjusted to 2 µg with empty vector. Total fluorescence and luminescence were used as relative measures of total expression of the acceptor and donor proteins, respectively. Total fluorescence was determined with Victor V microplate analyzer using an excitation filter at 405 nm and an emission filter at 515 nm. After the fluorescence measurement, total luminescence of the same cells was measured after the addition of DeepBlue C.


   ACKNOWLEDGMENTS
 
We thank Dr. Michael Mancini for his help with deconvolution microscopy experiments.


   FOOTNOTES
 
This work was supported by National Institutes of Aging Grants RO1AG18895 and RO1AG19230 (to R.G.S.).

First Published Online April 6, 2006

Abbreviations: AC, Adenylyl cyclase; Bis, bisindolylmaleimide I; BRET, bioluminescence resonance energy transfer; D1R, dopamine receptor subtype 1; GFP, green fluorescent protein; GHRH-R, GHRH receptor; GHS, GH secretagogue; GHS-R, GH secretagogue receptor; GPCR, G protein-coupled receptor; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PTX, pertussis toxin; Rluc, Renilla luciferase.

Received for publication February 15, 2005. Accepted for publication March 30, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Howard AD, Feighner SD, Cully DF, Arena JP, Liberator PA, Rosenblum CI, Hamelin M, Hreniuk DL, Palyha OC, Anderson J, Paress PS, Diaz C, Chou M, Liu KK, McKee KK, Pong SS, Chaung LY, Elbrecht A, Dashkevicz M, Heavens R, Rigby M, Sirinathsinghji DJ, Dean DC, Melillo DG, Patchett AA, Nargund R, Griffin PR, DeMartino JA, Gupta SK, Schaeffer JM, Smith RG, Van der Ploeg LH 1996 A receptor in pituitary and hypothalamus that functions in growth hormone release. Science 273:974–977[Abstract]
  2. Smith RG 2005 Development of growth hormone secretagogues. Endocr Rev 26:346–360[Abstract/Free Full Text]
  3. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K 1999 Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402:656–660[CrossRef][Medline]
  4. Cowley MA, Smith RG, Diano S, Tschop M, Pronchuk N, Grove KL, Strasburger CJ, Bidlingmaier M, Esterman M, Heiman ML, Garcia-Segura LM, Nillni EA, Mendez P, Low MJ, Sotonyi P, Friedman JM, Liu H, Pinto S, Colmers WF, Cone RD, Horvath TL 2003 The distribution and mechanism of action of ghrelin in the CNS demonstrates a novel hypothalamic circuit regulating energy homeostasis. Neuron 37:649–661[CrossRef][Medline]
  5. Sun Y, Wang P, Zheng H, Smith RG 2004 Ghrelin stimulation of growth hormone release and appetite is mediated through the growth hormone secretagogue receptor. Proc Natl Acad Soc USA 101:4679–4684[Abstract/Free Full Text]
  6. Smith RG, Van der Ploeg LH, Howard AD, Feighner SD, Cheng K, Hickey GJ, Wyvratt MJ Jr, Fisher MH, Nargund RP, Patchett AA 1997 Peptidomimetic regulation of growth hormone secretion. Endocr Rev 18:621–645[Abstract/Free Full Text]
  7. Selbie LA, Hill SJ 1998 G protein-coupled-receptor cross-talk: the fine-tuning of multiple receptor-signalling pathways. Trends Pharmacol Sci 19:87–93[CrossRef][Medline]
  8. Cheng K, Chan WW-S, Butler BS, Barreto A, Smith RG 1989 The synergistic effects of His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 on GRF stimulated growth hormone release and intracellular cAMP accumulation in rat primary pituitary cell cultures. Endocrinology 124:2791–2797[Abstract/Free Full Text]
  9. Koo GC, Huang C, Camacho R, Trainor C, Blake JT, Sirotina-Meisher A, Schleim KD, Wu TJ, Cheng K, Nargund R, McKissick G 2001 Immune enhancing effect of a growth hormone secretagogue. J Immunol 166:4195–4201[Abstract/Free Full Text]
  10. Murphy MG, Weiss S, McClung M, Schnitzer T, Cerchio K, Connor J, Krupa D, Gertz BJ; MK-677/Alendronate Study Group 2001 Effect of alendronate and MK-677 (a growth hormone secretagogue), individually and in combination, on markers of bone turnover and bone mineral density in postmenopausal osteoporotic women. J Clin Endocrinol Metab 86:1116–1125[Abstract/Free Full Text]
  11. Guan XM, Yu H, Palyha OC, McKee KK, Feighner SD, Sirinathsinghji DJ, Smith RG, Van der Ploeg LH, Howard AD 1997 Distribution of mRNA encoding the growth hormone secretagogue receptor in brain and peripheral tissues. Brain Res Mol Brain Res 48:23–29[Medline]
  12. Fremeau Jr RT, Duncan GE, Fornaretto MG, Dearry A, Gingrich JA, Breese GR, Caron MG 1991 Localization of D1 dopamine receptor mRNA in brain supports a role in cognitive, affective, and neuroendocrine aspects of dopaminergic neurotransmission. Proc Natl Acad Sci USA 88:3772–3776[Abstract/Free Full Text]
  13. Feighner SD, Howard AD, Prendergast K, Palyha OC, Hreniuk DL, Nargund R, Underwood D, Tata JR, Dean DC, Tan CP, McKee KK, Woods JW, Patchett AA, Smith RG, Van der Ploeg LH 1998 Structural requirements for the activation of the human growth hormone secretagogue receptor by peptide and non-peptide secretagogues. Mol Endocrinol 12:137–145[Abstract/Free Full Text]
  14. Wellman PJ, Davis KW, Nation JR 2005 Augmentation of cocaine hyperactivity in rats by systemic ghrelin. Regul Pept 125:151–154[CrossRef][Medline]
  15. Kandel ER, Schwartz JH 1982 Molecular biology of learning: modulation of transmitter release. Science 218:433–443[Abstract/Free Full Text]
  16. Mombaerts P, Wang F, Dulac C, Chao SK, Nemes A, Mendelsohn M, Edmondson J, Axel R 1996 Visualizing an olfactory sensory map. Cell 87:675–686[CrossRef][Medline]
  17. Cheng K, Chan WW-S, Butler BS, Barreto A, Smith RG 1991 Evidence for a role of protein kinase-C in His-D-Trp-Ala-Trp-D-Phe-Lys-NH2-induced growth hormone release from rat primary pituitary cells. Endocrinology 129:3337–3342[Abstract/Free Full Text]
  18. Boute N, Jockers R, Issad T 2002 The use of resonance energy transfer in high-throughput screening: BRET versus FRET. Trends Pharmacol Sci 23:351–354[CrossRef][Medline]
  19. Angers S, Salahpour A, Joly E, Hilairet S, Chelsky D, Dennis M, Bouvier M 2000 Detection of ß2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc Natl Acad Sci USA 97:3684–3689[Abstract/Free Full Text]
  20. Couturier C, Jockers R 2003 Activation of the leptin receptor by a ligand-induced conformational change of constitutive receptor dimers. J Biol Chem 278:26604–26611[Abstract/Free Full Text]
  21. Mercier JF, Salahpour A, Angers S, Breit A, Bouvier M 2002 Quantitative assessment of ß1- and ß2-adrenergic receptor homo- and heterodimerization by bioluminescence resonance energy transfer. J Biol Chem 277:44925–44931[Abstract/Free Full Text]
  22. Percherancier Y, Berchiche YA, Slight I, Volkmer-Engert R, Tamamura H, Fujii N, Bouvier M, Heveker N 2005 Bioluminescence resonance energy transfer reveals ligand-induced conformational changes in CXCR4 homo- and heterodimers. J Biol Chem 280:9895–9903[Abstract/Free Full Text]
  23. Gutkind JS 1998 Cell growth control by G protein-coupled receptors: from signal transduction to signal integration. Oncogene 17:1331–1342[CrossRef][Medline]
  24. Gutkind JS 1998 The pathways connecting G protein-coupled receptors to the nucleus through divergent mitogen-activated protein kinase cascades. J Biol Chem 273:1839–1842[Free Full Text]
  25. Bhalla US, Iyengar R 1999 Emergent properties of networks of biological signaling pathways. Science 283:381–387[Abstract/Free Full Text]
  26. Werry TD, Wilkinson GF, Willars GB 2003 Mechanisms of cross-talk between G-protein-coupled receptors resulting in enhanced release of intracellular Ca2+. Biochem J 374:281–296[CrossRef][Medline]
  27. Smith RG, Cheng K, Schoen WR, Pong SS, Hickey G, Jacks T, Butler B, Chan WW, Chaung L-YP, Judith F, Taylor AM, Wyvratt Jr M, Fisher MH 1993 A nonpeptidyl growth hormone secretagogue. Science 260:1640–1643[Abstract/Free Full Text]
  28. Sunahara RK, Taussig R 2002 Isoforms of mammalian adenylyl cyclase: multiplicities of signaling. Mol Interv 2:168–184[Abstract/Free Full Text]
  29. Hanke S, Nurnberg B, Groll DH, Liebmann C 2001 Cross talk between ß-adrenergic and bradykinin B2 receptors results in cooperative regulation of cyclic AMP accumulation and mitogen-activated protein kinase activity. Mol Cell Biol 21:8452–8460[Abstract/Free Full Text]
  30. Rios CD, Jordan BA, Gomes I, Devi LA 2001 G-protein-coupled receptor dimerization: modulation of receptor function. Pharmacol Ther 92:71–87[CrossRef][Medline]
  31. Milligan G 2004 G protein-coupled receptor dimerization: function and ligand pharmacology. Mol Pharmacol 66:1–7[Abstract/Free Full Text]
  32. Angers S, Salahpour A, Bouvier M 2002 Dimerization: an emerging concept for G protein-coupled receptor ontogeny and function. Annu Rev Pharmacol Toxicol 42:409–435[CrossRef][Medline]
  33. Jordan BA, Trapaidze N, Gomes I, Nivarthi R, Devi LA 2001 Oligomerization of opioid receptors with ß2-adrenergic receptors: a role in trafficking and mitogen-activated protein kinase activation. Proc Natl Acad Sci USA 98:343–348[Abstract/Free Full Text]
  34. AbdAlla S, Lother H, Quitterer U 2000 AT1-receptor heterodimers show enhanced G-protein activation and altered receptor sequestration. Nature 407:94–98[CrossRef][Medline]
  35. Ciruela F, Escriche M, Burgueno J, Angulo E, Casado V, Soloviev MM, Canela EI, Mallol J, Chan WY, Lluis C, McIlhinney RA, Franco R 2001 Metabotropic glutamate 1{alpha} and adenosine A1 receptors assemble into functionally interacting complexes. J Biol Chem 276:18345–18351[Abstract/Free Full Text]
  36. Patel RC, Lange DC, Patel YC 2002 Photobleaching fluorescence resonance energy transfer reveals ligand-induced oligomer formation of human somatostatin receptor subtypes. Methods 27:340–348[CrossRef][Medline]
  37. Grant M, Collier B, Kumar U 2004 Agonist-dependent dissociation of human somatostatin receptor 2 dimers: a role in receptor trafficking. J Biol Chem 279:36179–36183[Abstract/Free Full Text]
  38. Rocheville M, Lange DC, Kumar U, Sasi R, Patel RC, Patel YC 2000 Subtypes of the somatostatin receptor assemble as functional homo- and heterodimers. J Biol Chem 275:7862–7869[Abstract/Free Full Text]
  39. Kroeger KM, Pfleger KD, Eidne KA 2003 G-protein coupled receptor oligomerization in neuroendocrine pathways. Front Neuroendocrinol 24:254–278[CrossRef][Medline]
  40. George SR, Lee SP, Varghese G, Zeman PR, Seeman P, Ng GY, O’Dowd BF 1998 A transmembrane domain-derived peptide inhibits D1 dopamine receptor function without affecting receptor oligomerization. J Biol Chem 273:30244–30248[Abstract/Free Full Text]
  41. Baneres JL, Parello J 2003 Structure-based analysis of GPCR function: evidence for a novel pentameric assembly between the dimeric leukotriene B4 receptor BLT1 and the G-protein. J Mol Biol 329:815–829[CrossRef][Medline]
  42. Kimura K, White BH, Sidhu A 1995 Coupling of human D-1 dopamine receptors to different guanine nucleotide binding proteins. Evidence that D-1 dopamine receptors can couple to both Gs and Go. J Biol Chem 270:14672–14678[Abstract/Free Full Text]
  43. Sidhu A, Sullivan M, Kohout T, Balen P, Fishman PH 1991 D1 dopamine receptors can interact with both stimulatory and inhibitory guanine nucleotide binding proteins. J Neurochem 57:1445–1451[Medline]
  44. Morgan DG, May PC, Finch CE 1987 Dopamine and serotonin systems in human and rodent brain: effects of age and neurodegenerative disease. J Am Geriatr Soc 35:334–345[Medline]
  45. Bannon MJ, Poosch MS, Xia Y, Goebel DJ, Cassin B, Kapatos G 1992 Dopamine transporter mRNA content in human substantia nigra decreases precipitously with age. Proc Natl Acad Sci USA 89:7095–7099[Abstract/Free Full Text]



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