Neurocrine Biosciences, Inc., San Diego, California 92113
Address all correspondence and requests for reprints to: Rich Maki, Neurocrine Biosciences, Inc., 12790 El Camino Real, San Diego, California 92113. E-mail: rmaki{at}neurocrine.com.
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ABSTRACT |
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INTRODUCTION |
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Many studies of GPCR structure and function have mutated specific residues in GPCRs to identify domains responsible for ligand binding, G protein coupling, receptor desensitization, internalization, and down-regulation. Furthermore, such studies have also begun to elucidate the conformational changes that GPCRs undergo during their activation and inactivation and the positions of those residues that are critical for the correct folding of the polypeptide chain during synthesis in the ER. Among these residues, those located at or near the intracellular faces of TM3 and TM6 have been implicated as holding the correctly folded receptor in the inactive conformation adopted when the receptor is not bound to ligand. The recent elucidation of the crystal structure of rhodopsin revealed that these residues directly interact with each other through hydrogen bond formation between E134 and R135 of the (D/E)RY motif in TM3, and R135 with T251 and E247 in TM6 (2). The dry motif is highly conserved among GPCRs, and it has been shown that mutation of the central arginine, which corresponds to R135 of rhodopsin results in a relaxation of the conformation of the receptors, allowing them to couple to G proteins in the absence of bound ligand (3). These constitutively active receptors may also undergo constitutive internalization, as is the case with a naturally occurring mutant of the arginine-vasopressin V2 receptor (R137H) that is responsible for the development of nephrogenic diabetes insipidus (3). The observed reduction in cell surface expression of this receptor in the kidneys of patients causes Arg-vasopressin insensitivity, resulting in a failure to reabsorb water from the urine.
The region of intracellular loop 3 (i3) and TM6 surrounding the residues involved in interacting with the (D/E)RY motif of TM3 are also implicated in constraining GPCRs in inactive conformations. A loose consensus sequence at the end of i3 and into the amino terminus of TM6 has long been believed to regulate GPCR activity because mutations in this region can disrupt receptor coupling to G proteins or lead to coupling in the absence of bound ligand. Evidence from studies of the 2A-adrenergic,
1B-adrenergic, 5-hydroxytryptamine2A (5-HT2A) and 5-HT2C receptors have all shown that mutations in this region lead to the constitutive activation of the receptor (4, 5, 6, 7). The consensus sequence is described as being BBXXB, in which B is a basic residue and X is any residue. In many cases the second X is the serine (S) or threonine (T) that is predicted from the structure of rhodopsin to hydrogen bond with the R of the (D/E)RY in TM3. Whereas certain mutations at this position in the 5-HT1B receptor (T313 mutated to R, K, or Q) lead to the receptor becoming constitutively active and expressed at lower levels on the cell surface, most other substitutions of T313 fail to lead to constitutive activation of the receptor but still reduce its cell surface expression (8). Thus, there are likely alternative mechanisms other than constitutive internalization that result in limited cell surface expression.
Recently, a number of mutations in GPCRs have been identified that result in a low level of cell surface expression due to misfolding and retention in the ER, followed by ER-associated degradation (9). Some of these mutations have also been shown to cause human diseases, including mutations in rhodopsin that result in retinitis pigmentosa (10), mutations in the GnRH receptor that cause hypogonadotropic hypogonadism (11), and mutations in the melanocortin 4 receptor that are linked to familial obesity (12). Whereas some such mutations could be dismissed as simply leading to gross misfolding of the receptor protein, it has been shown recently that in other examples in which mutant receptors are retained in the ER, they can be functionally rescued by the addition of pharmacological chaperones (13). These agents permeate the cell and bind to the mutant receptor protein, stabilizing it in a more native conformation and allowing the protein to pass from the ER into the secretory pathway, and then to the cell surface where it functions normally. Therefore, the positions at which these types of mutations occur may represent key residues involved in maintaining the GPCR in a conformation that allows the receptor to be recognized as correctly folded and suitable for export from the ER.
The type 1 melanin concentrating hormone receptor (MCHR1, also known as SLC-1 or GPR24) is a Class A (rhodopsin-like) GPCR the natural ligand of which is melanin-concentrating hormone (MCH) (14, 15). Binding of MCH activates the receptor, which couples to Gq/11 and Gi/o proteins to stimulate multiple intracellular signaling pathways (16). The MCH system has been linked to a variety of physiological functions including the regulation of feeding, energy metabolism (17), and the response to stress (18) and is thus an exciting new target for the treatment of metabolic disorders, anxiety, and depression. The MCHR1 contains a putative BBXXB consensus at the junction of i3 and TM6 that encompasses T255 (residues 252256 in human (h) MCHR1: KRVTR). T255 may be equivalent to residue T313 in the 5-HT1B receptor, mutation of which reduces cell surface expression of the receptor without leading to constitutive activation, and therefore this residue may also play a role in the trafficking of hMCHR1 to the cell surface. To investigate this further, we mutated residue T255 to alanine in hMCHR1 and studied the localization and signaling properties of this receptor.
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RESULTS |
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The Lack of Cell Surface Expression of MCHR1-T255A Is Not Due to Constitutive Internalization
The intracellular retention of MCHR1-T255A suggested that the receptor was either constitutively internalized or that the protein was misfolded and trapped in the ER and thus unable to reach the cell surface. Dynamin2-K44A is a dominant negative mutant of dynamin2 that, when expressed in cells, is able to inhibit the internalization of GPCRs, both in response to agonist and as a result of constitutive internalization through mutation (19). Thus, dynamin2-K44A can be used to determine whether a receptor that is confined to an intracellular compartment is the result of receptor internalization, because dynamin2-K44A inhibits this mechanism and results in an accumulation of the receptors at the cell surface. HEK-293 cells stably expressing either MCHR1-GFP or MCHR1-T255A-GFP were transiently transfected with dynamin2-K44A. After 48 h the cells were either treated with 100 nM MCH peptide or left untreated, and then fixed and stained with anti-dynamin2 antibody and examined by fluorescence microscopy (Fig. 3). In cells expressing MCHR1-GFP and not treated with MCH the GFP fluorescence was seen at the cell surface (Fig. 3
, first row, MCHR1-GFP, NT). In those cells expressing dynamin2-K44A (Fig. 3
, first row, Dynamin2-K44A, NT) there was no apparent change in the cell surface expression of MCHR1-GFP (Fig. 3
, first row, overlay). When the MCHR1-GFP-expressing cells were treated with MCH there was an accumulation of punctate GFP fluorescence within the cytosol in those cells not expressing dynamin2-K44A (presumably representing clusters of receptor molecules in endosomes) (Fig. 3
, second row, MCHR1-GFP, +MCH, arrow). In contrast, in cells expressing both MCHR1-GFP and dynamin2-K44A, agonist-driven internalization of MCHR1 was dramatically reduced, with most of the GFP fluorescence remaining at the cell surface and only a few small punctate sites formed within the cytosol (Fig. 3
, second row, overlay). Transfection of dynamin2-K44A into cells expressing MCHR1-T255A-GFP did not alter the pattern of receptor distribution with or without the addition of agonist treatment (Fig. 3
, third and fourth rows). Notably, the receptor was predominantly in an intracellular compartment even in cells expressing dynamin2-K44A (Fig. 2
, third and fourth rows). Therefore, it is unlikely that the lack of cell surface expression and the intracellular distribution of MCHR1-T255A-GFP are caused by the receptor being internalized, but rather, through failure of the mutant receptors to be trafficked to the plasma membrane.
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Calnexin is a membrane-associated lectin protein in the ER that binds to oligosaccharide groups on newly synthesized glycoproteins and functions to aid in protein folding, retention of misfolded proteins in the ER and, in some cases, the degradation of these misfolded proteins (21). To investigate whether MCHR1-T255A is retained in the ER by a calnexin-mediated mechanism, we studied the association between FL-MCHR1 and FL-MCHR1-T255A with calnexin (Fig. 4B). FL-MCHR1 or FL-MCHR1-T255A expressed in HEK-293 cells was immunoprecipitated with anti-FLAG (M2) antibody and resolved by SDS-PAGE; both receptor and associated calnexin were then quantified by Western blotting. The amount of calnexin that coimmunoprecipitated with MCHR1-T255A was twice the amount observed with FL-MCHR1, even though the estimated total amount of FL-MCHR1-T255A was about half that of FL-MCHR1 (as measured by band densitometry). Thus, the reduced level of glycosylation of MCHR1-T255A compared with MCHR1, the colocalization of MCHR1-T255A with the ER marker KDEL proteins, and the greater amount of calnexin associated with MCHR1-T255A strongly suggests that the majority of MCHR1-T255A is being retained in the ER.
MCHR1-T255A Can Be Rescued to the Cell Surface by a Small Molecule Antagonist of MCHR1
It has been reported that some mutated GPCRs that are retained as misfolded proteins in the ER can be rescued to the cell surface through interaction with stabilizing chemical chaperones. We investigated whether the defect in MCHR1-T255A folding and localization could also be rescued by cell-permeable antagonists of MCHR1. The small molecule antagonist of MCHR1, NBI-A, was incubated overnight with HEK-293 cells that had been transiently transfected with either FL-MCHR1 or FL-MCHR1-T255A. The inhibition constant (Ki) of NBI-A for FL-MCHR1 was 2.7 ± 0.3 nM, and the IC50 in a FLIPR assay was 111.35 ± 8.46 nM whereas the Ki of NBI-A for FL-MCHR1-T255A was 43.97 ± 12.68 nM (Table 1). Because of the low level of MCHR1-T255A, cell surface expression and, consequently, a low functional response to the MCH ligand before rescue, the IC50 of NBI-A could not be accurately determined. The expression level of both receptors was evaluated by monitoring the cell surface expression of the FLAG-tagged receptors using FACS (Fig. 5A
). As observed previously (Fig. 2A
), the cell surface expression level of wild-type FL-MCHR1 was approximately 5-fold higher than that of FL-MCHR1-T255A. After incubation with 1 µM NBI-A, the cell surface expression of FL-MCHR1-T255A increased 5-fold to a level comparable to that of FL-MCHR1, whereas the level of FL-MCHR1 increased only slightly (Fig. 5A
). This increase in cell surface expression was also observed by fluorescence microscopy as a dramatic change in the distribution of MCHR1-T255A-GFP from being primarily intracellular (Fig. 5B1
) before treatment, to being almost completely at the cell surface after treatment with NBI-A (Fig. 5B2
). In contrast, treatment with an equivalent concentration of a structurally very similar compound that lacks affinity for the MCHR1 (NBI-B) failed to elicit the same redistribution of receptor molecules (Fig. 5B3
), demonstrating that the effect of NBI-A is likely dependent on its ability to bind MCHR1 and not through a nonspecific effect on the cellular trafficking machinery. The dose dependence and time course of the redistribution of FL-MCHR1-T255A by NBI-A were then determined by monitoring increased cell surface expression by FACS (Fig. 5
, C and D); the logEC50 for redistribution was determined to be 6.474 ± 0.039 (336 nM) with a t1/2 of the maximum response of 4 h, reaching a maximum cell surface expression after approximately 12 h.
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MCHR1-T255A Is Functional Once It Has Been Rescued to the Cell Surface
To determine whether the rescued MCHR1-T255A was still capable of coupling to G proteins and signaling, we evaluated the ability of the receptor to mobilize intracellular Ca2+ in response to MCH before and after treatment with NBI-A (Fig. 6). HEK-293 cells stably expressing FL-MCHR1 or FL-MCHR1-T255A were treated with the antagonist NBI-A or left untreated for comparison. The following day the cells were washed thoroughly to remove most of the antagonist before their responsiveness to MCH peptide was determined by FLIPR assay. Without the addition of NBI-A, the maximum response of MCHR1-T255A to MCH peptide was 17% of that response generated by MCHR1. After rescue by NBI-A, the maximum response of MCHR1-T255A increased to a level similar to that of wild-type MCHR1 treated with NBI-A, which was 60% of the response observed for MCHR1 before NBI-A treatment. This difference between the maximum responses of MCHR1 before and after NBI-A treatment is most likely due to the incomplete removal of the antagonist from the cells. In all cases the logEC50 values were similar: FL-MCHR1, 7.541 ± 0.069; FL-MCHR1-T255A, 7.425 ± 0.169; FL-MCHR1 + NBI-A, 7.28 ± 0.083; and FL-MCHR1-T255 + NBI-A, 7.339 ± 0.110 (Table 1
).
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DISCUSSION |
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Proteins that are destined for secretion or the cell surface are translated into the ER and exported to the Golgi where additional maturation takes place. In the ER these proteins undergo N-glycosylation and disulfide bridge formation and are folded correctly (9). Those proteins that are incorrectly folded are recognized by regulatory mechanisms in the ER and are targeted for ER-associated degradation. Calnexin, a chaperone associated with the ER, binds to monoglycosylated glycans on newly synthesized proteins and aids in the folding process (21). Proteins that are misfolded are frequently associated with calnexin for longer periods of time and are targeted for degradation. We showed that both MCHR1 and MCHR1-T255A associated with calnexin, but considerably more of the mutant protein was associated with calnexin than wild-type protein. In a similar example, it was shown that both the wild-type V2 vasopressin receptor and a mutant receptor that is retained in the ER associate with calnexin. However, the mutant receptor was shown to remain associated with calnexin for longer than the wild-type receptor (25).
Two additional lines of evidence support the idea that the hMCHR1-T255A is retained in the ER. First, we showed that hMCHR1-T255A colocalized with KDEL-containing proteins. The KDEL sequence located at the carboxyl-terminal end of proteins serves as a signal for these proteins to be retained in the ER (20). Thus, the colocalization of a protein with KDEL-containing proteins is a good indication that the protein in question is in the ER. Second, we found that the glycosylation pattern of hMCHR1-T255A resembles a protein that is incompletely glycosylated (41 kDa) compared with the wild-type receptor (55 kDa). GPCRs undergo N-linked glycosylation in the ER and O-linked glycosylation in the Golgi (26, 27); therefore it is likely that hMCHR1-T255A has undergone some N-glycosylation, but due to its inability to move to the Golgi, addition of O-glycosylation cannot take place. The association with calnexin, the colocalization with KDEL proteins, and the immature state of glycosylation all point to the conclusion that hMCHR1 T255A is being retained in the ER.
The results presented here are not consistent with MCHR1-T255A being constitutively internalized. This is based on several lines of evidence. First, MCHR1-T255A-GFP was found primarily in an intracellular compartment, and treating cells with MCH did not significantly alter this pattern. In contrast, the addition of MCH to cells expressing the wild-type receptor resulted in visible internalization of the GFP-tagged receptor. Second, we expressed a dominant negative dynamin2 mutant (K44A) protein in cells expressing the wild-type or mutant MCHR1. Dynamin2 is a GTPase involved in trafficking of proteins at the cell surface (28), and dominant negative mutants of dynamin2 have been shown to inhibit receptor-mediated endocytosis. Thus, expression of K44A dynamin2 should block internalization of constitutively internalized receptors, increasing the level of cell surface expression. The finding that expression of dominant negative dynamin-2 K44A inhibited agonist-induced internalization of wild-type hMCHR1 but did not change the pattern of intracellular localization of hMCHR1-T255A indicates that hMCHR1-T255A is not constitutively internalized.
We observed that a membrane-permeable small molecule antagonist to the MCH receptor was able to rescue a range of mutant MCH receptors to the cell surface: T255A, 321, R319Q/K320Q, and N13, 16, 23Q. Interestingly, these mutations span almost the entire length of the protein, N13, 16, 23Q being near the N terminus, T255A at the junction of i3 and TM6, and R319Q/K320Q and
321 being located near the C terminus, yet all can be rescued to the cell surface by the same small molecule antagonist. This suggests that they all are likely rescued through a common mechanism, i.e. the small molecule enters the cell and associates with the binding pocket of the misfolded protein and, in doing so, enables it to fold in a more native conformation that can then be trafficked to the cell surface. Such a mechanism is supported by the several other small molecule pharmacological chaperones that have been described elsewhere (13, 29). Pharmacological chaperones have been used to demonstrate that both mutant GPCRs as well as some wild-type receptors can be rescued to the cell surface. For many mutants of GPCRs for which cell surface expression is low or absent, including the V2, GnRH,
-and µ-opioid receptors, the addition of antagonists to cells expressing the respective receptor results in an increase in the level of cell surface expression (11, 30, 31, 32). Like the mutations we describe here in MCHR1, mutations in other GPCRs that cause their retention in the ER but which are rescued by pharmacological chaperones can be located at a variety of sites in the receptor. However, all the molecules that work as chaperones have the ability to interfere with the binding of the natural ligand to the receptor and thus must affect the region of the binding pocket. Results from studies using small molecule antagonists as pharmacological chaperones suggest that the small molecules play an active role in the folding of the receptors, perhaps by locking them into a specific, inactive conformation. In some cases inefficient processing of the wild-type receptor protein can also lead to retention in the ER. For example, only about 40% of wild-type
-opioid receptor reaches the cell surface when expressed in HEK-293 cells (27). When membrane-permeable opioid ligands are added to the cells, they facilitate the cell surface expression of the receptor. Interestingly, we noticed that the level of cell surface expression of wild-type MCHR1 also increased after the addition of NBI-A to the cells and may also be due to the small molecule enhancing the processing and trafficking of the wild-type receptor. This phenomenon has also been noted for the V2 vasopressin (31) and GnRH receptors (11).
It has been proposed that correct glycosylation of the MCHR1 is required for it to be efficiently trafficked from the ER to the cell surface (23); however, our data are not consistent with a mechanism by which NBI-A rescues MCHR1-T255A simply by promoting its full glycosylation. Whereas rescue of FL-MCHR1-T255A by NBI-A does lead to the mutant receptor becoming glycosylated to a similar extent to the wild-type receptor, NBI-A also rescues the N13, 16, 23Q mutant receptor, which cannot undergo N-glycosylation. Thus, full glycosylation of MCHR1-T255A is most likely a consequence of binding NBI-A, which allows its efficient trafficking through the ER and Golgi, and not the mechanism of rescue per se.
One possible model for the lack of proper folding of the MCHR1-T255A is suggested by examining the crystal structure of bovine rhodopsin. The (D/E)R(Y/W) sequence is one of the most conserved sequence motifs found in GPCRs and is located at the junction of TM3 and intracellular loop 2 (i2). The crystal structure of bovine rhodopsin suggests that the ERY motif interacts with surrounding residues via hydrogen bonds. Arg135 in this motif interacts with Glu247 and Thr251 in the N terminus of the TM6 by hydrogen bonds, and these interactions are critical to keep rhodopsin in the inactive conformation (2). Thr251 is part of a conserved sequence in many GPCRs, the BBXXB motif. It has been thought for some time that this domain is involved in receptor-G protein coupling and that mutations within this domain can frequently lead to constitutively active receptors, as has been shown for the 1B-adrenergic (4) and 5-HT1B receptors (8). In the case of 5-HT1B receptor, the substitution of Thr313 in the motif RKATK (BBXXB) with a variety of amino acids including alanine results in a receptor with reduced cell surface expression. The existence of a similar motif (KRVTR) in MCHR1 at the corresponding position suggests an interaction between the DRY motif and the BBXXB motif in MCHR1 (Fig. 1
). Because Thr255 in MCHR1 is positioned at the junction of i3 and TM6 it is possible that Thr255 in MCHR1 interacts with Arg141 in the DRY motif of MCHR1 via hydrogen bonds. Disruption of the hydrogen bond between Arg141 and Thr255 may result in a disruption of the interaction between TM3 and TM6. This could then result in a protein that is folded incorrectly and can not exit the ER.
In conclusion, we have demonstrated that a single amino acid mutation in MCHR1 can result in the receptor being retained in the ER. The receptor can be rescued by treating the cells with a small molecule antagonist of the MCH receptor. This small molecule antagonist probably acts as a pharmacological chaperone by stabilizing the receptor in a more native conformation. These results add to our knowledge of receptor folding and transport from the ER and point to the use of small molecule antagonists as potential therapeutics for misfolded proteins.
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MATERIALS AND METHODS |
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Cell Culture and Transfection
HEK-293 cells were maintained in DMEM supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (FBS), 0.2 mM glutamine, 1 mM sodium pyruvate, and penicillin-streptomycin (50 I.U./ml and 50 µg/ml, respectively) at 37 C and 7% CO2. Transient transfections were performed using Effectene transfection kit (QIAGEN, Valencia CA). After 24 h of transfection, cells were harvested and distributed into appropriate plates, and experiments were performed the following day. Stable clones expressing MCHR1 constructs were selected by culturing the cells in the presence of G418 (400 µg/ ml) for 2 wk.
Radioligand Binding Saturation Assay
In radioligand saturation experiments total and nonspecific binding was measured in duplicate at varying radioligand concentrations. Nonspecific binding was measured in the presence of 1 µM of the MCHR1 small molecule antagonist NBI-A. Cells were harvested from culture dishes by the addition of PBS containing 10 mM EDTA and collected by centrifugation followed by washing the cells twice in ice-cold DMEM. Receptor binding assays were performed using [125I]MCH (NEN Life Sciences Products, Boston, MA). FL-MCHR1 or FL-MCH T255A expressing HEK-293 cells (20,00030,000 cells in 100 µl) were added to tubes containing 100 µl DMEM containing 0.1% BSA and 50.01 nM [125I]MCH (2-fold series dilution). The binding mixture was incubated for 30 min at room temperature. Cells were centrifuged at 9000 rpm for 5 min, and the amount of bound [125I]MCH in the cell pellet was measured in a COBRA II Auto-Gamma counter (Packard, Palo Alto, CA). Binding data were analyzed using PRIZM (GraphPad Software, San Diego, CA).
Measurement of Intracellular Calcium Mobilization
Stable cell lines of HEK-293 cells expressing MCHR1 or MCHR1-T255A were seeded in 96-well plates at a density of 1 x 105 cells per well with or without small molecule antagonist NBI-A (Zhu, F., manuscript in preparation) or NBI-B (1 µM), which was added 1 d before the assay was performed. The cells were washed twice with Hanks buffered saline and loaded for 1 h at 37 C, 7% CO2, with 1 µM Fluo-4 (Molecular Probes, Inc., Eugene, OR) diluted in DMEM containing 0.02% Pluronic (Molecular Probes, Inc.) and 2.5 mM probenecid (Sigma-Aldrich, St. Louis, MO). Cells were washed two times with 200 µl of Hanks buffered saline containing 20 mM HEPES and 2.5 mM probenecid and then resuspended in 150 µl of the same buffer. Mobilization of intracellular Ca2+ in response to different concentrations of MCH peptide was measured on a Fluorometric Imaging Plate Reader (FLIPR) (Molecular Devices, Sunnyvale, CA), and the data were analyzed using GraphPad PRIZM software (GraphPad Software).
Flow Cytometry
The cell surface expression levels of FL-MCHR1 and FL-MCHR1-T255A were evaluated by FACS. HEK-293 cells transiently or stably expressing FL-MCHR1 or FL-MCHR1-T255A were seeded in poly-D-lysine-coated six-well dishes (BIOCOAT, Fort Washington, PA) (Becton Dickinson Labware, Bedford, MA) the day before the assay, with or without addition of 1 µM of the antagonist NBI-A. Cells were washed twice with ice-cold DMEM and incubated with anti-FLAG M2 antibody (Sigma-Aldrich) diluted 1:500 in DMEM, for 1 h at 4 C. The cells were rinsed three times with ice-cold DMEM and incubated with 1:250 goat antimouse IgG antibody conjugated to AlexaFluor 488 dye (Molecular Probes) at 4 C for 30 min in the dark. Cells were washed with ice-cold PBS three times and detached from dishes with PBS containing 5 mM EDTA. The fluorescence intensity of 1 x 104 cells from each well was measured on a FACScan flow cytometer (BD Biosciences-CLONTECH).
Determination of Receptor Internalization by Cell Microscopy
Cell lines expressing MCHR1-GFP or MCHR1-T255A-GFP were seeded into 35-mm dishes containing glass coverslips coated with poly-D-lysine and collagen. After 24 h the cells were washed once in DMEM at 37 C and 100 nM MCH peptide was added (in DMEM at 37 C). The cells were incubated at 37 C, 7% CO2 for 30 min, washed with ice-cold Hanks buffered saline, and observed by inverted fluorescence microscopy on a Nikon Eclipse TE 300 microscope (Nikon, Melville, NY). Images were captured with a Spot-RT Digital Camera (Diagnostic Instruments, San Diego, CA) using a x40 objective lens and standard fluorescence microscopy filter sets, and analyzed using ImagePro Plus Analysis Software (Media Cybernetics, Carlsbad, CA). To investigate whether dynamin2-K44A inhibited internalization of MCHR1, dynamin2-K44A in pcDNA3 was transiently transfected into the cells expressing either MCHR1-GFP or MCHR1-T255A-GFP. The cells were grown in either a six-well dish or on glass coverslips for 48 h after transfection. MCH peptide (100 nM) was then added and the cells were incubated at 37 C, 7% CO2, for a further 30 min to stimulate internalization of MCHR1. Receptor localization was then observed as described above.
Immunocytochemical Staining
HEK-293 cells stably expressing MCHR1-GFP or MCHR1-T255A-GFP, and transfected with or without dynamin2-K44A, were grown overnight on glass coverslips coated with poly-D-lysine and collagen. In experiments measuring the effect of dynamin2-K44A on receptor internalization, the cells were stimulated with 100 nM MCH for 30 min at 37 C. The cells were then washed three times with PBS and fixed in methanol for 5 min at 20 C. After a further three washes with PBS, nonspecific antibody binding sites were blocked with 10% vol/vol FBS in PBS for 1 h. Monoclonal antibodies to KDEL proteins (Stressgen Bioreagents, San Diego, CA) or dynamin2 (Upstate Biotechnology, Inc., Lake Placid, NY) were diluted in PBS containing 10% FBS and applied to the cells for 1 h at room temperature. The cells were washed three times with PBS before incubation with the secondary antibody conjugated to AlexaFluor 594 (Molecular Probes) diluted 1:100 in PBS with 10% FBS (in the dark for 30 min). The cells were washed three times with PBS, mounted on glass microscope slides, and observed by fluorescence microscopy.
Immunoprecipitations and Western Blotting
HEK-293 cells transfected with FL-MCHR1 or FL-MCHR1-T255A were washed twice in ice-cold PBS, lysed on ice for 10 min with 1 ml lysis buffer [50 mM HEPES (pH 7.3); 0.5% Nonidet P-40; 250 mM NaCl; 10% glycerol; 2 mM EDTA; 100 µM Na3VO4; 1 mM NaF; and one Complete protease inhibitor tablet for each 50 ml buffer (Roche Applied Sciences, Indianapolis, IN)]. Supernatants were prepared by centrifugation at 14,000 rpm for 10 min; protein concentrations were normalized, and receptor molecules were immunoprecipitated by incubation with anti-FLAG M2 agarose conjugate (Sigma-Aldrich) at 4 C overnight. The agarose conjugates were pelleted by centrifugation at 1400 rpm for 10 min and washed twice at 4 C with lysis buffer. Immunoprecipitated receptors were deglycosylated with PNGase F (New England Biolabs, Beverly, MA) according to the manufacturers suggested conditions, except that the digestion was performed at room temperature for 1 h. All protein samples were solubilized in sodium dodecyl sulfate loading dye at 54 C for 10 min and resolved by SDS-PAGE on NuPAGE 12% Bis-Tris precast gels (Invitrogen, Carlsbad, CA). Proteins were transferred to polyvinylidine difluoride membranes (Invitrogen), blocked with 3% nonfat milk in TBST buffer (20 mM Tris-HCl, pH 7.4; 150 mM NaCl; 0.05% Tween 20) for 1 h, and incubated for 1-h with primary antibodies diluted in 3% milk-TBST. After three washes with TBST, filters were incubated for 1 h in horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences, Piscataway, NJ) diluted 1:3000 in 3% milk-TBST. After further washing, the proteins were visualized using SuperSignal West Pico Chemiluminescence Substrate (Pierce Chemical Co., Rockford, IL).
Molecular Modeling
The hMCHR1 receptor model was built using the crystal structure of bovine rhodopsin with 2.6Å resolution as the template (Protein Data Bank code: 1L9H). The sequence alignment was done using Point Accepted Mutation scoring matrix (Gap open penalty set at 10 and Gap extension penalty set at 0.05) with Chou Fasman secondary structure prediction method implemented in Insight II (33,34). Due to the low sequence homology between various GPCR class A receptors, especially in the loop regions, manual intervention was needed after the automatic alignment. The conserved residues throughout the seven TM domains were used as guidance without attempts to optimize the matches in the loop regions. The disulfide bond between TM3 and extracellular loop 2 was kept, and gaps were avoided in the seven TMs to minimize the disruption of the defined secondary structures.
A collection of 10 hMCHR1 homology models were generated using Modeler implemented in Insight II, a software package provided by Accelrys (San Diego, CA). In Modeler, protein structures were built by the satisfaction of multiple spatial restraints, which were incorporated into an objective function. Conjugate gradients technique was applied to optimize the objective function. The models were further refined using molecular dynamics with simulated annealing, and the model with the fewest violations after automatic protein structure validation and visual inspection was chosen for further investigation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: ER, Endoplasmic reticulum; FBS, fetal bovine serum; FLIPR, fluorometric imaging plate reader; FL-MCHR1, FLAG-tagged MCHR1: FU, fluorescent unit; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; HEK, human embryonic kidney; 5-HT, 5-hydroxytryptamine; i3, intracellular loop 3; KDEL, Lys-Asp-Glu-Leu; MCH, melanin-concentrating hormone; MCHR, melanin concentrating hormone receptor; PNGase F, peptide N-glycosidase F; TM, transmembrane.
Received for publication July 27, 2004. Accepted for publication May 24, 2005.
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REFERENCES |
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