From the Rudolf Magnus Institute of Neuroscience, Department of Pharmacology and Anatomy, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands
Received for publication, November 6, 2002 , and in revised form, March 30, 2003.
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ABSTRACT |
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INTRODUCTION |
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In humans, the melanocortin system is also involved in body weight regulation. For instance, mutations causing defects in synthesis or processing of pro-opiomelanocortin, which encodes the melanocortin agonist -MSH, lead to obesity (10). Also, four frameshift mutations, one nonsense mutation, and 24 missense mutations have been found in the MC4R. Most of these mutations were only identified in obese individuals (1120), suggesting that mutations in the MC4R predispose for development of obesity. The incidence among obese individuals was estimated to be as high as 4%, which would make it the most common monogenic form of obesity (16).
For several of the MC4R mutations, segregation of the mutant allele with obesity was investigated. For one frameshift mutation and one nonsense mutation, post hoc analysis yielded LOD scores of >3 (21). Yeo et al. (11) reported a LOD score of 1.5 for the same frameshift mutation. Although for the other mutants that were tested for segregation with obesity, most family members that carried these MC4R mutations were obese (1416), causality is still ambiguous. Determination of penetrance was limited by small pedigrees and the complexity of the trait. For instance, some families may already be predisposed for obesity or leanness due to environmental and/or other genetic factors (21). This could explain why phenotypes of carriers varied from no obesity to extreme obesity (1517), as is exemplified by the lack of cosegregation of a frameshift mutation that may be considered a loss of function mutation, with obesity (16). Also, in one study, deletions in the chromosomal region that contains the MC4R were not associated with obesity (22). Thus, although mutations in the MC4R predispose for development of obesity, not for every individual mutation is its role in the pathogenesis of obesity clear, and susceptibility to MC4R mutations may vary between individuals.
Pharmacological evaluation of mutant receptors gives more insight into the impact of a mutation on receptor function at the molecular level and may be helpful to determine the quantitative effect of a mutation in mutation carriers. Since most of the mutations in the MC4R result in nonconservative amino acid changes, it seems likely that these mutations affect receptor function rather than that they are in complete linkage disequilibrium with disease mutations elsewhere in the gene. As outlined above, loss of function is expected from MC4R mutations that cause obesity. Indeed, several mutant MC4Rs only found in obese individuals were shown to have impaired or no functionality in vitro (1416), whereas mutations that had also been found in nonobese individuals did not affect receptor function (16, 23). For most mutants that have been tested, however, only agonist binding and potency were determined. Since AgRP is an endogenous ligand for the MC4R, differences in its affinity can be expected to affect MC4R activity as well. In addition, for only two mutant receptors has the cell surface expression level been determined (14, 23), whereas this is often reduced as result of a mutation. Therefore, we extended the existing studies by assessing cell surface expression, affinity of AgRP and -MSH, and the biological response to
-MSH for 11 of the MC4R mutations.
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EXPERIMENTAL PROCEDURES |
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Construction of Mutant ReceptorsThe human-MC4R cDNA (GenBankTM L08603
[GenBank]
) was used as a template. The receptor cDNA was cloned in pcDNA3 (Invitrogen) and maintained in the Escherichia coli DH5 strain. The mutations were introduced with a PCR strategy using Pfu polymerase (Stratagene, La Jolla, CA) and Pfu buffer (Stratagene). Per reaction, two complementary primers that contained the mutations were used. The reactions yielded complete copies of both complementary strands of the plasmid, which readily can be transformed into bacteria. The primers used were as follows (per mutation, only the primer homologous to the coding strand is given from 5'- to 3'-end): S30F, CTGCACAGCAACGCGTCTGAGTTCCTTGGAAAAGGC; P78L, CTGCATTCACTCATGTACTTTTTCATATGCAGCTTGG; T112M, CTATTAAACAGTACTGATATGGATGCACAG; R165W, CCATAACATCATGACAGTTAAGTGGGTTGGGATC; R165Q, CCATAACATCATGACAGTTAAGCAGGTTGGGATC; I170V, GGTTGGGATCATCGTAAGTTGTATCTGGG, G252S, GCGATTACGTTGACCATCCTGATTAGCGTCTTTGTTGTCTGCTG; I317T, CTGAGGAAAACCTTTAAAGAGATCACCTGTTGCTATCCCCTGGG; I137T, CCTTGCTTGCATCCACCTGCAGCCTGCTTTCAATTGC; L250Q, CCTTGACCATCCAGATCGGCGTCTTTGTTGTCTGC; I301T, CAATCATCGATCCTCTGACTTATGCATTACGGAGTCAAG; V253I, GACCATCCTGATCGGCATCTTTGTTGTCTGC. Reaction mixtures (40-µl end volume) consisted of 20 ng of template DNA, 35 ng of DNA of each primer, 500 µM dNTPs (Amersham Biosciences), and 3 units of Pfu polymerase (Stratagene) in the appropriate buffer concentration. The cycling parameters were 95 °C for 30 s, followed by 16 cycles of denaturation at 95 °C for 30 s, annealing at 50 °C for 1 min, and extension at 60 °C for 13 min, and at the end of the reaction an extension step at 68 °C for 20 min. Reaction samples were then cut with DpnI to fragment methylated template DNA and subsequently transformed into E. coli DH5
. The entire coding region of the receptor and upstream sequences of each construct were verified by sequence analysis.
Construction of HA-tagged ReceptorsThe WT and three of the mutant receptors were N-terminally tagged with an HA tag using a PCR strategy. Sequences of the primers that were used are as follows (5' to 3'): 1, CCATATGATGTTCCAGATTATGCTATGGTGAACTCCACCCACCG; 2, AGTCTCGAGACCTGCGTTAATATCTGCTAGACAAG; 3, ATCAGAATTCGGCCACCATGTATCCATATGATGTTCCAGATTATGC.
Two consecutive PCRs were performed, the first using WT or mutant receptor DNA as template and primers 1 and 2. The second PCR used the product of the first PCR as template and primers 2 and 3. The reaction mixtures (20 µl) contained 10 ng of template DNA, 30 ng/primer, 200 µM dNTPs (Amersham Biosciences), and 2 units of Pfu polymerase (Stratagene). The cycling parameters were as follows: 95 °C for 5 min, 30 cycles of 95 °C for 30 s, 50 °C for 30 s, and 72 °C for 2 min, followed by 72 °C for 10 min. These reactions insert an HA tag immediately upstream of the start codon of the MC4R gene, preceded by a consensus Kozak sequence and an EcoRI site. N-terminally, a BspMI and XhoI site are inserted after the stop codon of the MC4R gene. The PCR products were cloned in pcDNA3 (Invitrogen) using the EcoRI and XhoI sites and subsequently verified by sequence analysis.
Cell Culture and TransfectionHEK293 and BHK cells were grown in Dulbecco's modified Eagle's medium, supplemented with 10% fetal calf serum (Integro, Zaandam, The Netherlands), 2 mM glutamine (Invitrogen), and nonessential amino acids (Invitrogen). In order to avoid differences between cells not related to MC4r expression, transient expression of different receptors was performed in one batch of 293 cells for each experiment. Each experiment was performed at least three times. All transfections were done using standard calcium phosphate precipitation. For binding experiments and the adenylate cyclase assay, receptors were transiently expressed in HEK293 cells by transfecting cells growing in 10-cm dishes with 7 µg of DNA. For the reporter gene assay, cells growing in 10-cm dishes were co-transfected with 200 ng of receptor DNA and 7 µg of CRE-lacZ construct (24). The immunocyotochemical studies used BHK cells that were transfected with 1.4 µg of DNA/well of a six-well plate.
To allow determination of transfection efficiency, cells were cotransfected with a lacZ construct. Two days later, the cells were fixed in 2% paraformaldehyde, 0.2% glutaraldehyde for 15 min and washed with phosphate-buffered saline (PBS). By adding freshly prepared substrate solution (1 mg/ml 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-gal), 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2 in PBS),
-galactosidase-positive cells were stained.
Binding AssayIC50 values were determined with 125I-[Nle4-D-Phe7]-MSH as tracer. [Nle4-D-Phe7]-
-MSH was iodinated using bovine lactoperoxidase (Calbiochem) and 125I-Na (ICN, Aurora, OH) according to Oosterom et al. (25) and subsequently high pressure liquid chromatography-purified on a C18 column (µBondapak 3.9 x 300 mm; Waters, Milford, MA).
48 h after transfection, cells growing in 24-well plates were washed with Tris-buffered saline supplemented with 2.5 mM calcium chloride and incubated for 30 min at room temperature with peptides and tracer diluted in Ham's F-10 medium (Invitrogen) supplemented with 2.5 mM calcium chloride, 0.25% bovine serum albumin (ICN, Aurora, OH), and 200 KIU/ml aprotinin (Sigma). After two washes with ice-cold Trisbuffered saline (plus 2.5 mM calcium chloride) to remove nonbound tracer, the cells were then lysed in 1 M sodium hydroxide, and samples were counted in a -counter.
Adenylyl Cyclase AssayAdenylyl cyclase activity was determined using a modified method of Salomon (26). Cells were grown in 24-well plates and incubated for 2 h with 2 µCi/ml [3,8-3H]adenine (21.7 Ci/mmol; PerkinElmer Life Sciences) in assay medium (Dulbecco's modified Eagle's medium (Invitrogen) containing 0.2% bovine serum albumin (ICN), 2 mM L-glutamine (Invitrogen), and nonessential amino acids. Subsequently, the cells were washed with assay medium containing 0.25 mM isobutylmethylxanthine (Sigma) and incubated for 20 min with compounds diluted in assay medium with 0.25 mM isobutylmethylxanthine. Then 1 ml of cold stop solution (5% trichloric acid (Merck), 1 mM cAMP (Roche Applied Science), 1 mM ATP (Roche Applied Science)) per well was added, and the plates were centrifuged at 250 x g. Finally, ATP and cAMP fractions were separated on Dowex (AG-50W-X4; Bio-Rad) and alumina (WN-3; Sigma) columns, respectively. ATP and cAMP fractions were dissolved in scintillation mixture (Ultima GoldTM; Packard Instrument Co.) and counted in a -counter.
The equation [3H]cAMP/([3H]cAMP + [3H]ATP) was used to calculate adenylate cyclase (AC) activity as the percentage of [3H]ATP that is converted to [3H]cAMP.
Reporter Gene AssayIn this assay, lacZ is used as a reporter gene (24). The cells were dispensed into 96-well plates (BectonDickinson) and 2 days later incubated with peptides at the appropriate concentrations in serum-free medium (Dulbecco's modified Eagle's medium containing 0.2% bovine serum albumin (ICN) glutamine (Invitrogen) and nonessential amino acids (Invitrogen)). After 56 h of incubation, the assay medium was aspirated, and 40 µl of lysis buffer (PBS containing 0.1% Triton X-100 (Roche Applied Science)) was added. The plates were stored at 20 °C, and after thawing, 80 µl of substrate mix (0.1 M phosphate buffer, pH 7.4, containing 1.6 g/liter o-nitrophenyl -D-galactopyranoside (Molecular Probes, Leiden, The Netherlands), 67.5 mM
-mercaptoethanol (Merck), and 1.5 mM magnesium chloride) was added. Absorbance at 405 nm was determined in a Victor2 microplate reader (PerkinElmer Life Sciences).
ImmunocytochemistryFor immunocytochemical experiments with HA-tagged receptors, BHK cells were used, since they attached better to glass coverslips as compared with HEK293 cells, which were washed away during the procedures. Cells growing on glass coverslips were transfected overnight with receptor DNA, and after 48 h they were fixed in 4% paraformaldehyde and washed quickly two times in PBS. After incubation (30 min at room temperature) in TNB blocking buffer (provided with Tyramide Signal Amplification kit; PerkinElmer Life Sciences), cells were incubated overnight at 4 °C with horseradish peroxidase-conjugated rabbit anti-HA antibodies (Roche Applied Science) diluted in TNB buffer supplemented with 0.05% Triton TX-100. The samples were washed three times for 5 min with PBS containing 0.05% Tween 20. The Tyramide Signal Amplification kit with fluorescein isothiocyanate-labeled streptavidin was used according to the manufacturer's instructions to stain positive cells. The coverslips containing the cells were mounted onto glass slides with 1,4-diazabicyclo(2,2,2)-octane/mowiol and viewed with a TCS NT confocal laser-scanning microscope (Leica, Heidelberg, Germany).
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RESULTS |
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Cell Surface ExpressionCells transfected with mutant receptors showed lower specific binding of 125I-[Nle4,D-Phe7]-MSH as compared with cells expressing the WT receptor (Fig. 1). Only the T112 M mutant showed specific binding that resembled that of the WT receptor. We observed no differences in transfection efficiencies or cell growth between cells transfected with WT or with mutant receptor constructs.
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The decreased specific binding could reflect lower cell surface expression of the mutant receptors but also lower affinity of 125I-[Nle4,D-Phe7]-MSH. Therefore, the affinity of [Nle4,D-Phe7]
-MSH for the receptors was determined (Table II). For most mutants, the affinity of [Nle4,D-Phe7]
-MSH was similar to that of the WT receptor, indicating that for these mutants the lower specific binding as shown in Fig. 1 is due to lower cell surface expression. The T112M, I301T, and L250Q mutants even showed increased affinity for [Nle4,D-Phe7]
-MSH. For these mutants, relative cell surface expression would be lower as was indicated in Fig. 1. Therefore, to determine cell surface expression of the L250Q and the T112M mutants more directly, saturation binding experiments were performed with these mutants (Fig. 2). Both mutants showed a decreased Bmax (16 and 41% of WT receptor expression for the L250Q and T112M mutant, respectively), whereas the Kd values are in agreement with the IC50 values obtained from the displacement studies. Thus, all of the mutant receptors have decreased cell surface expression as compared with the WT receptor.
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Gu et al. (14) reported decreased affinity of 125I-[Nle4,D-Phe7]-MSH and no changes in cell surface expression for the I137T mutant, whereas our results indicate that affinity is not changed but rather cell surface expression is impaired for this mutant. To confirm our data, saturation binding experiments were also performed with the I137T mutant. We found no difference in affinity as compared with the WT receptor, but the Bmax was reduced for the I137T mutant (Fig. 3).
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Affinities of -MSH and AgRP-(83132)For most mutants, the IC50 of
-MSH was similar to that of the WT receptor. For the T112M and I301T mutants, the IC50 of
-MSH was decreased 2-fold, and it was decreased 10-fold for the L250Q mutant, indicating higher affinity of
-MSH. Since AgRP is an endogenous ligand for the MC4R, the effect of the mutations on AgRP affinity was also determined. AgRP-(83132) was used, because it has been shown before that this fragment contains the pharmacologically active sequence of AgRP-(2830). Like for
-MSH, the affinity of AgRP-(83132) was similar to that of the WT receptor for most mutants, but decreased IC50 values were obtained for the L112M, I301T, and L250Q mutants. The ratio IC50(MSH)/IC50(AgRP) remained constant for most mutants, except for the L250Q and S30F/G252S mutants, which showed a decreased and increased ratio, respectively.
Activation StudiesThe MC4R couples to Gs and activates the AC/cyclic AMP pathway (31). First, the response of the mutant receptors to
-MSH was measured with an AC assay. For each receptor, the cells used in the AC assay were from the same transfection batch that was used to determine specific 125I-[Nle4,D-Phe7]
-MSH binding. Dose-response curves were obtained for all mutants, except for the P78L mutant, which did not show any activation by
-MSH at concentrations up to 1 µM. A decrease in potency of
-MSH was found for the I137T and S30F/G252S mutants (10-fold increase in EC50 value), but the other mutants had no or only minor differences in EC50 value as compared with the WT receptor (Table III). However, all mutants showed a decreased maximal response to
-MSH (Emax). There was a correlation between the relative cell surface expression of the receptors and the Emax as measured in the AC assay (Fig. 4).
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The EC50 value that is obtained depends on which assay is used to measure receptor activation (e.g. measuring genomic response or a more direct G-protein/effector response). Also, a receptor reserve may exist in a more sensitive assay, which limits the ability to measure differences in the number of maximally activated receptors. Therefore, we also used a sensitive lacZ reporter gene assay (24) to measure receptor activation. Most mutants showed an EC50 value that was comparable with that of the WT receptor but similar to the AC assay; the I137T and S30F/G252S mutants showed an increased EC50 value (4- and 3-fold increase, respectively) (Table III). The L250Q mutant was constitutively active, with basal activity that was almost as high as the maximal -MSH response of the WT receptor (Fig. 5). Further increase of activity by
-MSH was too small to obtain accurate EC50 values for the L250Q mutant. In contrast to the AC assay, the maximal response of the mutants to
-MSH did not differ from that of the WT receptor, although binding studies demonstrated that in the lacZ assay the mutant receptors had impaired cell surface expression (data not shown). However, expressing different amounts of WT receptor also did not affect the maximal
-MSH response (Fig. 6), showing that the lacZ assay is not suitable to relate cell surface expression to Emax.
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ImmunocytochemistryWe further explored the cellular distribution of HA-tagged forms of the WT receptor and the P78L, R165W, and R165Q mutants, which showed the largest decrease in cell surface expression (Fig. 7). The WT receptor showed a diffuse staining, which was also present at the cell surface. In contrast, the three mutants showed a staining that was much more concentrated around the nucleus, and the staining at the cell surface was absent.
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DISCUSSION |
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The decreased maximal response to -MSH of the mutants as measured in the AC assay most likely resulted from the lower cell surface expression, as was supported by the correlation between cell surface expression level and maximal
-MSH stimulation. Only for two mutants was there evidence for impaired intrinsic ability to respond to
-MSH (higher EC50 value), and two mutants showed increased affinity for
-MSH or AgRP-(83132). Therefore, the predominant effect of MC4R mutations that are associated with obesity appears to be impairment of cell surface expression, leading to lower
-MSH responses. Although in the lacZ assay lower cell surface expression did not result in decreased maximal
-MSH stimulation, this is most likely due to inability of the assay to reflect differences in receptor expression level. Indeed, expressing different levels of WT receptor did not affect the maximal
-MSH response. The EC50 values in the lacZ assay were lower than those obtained in the AC assay, suggesting that at lower receptor occupancy, a maximal response was already reached. This indicates the existence of a receptor reserve in the lacZ assay, which is probably due to its high sensitivity, and this could well explain the lack of correlation between cell surface expression and maximal receptor activation in the lacZ assay.
Decreased cell surface expression may therefore be an important mechanism underlying the increased risk for obesity of individuals carrying a MC4R mutation. The observation that mice lacking one MC4R allele have intermediate body weight as compared with wild type and homozygous knock-out animals also suggests that MC4R expression is important for maintaining normal body weight. Still, a dominant negative mechanism cannot be ruled out, although Ho et al. (23) showed that two frameshift mutations that are retained intracellularly did not affect wild type receptor function when expressed in the same cells. Furthermore, only the cAMP signal transduction route was investigated here. Other signal transduction pathways may potentially be involved in MC4R signaling. We cannot exclude the possibility that MC4R mutations affect these pathways and that this may contribute to the obesity phenotype as well.
Our results demonstrate the importance of determining cell surface expression level when analyzing the pharmacology of mutant receptors, since it is a parameter that is often affected, and it can influence the outcome of activation studies. Impaired cell surface expression resulting from mutations that are linked with diseases are also seen in other receptor systems, indicating that the total amount of cell surface receptors is indeed important for normal function. For instance, the majority of mutant vasopressin-2 receptors that are found in patients with nephrogenic diabetes insipidus fail to reach the cell surface (32, 33).
In addition to lower cell surface expression, resulting in lower Emax values, both activation assays showed increased EC50 values of -MSH for the I137T and S30F/G252S mutants. Since affinity of
-MSH was not decreased for these receptors (for the I137T mutant,
-MSH affinity was actually higher), G-protein coupling seems impaired for these mutants. In addition, the S30F/G252S mutant showed increased affinity for the inverse agonist AgRP-(83132). Thus, for these mutants, changes in pharmacology for the endogenous ligands may be an additional mechanism whereby melanocortin signaling is decreased. Interestingly, the ratio of the affinities for
-MSH and AgRP-(83132) remained constant for most mutants, including the T112M and I301T mutants, whose affinities differed 2-fold from those of the WT receptor. These data suggest that the binding of
-MSH to the MC4R is affected similarly to that of AgRP-(83132) by these mutations. Similar results were previously obtained for binding of a selective agonist and the antagonist Agouti protein to the MC4R (34).
In contrast to the present study, Vaisse et al. (16) reported increased EC50 values for the R165W, I170V, and I301T mutants. As already indicated by Vaisse et al. (16), the effects on the EC50 values could be due to reduced expression of the mutants. Indeed, these authors reported lower specific binding for the mutant receptors as compared with the WT receptor, and a receptor threshold in their assay could explain the differences with our results. Gu et al. (14) found that for the T112M mutant, the EC50 value of -MSH was not affected. Here this mutant was shown to be poorly expressed, and it should thus be considered a loss of function mutant. In addition, Gu et al. (14) reported decreased affinity of the I137T mutant for [Nle4,D-Phe7]
-MSH, whereas we found decreased expression rather than differences in [Nle4,D-Phe7]
-MSH affinity. There is no obvious explanation for these differences, but since both studies found decreased potency of
-MSH for this mutant, it may be concluded that it has an impaired
-MSH response.
Consistent with the data of Vaisse et al. (16), the L250Q mutant displayed high constitutive activity in the lacZ assay. As for many constitutively active receptors, this mutant has increased affinity for agonists. Although the inverse agonist AgRP-(83132) (35) had also increased affinity for the L250Q mutant, this was less pronounced than for the agonists -MSH and 125I-[Nle4,D-Phe7]
-MSH. The enhanced basal activity and increase in agonist affinity of this receptor may be considered gain of function, which would not fit with an obese phenotype. However, as for many CAM receptors (36), cell surface expression level of the L250Q mutant was decreased. Even more, the constitutive activity could only be detected in the sensitive lacZ assay and not in the AC assay and may actually be low and physiologically insignificant. Therefore, the net result of this mutation can still be impairment of melanocortin signaling.
Quantification of impairment in receptor function due to mutations may help to determine the influence of these mutations on body weight. Of the mutations tested here, clinical data have presently been reported for carriers with the R165W, I170V, L250Q, and I301T mutants (16), and family studies have been carried out for the I170V, I137T, R165W, and I301T mutants (14, 16, 17). However, more phenotypical data of mutation carriers are needed before correlations between receptor function and phenotype can be made, and it should be kept in mind that aspects of receptor function that have not been tested yet may correlate better with phenotype.
The immunocytochemical data suggest that the P78L, R165W, and R165Q mutants are expressed but are retained intracellular. This may also be the case for the other mutant MC4Rs that were tested here, since two frameshift mutations were also shown to be expressed but failed to reach the cell surface (23). Many mutant receptors that have poor cell surface expression, including CAM receptors, can be rescued to the cell surface upon ligand binding (3638). Pharmacological interventions aimed at treating the obesity of individuals with MC4R mutations may thus be directed toward stabilization of MC4R at the cell surface, preferably combined with receptor activation. Also for the development of MC4R agonists to treat obesity in general, screening for stabilization of the MC4R at the cell surface may be important, since our data suggest that MC4R mutations associated with obesity show decreased MC4R expression at the cell surface.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 31-30-2538517; Fax: 31-30-2539032; E-mail: R.A.H.Adan{at}med.uu.nl.
1 The abbreviations used are: MC4R, melanocortin-4 receptor; AgRP, Agouti-related protein; HA, hemagglutinin; PBS, phosphate-buffered saline; WT, wild type; AC, adenylate cyclase; -MSH,
-melanocytestimulating hormone.
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REFERENCES |
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