From Pfizer Global Research and Development, Ann
Arbor, Michigan 48105 and ¶ Wayne State University School of
Medicine, Detroit, Michigan 48201
Received for publication, November 12, 2002, and in revised form, January 15, 2003
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ABSTRACT |
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Loss-of-function mutations in the human
melanocortin-4 receptor (MC4R) are associated with obesity. Previous
work has implicated a C-terminal di-isoleucine motif at residues
316/317 in MC4R cell surface targeting. It was therefore of interest to
examine function and cell surface expression of an MC4R mutation found
in an obese proband in which one of these isoleucines was substituted
by threonine (I317T). Single mutant (I316T or I317T) and double mutant
(I316T,I317T) forms of MC4R were constructed by
oligonucleotide-directed mutagenesis and tested for function and cell
surface expression in transfected cells. Function was assessed using
assays for agonist,
[Nle4-D-Phe7] Monogenic forms of obesity have been identified in mouse and man
(1-8). These genes are either expressed in brain or their products act
on brain, indicating the prominent role of this tissue in energy
homeostasis. Neurons that express these genes are found in the arcuate
and paraventricular nuclei of the hypothalamus and form a neural
circuit capable of sensing signals associated with metabolic state and
coordinating behavioral and metabolic responses to the metabolic
demands of the organism. This neural circuit is composed of at least
three distinct neuronal subtypes. One subtype expresses the G
protein-coupled melanocortin-4 receptor (MC4R).1 MC4R is a
Gs-coupled receptor, and its stimulation leads to
generation of intracellular cAMP. The other two subtypes provide the
MC4R agonist, MC4R mutations are relatively common and have been estimated to account
for as much as 5% of the obesity found within certain populations (3,
4, 14, 15). Frameshift mutations in the gene resulting in truncated
MC4R protein can exhibit a dominant inheritance pattern, although one
example of severe truncation demonstrates variable penetrance (15).
Some of the truncated forms have been functionally evaluated, and they
neither bind ligand nor become associated with the outer surface of the
plasma membrane (16). The results from transfection experiments of cultured cells indicate that the truncated receptor forms do not appear
to have a dominant negative function (16), supporting simple
haploinsufficiency as an explanation for the dominant inheritance pattern when it occurs (12). MC4R haploinsufficiency is consistent with
MC4R heterozygotic knockout mice, which exhibit obesity intermediate to
wild-type and homozygotic knockout mice (11). MC4R haploinsufficiency is not supported in a report describing deletions of human chromosome 18q, which harbors MC4R, resulting in MC4R hemizygosity without obesity
(17).
In the case of MC4R missense mutations, the impact on phenotype
generally follows the severity of the impact on receptor function or
expression. Notably, the receptor with the N62S mutation was shown to
support a partial cAMP response to receptor stimulation and is
associated with a recessive inheritance pattern of obesity within the
pedigree (14). Results from these genetic analyses indicate little or
no receptor reserve of MC4R in human or mouse brains given the
sensitivity of the phenotype to reductions in MC4R receptor number
or function.
The C termini of G protein-coupled receptors contain a conserved,
dihydrophobic sequence composed of leucine, isoleucine, phenylalanine,
valine, or methionine and preceded by an acidic glutamate or aspartate
(18). Mutation of the conserved dileucine motifs in the vasopressin
V2 and lutropin/choriogonadotropin receptors have been
shown to limit membrane targeting of these receptors (18-20). In the
case of MC4R, the motif is composed of di-isoleucines at codons
316/317. We have shown that MC4R truncated proximal to the
di-isoleucines is not expressed on the cell surface, whereas MC4R
truncated two residues distal to this site is expressed (16). It was
therefore of interest to note the report of a human mutation, I317T, in
an obese proband (21). In the present report, we make the mutant MC4Rs
containing threonine substitutions at either codon 316 or 317, or both,
and test the mutants for cell surface expression and function.
Materials--
[Nle4-D-Phe7] Oligonucleotide-directed Mutagenesis--
To construct MC4R
sequence variants, wild-type MC4R was modified by adding a Kozak
sequence (GCCGCCGCC) and FLAG tag to the N termini of the MC4R coding
sequence by conventional PCR-based techniques. PCR products were cloned
into the vector pCR3.1 (Invitrogen). PCR products were treated with
Taq polymerase (Invitrogen) at 72 °C for 10 min to add 3'
overhanging Ts; subsequent cloning was carried out according to the
manufacturer's instruction. The modifications were confirmed by
sequencing. Site-directed mutagenesis of the FLAG-MC4R plasmid was
accomplished using the QuikChangeTM site-directed
mutagenesis kit (Stratagene). Primer pairs (5'-3') were designed to
introduce the desired mutations in the MC4R coding sequence: I316T
upstream (CTTCAAAGAGACCATCTGTTG) and downstream (CAACAGATGGTCTCTTTGAAG) primers; I317T upstream
(ACCTTCAAAGAGATCACCTGTTGCTATCCCCTG) and downstream
(CAGGGGATAGCAACAGGTGATCTCTTTGAAGGT) primers; and I316T,I317T upstream
(CTTCAAAGAGACCACCTGTTG) and downstream (CAACAGGTGGCTTCTTTGAAG) primers.
Primer pairs (LifeTech) with the desired mutations were annealed to the
wild-type expression construct. The plasmid-primer mixture was
amplified in a PCR reaction with Pfu turbo DNA polymerase following the manufacturer's recommendations. After amplification the
products were treated with the restriction enzyme DpnI. The products of the restriction digest were used to transform competent Escherichia coli, strain XL1-Blue, following the procedure
recommended by the manufacturer (Stratagene). Plasmids isolated from
the resulting bacterial colonies were screened by DNA sequencing to
identify clones with the desired mutations. The entire MC4R coding
region of plasmids with mutations was sequenced to confirm the absence of unwanted mutations that might have been introduced during the PCR.
Cell Culture and Transfection--
293T and COS-7 cells were
maintained in Dulbecco's modified Eagle's medium (with glutamine;
Invitrogen) supplemented with 10% fetal bovine serum, 100 units/ml
penicillin, and 100 µg/ml streptomycin (Invitrogen). Cells were
incubated at 37 °C in humidified air containing 5% CO2.
Cells were generally at 70-80% confluence on the day of transfection,
and transfections of FLAG-tagged WT or mutant MC4R plasmids were
carried out using LipofectAMINE reagent according to the
manufacturer's instruction (Invitrogen).
Whole-cell Receptor Binding Assays--
Receptor binding assays
were carried out on monolayers of 293T cells transiently transfected
with MC4R mutants. HEK-293T cells were transfected in 150 × 25-mm
dishes and allowed to incubate overnight at 37 °C in humidified air
containing 5% CO2. The cells were detached from plates
with PBS, 0.02% EDTA and collected by centrifugation. The cells were
plated into 24-well culture dishes at a density of 2.5 × 104 cells/well and allowed to attach overnight. The binding
was carried out in a total volume of 200 µl containing Dulbecco's
modified Eagle's medium and 1 mg/ml BSA, 50 pM
[125I]NDP- cAMP Accumulation Assays--
48 h after transfection, 293T
cells were washed once with PBS and then detached from the plate with
PBS containing 0.02% EDTA (Sigma). The detached cells were harvested
by centrifugation and resuspended in Hanks' balanced salt solution
(Invitrogen) containing 0.5 mM IBMX, 2 mM
HEPES, pH 7.5 (IBMX buffer). After incubation at 37 °C for 15 min to
allow for IBMX uptake, 0.4 ml of cell suspension (~5 × 105 cells/ml) was added to 0.1 ml of IBMX buffer containing
various concentrations of agonists or 10 µM forskolin.
The cells were subsequently incubated at 37 °C for 15 min to allow
for cAMP accumulation. The activity was terminated by adding 0.5 ml of
5% trichloroacetic acid, and cAMP released from lysed cells was
assayed by the cAMP 125I scintillation proximity assay
system (Amersham Biosciences). EC50 values were calculated
with a 95% confidence interval using GraphPad Prism software (using
nonlinear regression analysis fitted with a sigmoidal dose-response
curve with variable slope).
Immunofluorescence Staining--
COS-7 cells were chosen for
this study as they were more adherent than 293T cells during the
staining process. 24 h after transfection, COS-7 cells were seeded
onto 25 mM circular microscope coverslips, size 1 (Fisher
Scientific), in six-well culture plates (Corning-Costar) and incubated
overnight. The next day, the cells were washed four times with DPBS and
fixed for 20 min in 4% paraformaldehyde in DPBS followed by four
washes with DPBS. Cells were then incubated for 5 min either with DPBS
alone for nonpermeabilized staining or with DPBS containing 0.2%
Triton X-100 for permeabilization. Following blocking for 30 min with
DPBS containing 10% fetal bovine serum and 2% BSA (blocking buffer),
cells were incubated for 1 h in blocking buffer containing 1 µg/ml anti-FLAG monoclonal antibodies M2 (Sigma) followed by three
washes each for 10 min with blocking buffer. Cells were then incubated
for 30 min in blocking buffer containing a 1:1000 dilution of Oregon
Green 488 goat anti-mouse IgG conjugate (highly cross-absorbed;
Molecular Probes). After being washed twice for 10 min with
DPBS, cells were mounted an glass slides using Aqua-PolyMount
(Polysciences, Inc.) and viewed using a confocal laser scanning
microscope (Fluoview, Olympus).
Flow Cytometry--
48 h post-transfection, HEK-293 cell
monolayers were washed with incubation buffer (DPBS containing 20 mM HEPES, pH 7.4, and 1 mg/ml BSA). The cells were detached
from the plates by incubation in DPBS with 0.02% EDTA (Sigma, catalog
No. E8008). The cells were collected by centrifugation, washed in
incubation buffer, and then suspended to a concentration of 4.0 × 107 cells/ml in incubation buffer. 50 µl of cells were
incubated with 50 µl of 10 µg/ml murine anti-FLAG M2 monoclonal
antibody (Sigma, catalog No. 316) in incubation buffer for 30 min at
room temperature. The cells were collected by centrifugation and washed three times in incubation buffer. The cell pellets were suspended in
100 µl of incubation buffer containing 1 µg/ml Alexa
Fluor® 488 goat anti-mouse IgG (H+L) (Molecular Probes,
catalog No. A-11001) and incubated at room temperature for 30 min. The
cells were collected by centrifugation and washed three times with
incubation buffer. The final cell pellets were suspended in incubation
buffer to a concentration of 2.0 × 106 cells/ml.
Propidium iodide was added to a final concentration of 1 µg/ml, and
the cell suspensions were passed through a 100-µm filter prior to
flow cytometry. Flow cytometry was performed on a
fluorescence-activated cell sorter (FACS Vantage S.E., BD
Biosciences). Single live cells were gated by scatter and negative propidium iodide fluorescence, 488 nM excitation
wavelength, 675 nM emission wavelength. For each
transfection, the fluorescence of 25,000 single live cells was measured
at 488 excitation wavelength, 530 nM emission wavelength,
to measure the expression of the FLAG antigen on the surface of the cells.
Real-time Quantitative RT-PCR (TaqMan®)--
48 h
post-transfection, total RNA was isolated from cells using TriSol
reagent (Invitrogen) following the manufacturer's recommended protocol. RNA was reverse transcribed using TaqMan® reverse
transcription reagents (PerkinElmer Life Sciences). 400 ng of total
RNA was transcribed in a 50-µl reaction. Reaction mixture contained
5.5 mM MgCl2, 500 µM each dNTP,
2.5 µM random hexamers, 200 units of RNase inhibitor, and
62.5 units of MultiScribe reverse transcriptase. Reverse transcription
reactions were incubated at 25 °C for 15 min, 48 °C for 30 min,
and 95 °C for 5 min. Multiplex PCR reactions were run on reverse
transcribed RNAs to quantitate the relative abundance of MC4R RNA in
the original RNA samples. Human MC4R primers and a FAM-Fluorescent
probe to a region near the 3'-end of the coding region of the MC4R
sequence, conserved in the wild-type and mutant sequences, were
designed using the Primer Express software (PerkinElmer Life Sciences)
and synthesized (PerkinElmer Life Sciences). The real-time quantitative
RT-PCR, using 18 S ribosomal RNA (rRNA) for normalization, was
performed using TaqMan® Universal PCR Master Mix reagents and
TaqMan® Ribosomal RNA control reagents (VIC Dye) (PerkinElmer Life
Sciences) essentially following the manufacturer's protocol. Briefly,
each reaction mixture contained 5.5 mM MgCl2,
500 µM dNTP, 600 nM both forward and reverse
18 S rRNA primers, 200 nM 18 S rRNA-VIC probe, 600 nM both forward and reverse MC4R primers, 200 nM MC4R-FAM probe, and cDNA in a final volume of 25 µl. Reactions were run and analyzed using ABI PRISM 7700 sequence
detection system (PerkinElmer Life Sciences). MC4R mRNA levels were
normalized to 18 S rRNA, and the relative copy number was determined
using the standard curve method for multiplex PCR as detailed in User
Bulletin No. 2 for the ABI Prism 7700 sequence detection system
(Applied Biosystems).
Substitutions I316T or I317T Decrease Cell Surface Expression of
MC4R as Determined by Receptor Binding in Transfected HEK-293
Cells--
To assess the impact of threonine substitution at
isoleucines 316 and 317 on cell surface expression of MC4R, FLAG-tagged WT or mutated MC4R was transfected into HEK-293 cells and tested for
whole-cell binding of 125I-labeled MC4R agonist. Compared
with cells expressing FLAG-tagged wild-type MC4R, whole-cell binding of
a subsaturating concentration (50 pM) of
[125I]NDP- Substitutions I316T or I317T Decrease Agonist-stimulated cAMP
Accumulation in Transfected HEK-293 Cells--
Compared with HEK-293
cells transfected with FLAG-tagged WT MC4R, cells expressing the singly
substituted receptor at I316T or I317T showed 29 and 37% reductions,
respectively, in maximal agonist-stimulated cAMP accumulation
(Fig. 2, Table I). In cells transfected
with the double mutant, this measure was further reduced to 31% of WT
values (Fig. 2 and Table I). Cells transfected with control GFP
exhibited no agonist-stimulated cAMP accumulation (Fig. 2). The
EC50 values for NDP- Effect of I316T and I317T Mutations on Cell Surface
Targeting of MC4R as Evaluated by Fluorescence Microscopy--
To
visualize the cell surface expression of FLAG-tagged WT and mutated
MC4R, COS-7 cells were transfected with the appropriate expression
constructs and the cellular localization was analyzed by
immunofluorescence microscopy. To distinguish receptors on the cell
surface from intracellular receptors, a monoclonal anti-FLAG antibody
was used with non-permeabilized and permeabilized cells, respectively.
Threonine substitutions of the isoleucines at positions 316 and 317 had
no effect on immunofluorescent labeling of receptor in permeabilized
cells (Fig. 4). In non-permeabilized
cells, it appeared that more WT receptors were targeted to the cell
surface than either of the I316T or I317T mutants and that cell surface targeting was further reduced in the double mutant (Fig. 4).
Effect of I316T and I317T Mutations on Cell Surface
Targeting of MC4R as Evaluated by Fluorescence-activated Cell
Sorting--
To quantitate immunofluorescent detection of FLAG-tagged
WT or mutant MC4R, HEK-293 cells transiently expressing these receptor forms were detected and counted by fluorescence-activated cell sorting.
Twenty-five thousand cells from each transfection were sorted, and the
percentage of cells having a fluorescence intensity signal above a
previously determined cutoff value was determined (Fig.
5). Forty-five percent of cells
expressing the FLAG-tagged WT MC4R (mean fluorescence intensity
signal = 61) met this criterion, whereas only 26% of cells
expressing either I316T or I317T (mean fluorescence intensity
signals = 31 and 32, respectively) and 6% of cells expressing the
double mutant exhibited fluorescence intensity (mean fluorescence
intensity signal = 14) above the cutoff value (Fig. 5).
Deficient Cell Surface Expression of MC4R Mutants Is Not Due to
Lower Transcript Levels--
To determine whether the decreased cell
surface expression of MC4R mutants was due to decreased transcript
levels mutant mRNA, because of either to decreased transcription
or stability, the transcript levels of WT and mutant receptor forms
were measured by TaqMan® quantitative RT-PCR. Reversed transcribed
RNA from each transfection was serially diluted and transcript level of MC4R determined as described under "Experimental Procedures." Results obtained from the dilution experiments indicate that the technique is sufficiently sensitive to detect 2-fold differences in
transcript level (Fig 6A). To
show the specificity of the MC4R primers and probes, cells were
transfected with a control expression construct for the 5-HT7 receptor
and probed for MC4R transcript. RNA collected and reverse transcribed
from these cells and probed for MC4R transcripts required many more
amplification cycles to generate a signal (Fig 6A). Cycle
threshold number for 5-HT7-transfected cells was equal to
the background signal seen using herring sperm DNA as input (data not
shown). Normalization of the data to 18 S rRNA revealed that the
transcript levels of wild-type and single mutant forms were not
different from each other (Fig 6B). However, the transcript
level for the double mutant was greater. Relative to wild-type levels,
double mutant transcript copy numbers were 2.2- and 2.4-fold greater at
the 20 and 10 ng input levels, respectively (Fig. 6B).
Previous work has indicated that the di-isoleucine pair at amino
acid residues 316 and 317 in the C terminus of the MC4R might be
important for cell surface targeting (16). An MC4R transition mutation
has been described in an obese human proband in which thymine is
replaced by cytosine at base position 950, resulting in the
substitution of threonine for the isoleucine at 317 (21). Results
obtained from the present study indicate that these C-terminal isoleucines are important for normal cell surface expression of MC4R in
transiently transfected cells and that substitution of isoleucine by
threonine at position 316 or 317 lowers MC4R cell surface expression.
Double substitutions by threonine are additive.
I316T or I317T each lowers the binding of the 125I-labeled
MC4R agonist NDP- This work establishes the importance of the C-terminal di-isoleucine
pair for normal cell surface expression of MC4R. Previous work (18) has
indicated that the di-leucine motif in the C terminus of the
vasopressin V2 receptor is important in the exiting of the
receptor from the endoplasmic reticulum. In that study, single substitutions of hydrophobic leucines by hydrophobic isoleucine had no
effect on V2 receptor cell surface expression (18);
however, substitution of either leucine by the polar threonine
decreased cell surface expression, and the double substitution
completely suppressed it.
An examination of the C terminus of the family of melanocortin
receptors indicates high sequence conservation only through the first
15 residues (Fig. 7). The dihydrophobic
motif, at C-terminal residues 12 and 13, is completely conserved in
this receptor family, although it is composed of various
hydrophobic amino acids(Fig. 7). A survey of C termini from 384 G
protein-coupled receptors (GPCRs) from the Swiss Protein Database
(European Molecular Biology Laboratory, Heidelberg, Germany) showed
that 180 GPCRs contained a C-terminal dihydrophobic pair consisting of
II, LL, IL, or LI within the first 45 residues and that the most
frequently occurring starting position of these pairs was position 12 (Fig. 8).
-melanocyte-stimulating
hormone (NDP-
-MSH) or forskolin-stimulated cAMP accumulation. Cell
surface expression was determined by whole-cell binding of
[125I]NDP-
-MSH, fluorescence immunocytochemistry and
fluorescence-activated cell sorting. Maximal cAMP generation of the
single mutants was reduced by 40% of wild-type receptor; the double
mutant further reduced function to 40% of control, effects that were
mirrored by decreases in cell-surface expression. Quantitative RT-PCR
showed that, relative to wild-type receptor, transcript levels for the mutated receptors were not reduced. The results further implicate the
C-terminal di-isoleucines in cell surface expression of MC4R and
suggest that mutations of residues 316 or 317 would predict MC4R hypofunction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-melanocyte-stimulating hormone (
-MSH), derived
from pro-opiomelanocortin, or the MC4R antagonist ligand, AGRP
(agouti-related peptide) (9, 10). The hormone leptin, secreted from
adipose tissue, circulates in the blood and regulates appetite by
direct action on the neurons that supply the ligands for MC4R.
Activation of MC4R suppresses appetite, and disruption of the gene in
mouse and man results in hyperphagia and obesity (11-13).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-MSH
(NDP-
-MSH) was purchased from Peninsula Laboratories.
-MSH and
forskolin was purchased from Calbiochem. 3-Isobutyl-methylxanthine
(IBMX) was from Sigma, and [125I]NDP-
-MSH was from
Amersham Biosciences.
-MSH, and increasing concentrations of cold
NDP-
-MSH. After a 90-min incubation, the medium was aspirated and
the wells were washed three times with 0.5 ml/well of Dulbecco's
phosphate-buffered saline (DPBS, Invitrogen) containing 20 mM HEPES, pH 7.4, and 0.1 mg/ml BSA. 100 µl of 0.1 N NaOH was added to the wells, and the cell lysates were
transferred to 20-ml glass vials containing 15 ml of
scintillation mixture. The samples were counted in a Packard Tri-Carb
liquid scintillation counter. Nonlinear regression analysis was
performed using GraphPad Prism software (GraphPad Software, Inc., San
Diego, CA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-MSH was decreased in cells
expressing either of the singly substituted mutants I316T or I317T by
41 and 34%, respectively (Fig. 1, Table
I). Transfection of the double mutant (DMut), in which the isoleucines
at both positions 316 and 317 were changed to threonines, resulted in
no detectable specific binding (Fig. 1).
The affinity (Ki) of MC4R for the agonist,
NDP-
-MSH, was not affected by either I316T or I317T (Table
I). Specific whole-cell binding of the label to control HEK-293 cells
transfected with an expression construct for green fluorescent protein
(GFP) was undetectable and did not differ from untransfected cells
(data not shown).
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Fig. 1.
Substitution of the di-isoleucines by
threonine at residues 316 and 317 of MC4R decreases whole-cell binding
of 125I-labeled
NDP- -MSH without reducing affinity of the
receptor for NDP-
-MSH. Transfection of
HEK-293 cells and whole-cell binding were performed as described under
"Experimental Procedures." Each point is the mean of triplicate
values obtained from three independent experiments. Competitive binding
curves were fit as described under "Experimental Procedures."
Values and statistical analyses of the binding parameters are presented
in Table I.
Pharmacological properties of MC4R WT and mutants
-MSH-dependent cAMP
generation were not affected by threonine substitution of the
isoleucines at positions 316 and 317 (Table I). Stimulation of HEK-293
cells by 10 µM forskolin increased cAMP
accumulation by 5-fold, an effect that was greatly potentiated in cells
transfected with WT MC4R (Fig. 3). The
ability of MC4R receptor transfection to potentiate forskolin-stimulated cAMP generation appeared lower in the single mutants, although differences between the WT and single mutant group
means were not statistically significant (Fig. 3). Transfection of the double mutant resulted in significantly lower
forskolin-stimulated cAMP production than in the WT or single mutants
and was not different from cells expressing control GFP (Fig. 3).
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Fig. 2.
Substitution of the di-isoleucines by
threonine at residues 316 and 317 of MC4R decreases maximal
agonist-stimulated cAMP accumulation in transfected HEK-293 cells.
Cell transfection and whole-cell cAMP accumulation were performed as
described under "Experimental Procedures." Each point is the mean
of triplicate values obtained from three independent experiments.
Dose-response curves were fit as described under "Experimental
Procedures." Values and statistical analyses of the dose-response
parameters are presented in Table I.
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Fig. 3.
Forskolin-stimulated cAMP accumulation in
HEK-293 cells transiently transfected with wild-type or mutated
MC4Rs. Transfection and assay of cAMP accumulation were performed
as described under "Experimental Procedures." Values are means ± S.E., n = 7-9/group. Statistical differences
between means determined by analysis of variance followed by the
Newman-Keuls test: , differs from all groups except DMut,
p < 0.05; *, differs from all groups except GFP,
p < 0.05.
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Fig. 4.
Immunofluorescence of COS-7 cells transfected
with N-terminal FLAG-tagged wild-type or mutated MC4Rs.
Cells were transiently transfected and grown in chamber slides
as described under "Experimental Procedures," either treated
(P) or not treated (N) with permeabilizing agent.
The tagged receptor was immunodetected as described under
"Experimental Procedures."
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Fig. 5.
Fluorescence-activated cell sorting of COS-7
cells transfected with N-terminal FLAG-tagged wild-type or mutated
MC4Rs. Cells were transfected and fluorescence-activated cell
sorting was performed as described under "Experimental Procedures."
The bar in each graph represents the gating set to exclude
95% of the background signal determined from cells transfected with
wild-type MC4R without an N-terminal FLAG tag (blank
histogram in top graph). Values above the gating
bar represent percentage of 2 × 105 cells expressing
N-terminal FLAG-tagged WT or mutated MC4Rs having fluorescent intensity
signals above the gate setting.
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Fig. 6.
Transcript levels of wild-type and mutated
MC4Rs in transfected cells. Quantitative RT-PCR was performed on
cells transfected with WT or mutated MC4R constructs, and the mRNA
for WT or mutated MC4R was determined as described under
"Experimental Procedures." A, for each construct, RNA
input levels of 2.5, 5, 10, and 20 ng were tested to show that the
method had sufficient sensitivity to detect differences in transcript
levels. A control plasmid carrying the cDNA for the 5-HT7 receptor
was used as a negative control. n = 3 wells/condition.
B, relative to wild-type levels, transcript copy level
normalized to 18 S RNA was ~2-fold higher for the double mutant form.
Bars represent means ± standard deviations of
triplicate determinations. Statistical differences between means were
determined by analysis of variance followed by the Newman-Keuls test
(*, p < 0.01).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-MSH to the cell surface but does not change
receptor affinity for the ligand. The reduced binding is reflected in
reduced agonist- as well as forskolin-stimulated cAMP accumulation. It is not clear whether the latter, agonist-independent activation of MC4R
is due to a meaningful constitutive MC4R activity (22, 23) or whether
it is an artifact of receptor overexpression. Regardless, it does
appear to report changes in receptor cell surface expression.
Qualitative imaging by immunofluorescence microscopy of changes in cell
surface expression suggests reduced cell surface expression of the
single mutants, although the differences in this measurement
were more definitive using the quantitative approach afforded by
fluorescence-activated cell sorting.
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Fig. 7.
Conservation of di-hydrophobic motif in human
melanocortin receptors at positions 12 and 13 of the cytoplasmic
tail.
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Fig. 8.
Stacked histogram of the amino acid position
number of leucine or isoleucine pairs in the C terminus of 195 GPCRs. 334 randomly selected human GPCRs were scanned for the
occurrence of a leucine or isoleucine pair (LL,
LI, IL, or II) within the first
45 residues of the C-terminal tail, and the position numbers of
the pairs were recorded. 180 GPCRs contained at least one pair and, as
shown in the figure, the position of the pairs peaked 10-13 residues
from the end of the seventh transmembrane (TM) domain.
Results from the present work do not indicate how the di-isoleucine
pair functions to regulate cell surface expression of MC4R or even
whether the motif operates by affecting transit to or from the plasma
membrane. Acidic dihydrophobic motifs, in particular di-leucine pairs
preceded by a cluster of acidic amino acids, have been shown to be
sorting receptor binding sites for the GGA (Golgi-localizing, -adaptin ear homology
domain, ARF-interacting) proteins required for the
transport of protein cargo from the trans-Golgi network to
endosomes (24, 25). The C-terminal dihydrophobic motif that appears in
GPCRs is preceded, in many GPCRs, by a conserved acidic amino acid
(18). Moreover, substitution of this residue (glutamate) by glutamine
in the vasopressin V2 receptor also blocks cell surface
expression of the receptor (18). It is possible that the GPCR
dihydrophobic motif serves as an intramolecular binding site necessary
for protein folding or an intermolecular binding site for an
adaptor-related protein required for appropriate membrane trafficking.
On the basis of results from the present study, we would predict that
the MC4R mutation I317T would decrease MC4R cell surface expression and
function. The MC4R haploinsufficiency seen in human carriers of
truncation mutations (3, 4) and in heterozygotic MC4R knockout
mice (11) suggest that a 50% decrease in receptor number might support
an obesity phenotype. Extrapolating from the present cell culture data,
a heterozygotic carrier of the I317T mutation might be expected to have
at least 80% normal MC4R expression. The reported obese proband was
heterozygotic for I317T and was extremely obese (above the 99th
percentile, body mass index = 36.5 kg/m2). If this
were the sole obesity-related mutation for the proband, it would
suggest an extreme sensitivity to the loss of MC4R. Further genetic
investigation of this pedigree and of others with mutations of the
C-terminal di-isoleucines 316/317 is required.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Current address: Millennium Pharmaceuticals, Cambridge, MA 02139.
To whom correspondence should be addressed: Dept. of
Psychiatry and Behavioral Neurosciences, Wayne State University School of Medicine, 510 E. Canfield, Detroit, MI 48201. Tel.: 313-577-0677; Fax: 313-993-4269; E-mail: rmackenz@med.wayne.edu.
Published, JBC Papers in Press, February 19, 2003, DOI 10.1074/jbc.M211546200
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ABBREVIATIONS |
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The abbreviations used are:
MC4R, melanocortin-4
receptor;
WT, wild type;
NDP--MSH, [Nle4-D-Phe7]
-melanocyte-stimulating
hormone;
IBMX, 3-isobutyl-methylxanthine;
PBS, phosphate-buffered
saline;
DPBS, Dulbecco's phosphate-buffered saline;
BSA, bovine serum
albumin;
GFP, green fluorescent protein;
RT-PCR, reverse transcriptase
PCR;
GPCR, G protein-coupled receptor;
DMut, double mutant.
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