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Research Article
Molecular Mechanism of the Constitutive
Activation of the L250Q Human Melanocortin-4
Receptor Polymorphism? ?
Bettina Proneth1? ?, Zhimin Xiang1? ?, Irina D.
Pogozheva2, Sally A. Litherland3, Oleg S.
Gorbatyuk1, Amanda M. Shaw4, William J.
Millard4, Henry I. Mosberg2and Carrie
1Department of Medicinal Chemistry, University of Florida, PO Box
100485, Gainesville, FL 32610-0485, USA
2Department of Medicinal Chemistry, University of Michigan, Ann
Arbor, MI 48109, USA
3Department of Pathology, Immunology and Laboratory Medicine,
University of Florida, Gainesville, FL 32610, USA
4Department of Pharmacodynamics, University of Florida,
Gainesville, FL 32610, USA
*Corresponding author: Carrie Haskell-Luevano, firstname.lastname@example.com
The Melanocortin-4 Receptor is a G-protein cou-
pled receptor that has been physiologically linked
to participate in the regulation of energy homeos-
tasis. The Melanocortin-4 Receptor is stimulated
from the pro-opiomelanocortin
and antagonized by the endogenous antagonist
agouti-related protein. Central administration of
melanocortin agonists has been demonstrated to
decrease food intake and conversely, treatment
with antagonists resulted in increased food intake.
Deletion of the Melanocortin-4 Receptor gene
from the mouse genome results in an obese and
hyperphagic phenotype.
human Melanocortin-4-Receptor have been found
in severely obese individuals, suggesting that Mel-
involved in human obesity and obesity-associated
diabetes. Herein, we have performed experiments
to understand the molecular mechanisms associ-
ated with the L250Q human Melanocortin-4-Recep-
obese woman. This L250Q human Melanocortin-4-
Receptor has been pharmacologically character-
ized to result in a constitutively active receptor.
The fact that a constitutively active human Mela-
nocortin-4-Receptor mutation was found in an
obese person is a physiologic contradiction, as
gene transcript
Polymorphisms of the
chronic activation of the human Melanocortin-4-
Receptor and subsequently high cyclic adenosine
monophosphate levels should theoretically result
in a normal or lean phenotype. In this study, we
demonstrated that agouti-related protein acts as
an inverse agonist at this constitutively active
receptor, and we propose a mechanism by which
agouti-related protein might contribute to the
obese phenotype in the L250Q patient. In addition,
using receptor mutagenesis, pharmacology, and
computer modeling approaches, we investigated
the molecular mechanism by which modification
of the L250 residue results in constitutive activa-
tion of the human Melanocortin-4-Receptor.
Key words: agouti-related protein, constitutive
activation, G-protein coupled receptor, melanotro-
pin, obesity, AGRP, POMC
Received 18 January 2006, revised and accepted
for publication 2 February 2006
The melanocortin system consists of five melanocortin receptors
(MCRs) that are expressed differentially throughout the body and
belong to the superfamily of G-protein coupled receptors (GPCRs)
that stimulate the cyclic adenosine monophosphate (cAMP) signal
transduction pathway (1–7). The melanocortin pathway includes
endogenous agonists and the only two known endogenous antago-
nists of GPCRs, agouti and agouti-related protein (AGRP) (8,9). The
melanotropin peptides, a-, b- and c-melanocyte-stimulating hor-
mone (MSH) and adrenocorticotropin hormone (ACTH), are the
endogenous agonists of the MCRs that are derived by posttransla-
tional processing of the precursor hormone pro-opiomelanocortin
(POMC) (10,11). Physiologically, the melanocortin system has been
identified to participate in the regulation of feeding behavior, obes-
ity, and energy homeostasis in rodents as well as in humans (12–
18). It has been demonstrated that targeted disruption of the Mel-
anocortin-4 Receptor (MC4R) gene in mice results in hyperphagia,
obesity, increased linear growth, and hyperinsulinemia (16). Addi-
tionally, pharmacologic studies of intracerebroventricular (ICV) injec-
tion of the synthetic melanocortin agonist MTII (19) inhibits feeding
in mice, and this effect can be blocked by co-administration of the
MC3R and MC4R antagonist SHU9119 (14,20). The endogenous
MC3R and MC4R antagonist AGRP (8,21) when administered cen-
trally, produces a sustained increase in food intake observed for
days (22,23). Ectopic expression of the AGRP protein in transgenic
mice results in hyperphagia and obesity (8,24). In vitro pharmacolog-
ic studies have demonstrated that AGRP is a competitive antagonist
of a-MSH at the centrally expressing MC3R and MC4R (8), and also
?These authors contributed equally to this study.
?This study is dedicated to Mrs Rozella May Haskell (lived between
September 10, 1922 and May 6, 2005) who passed away due to
complications of Type 2 Diabetes.
Chem Biol Drug Des 2006; 67: 215–229
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Munksgaard
doi: 10.1111/j.1747-0285.2006.00362.x
Page 2
possesses inverse agonist activity at the MC4R (25–27). The inverse
agonist activity of AGRP at the MC4R implicates an alternative
physiologic mechanism of AGRP function in addition to simple com-
petitive antagonism of a-MSH at MCRs.
In humans, more than 50 different single nucleotide polymorphisms
(SNPs) of the MC4R gene have been discovered (17,18,28–47). One
of these human MC4R polymorphisms, L250Q (GPCR residue i.d. ¼
6.40), is putatively located at the intracellular end of the transmem-
brane 6 (TM6) domain. The L250Q human Melanocortin-4-Receptor
(hMC4R) was identified in an extremely obese French female
patient and when characterized pharmacologically in vitro, resulted
in the discovery of the first constitutively active hMC4R (17). Consti-
tutive activation of the L250Q hMC4R resulted in elevated basal
cAMP levels of the receptor in the absence of agonist stimulation
(17). The fact that a constitutively active hMC4R mutation was
found in an obese person is a paradox, because chronic activation
of the hMC4R and subsequently high cAMP levels should theoretic-
ally result in a normal or lean phenotype. Thus, the molecular
mechanism by which this L250Q hMC4R polymorphism is involved
in human obesity is not yet understood at a molecular level and ini-
tiated the study reported herein.
Materials and Methods
Peptides used in this study were purchased from commercial sources, a-MSH, 4-norleu-
cine-7-D-phenylalanine (NDP)-MSH, MTII, ACTH(1–24), b-MSH, c2-MSH (Bachem, Ter-
rance, CA, USA), and human Agouti-related Protein [hAGRP(87–132); Peptides
International, Louisville, KY, USA]. The melanocortin tetrapeptide JRH887–9 (Ac-His-D-
Phe-Arg-Trp-NH2) was synthesized in our laboratory as previously reported (48).
hMC4R in vitro receptor mutagenesis
The human wild-type (WT) N-terminal Flag-tagged hMC4R cDNA was generously pro-
vided by Dr Robert Mackenzie (30), and was subcloned into the pBluescript plasmid
(Stratagene, La Jolla, CA, USA) for subsequent mutagenesis. In vitro hMC4R muta-
genesis was performed as described previously (49). Amino acid modifications of the
hMC4R were introduced by applying a polymerase chain reaction (PCR) strategy using
pfu turbo polymerase (Stratagene) and a complementary set of primers containing the
nucleotide mutation(s) resulting in the desired residue change. After completion of
the PCR (95 ?C for 30 s, 12 cycles of 95 ?C for 30 s, 55 ?C for 1 min, 68 ?C for
9 min) the product was purified (Qiaquick PCR Purification Kit, Qiagen, Valencia, CA,
USA) and eluted in water. Subsequently, the sample was cut with DpnI (Invitrogen,
Carlsbad, CA, USA) to linearize the WT template DNA leaving only nicked circularized
mutant DNA. The mutant hMC4R DNA was then transformed into competent DH5a
Escherichia coli cells. Single colonies were selected and the presence of the desired
mutant was verified by DNA sequencing. The DNA containing the mutant was then
excised and subcloned into the HindIII/XbaI restriction sites of the pCDNA3expres-
sion vector (Invitrogen). Complete Flag-hMC4R sequences were confirmed free of PCR
nucleotide base errors by DNA sequencing (University of Florida sequencing core
Generation of stable cell lines
Human embryonic kidney-293 (HEK-293) cells were maintained as described previously
(49) in Dulbecco's Modified Eagle's medium (DMEM) with 10% fetal calf serum and
seeded 1 day prior to transfection at 1 · 106cells/100-mm dish. Wild-type and mutant
DNA in the pCDNA3 expression vector (20 lg) were transfected using the calcium
phosphate method (50). Stable receptor populations were generated using G418 selec-
tion (0.7–1 mg/mL) for subsequent bioassay analysis.
cAMP-based functional bioassay
Human embryonic kidney-293 cells stably expressing WT and mutant receptors were
transfected with 4 lg of cAMP response element (CRE)/b-galactosidase reporter gene
as previously described (49,51). Briefly, 5000–15 000 post-transfection cells were plated
into collagen-treated 96-well plates and incubated overnight. Forty-eight hours post-
transfection, the cells were stimulated with 100 lL of peptide (10)6to 10)12M) for
a-MSH, NDP-MSH, and ACTH(1–24) and 100 lL of peptide (10)5to 10)11M) for c2-
MSH, b-MSH, and JRH887–9 or forskolin (10)4M) control in assay medium [DMEM
containing 0.1 mg/mL bovine serum albumin (BSA) and 0.1 mM isobutylmethylxanthine]
for 6 h. The assay media was aspirated and 50 lL of lysis buffer (250 mM Tris–HCl,
pH 8.0, and 0.1% Triton-X-100) was added. The plates were stored at )80 ?C over-
night. The plates containing the cell lysates were thawed the following day. Aliquots
of 10 lL were taken from each well and transferred to another 96-well plate for relat-
ive protein determination. To the cell lysate plates, 40 lL of phosphate-buffered saline
(PBS) with 0.5% BSA was added to each well. Subsequently, 150 lL of substrate buf-
fer [60 mM sodium phosphate, 1 mM MgCl2, 10 mM KCl, 5 mM b-mercaptoethanol,
2 mg/mL of o-nitrophenyl-b-D-galactopyranoside (ONPG)] was added to each well and
the plates were incubated at 37 ?C. The sample absorbance, OD405, was measured
using a 96-well plate reader (Molecular Devices, Sunnyvale, CA, USA). The relative pro-
tein was determined by adding 200 lL 1:5 dilution Bio-Rad (Hercules, CA, USA) G250
protein dye:water to the 10 lL cell lysate sample taken previously, and the OD595was
measured on a 96-well plate reader (Molecular Devices). Data points were normalized
both to the relative protein content and non-receptor-dependent forskolin values.
Assays were performed using duplicate data points and repeated in at least three
independent experiments. Data analysis, EC50, pA2 estimates, and their associated
standard errors of the mean, were determined by fitting the data to a non-linear least-
squares analysis using the PRISM program (v4.0, GraphPad Inc., San Diego, CA, USA).
The antagonistic and inverse agonist properties of hAGRP(87–132) were determined by
the ability of this ligand to competitively displace the MTII agonist in a dose-depend-
ent manner. The pA2values were generated using the Schild analysis method (52). Sta-
tistical analysis was performed using a Student's t-test.
Transient transfection bioassay
Human embryonic kidney-293 cells were maintained in DMEM with 10% fetal calf
serum and seeded 1 day prior to transfection at 2 · 106cells/100-mm dish. Mutant
and WT plasmid DNAs were transfected in a single experiment at different concentra-
tions, as shown in Table 1, using the calcium phosphate method (50). Cells were incu-
bated overnight at 35 ?C and 3% CO2, and the colorimetric reporter gene bioassays
were performed as described above, with the exception that dose–response curves of
compounds were omitted, only the basal and forskolin values were measured (49,53).
Competitive displacement-binding assays
NDP-MSH and hAGRP(87–132) iodination
125I-NDP-MSH and
method as previously described (54). Using 50 mM sodium phosphate buffer (pH 7.4) as
the reaction buffer,
USA) was added to 20 lg of NDP-MSH (Bachem) or 20 lg AGRP(87–132) (Peptides
International) in 5 lL buffer. To initiate the reaction, 10 lL of a 2.4 mg/mL solution of
chloramine-T (Sigma Chemical Co., St Louis, MO, USA) was added for 15 seconds with
gentle agitation. This reaction was terminated by the addition of 50 lL of a 4.8 mg/
mL solution of sodium metabisulfite (Sigma Chemical Co.) for 20 seconds with gentle
agitation. The reaction mixture was then diluted with 200 lL 10% BSA and the resul-
125I-AGRP(87–132) were prepared using a modified chloramine-T
125I-Na (0.5 mCi; Amersham Life Sciences, Inc., Piscataway, NJ,
Table 1: Concentration of DNA used in the transient transfection assay
DNA transfected
Concentration plotted (ng)
05 2550 75 100
Mutant/WT (ng)
pCDNA3plasmid (ng)
CRE/b-gal (lg)
05 25
100 950
WT, wild type; CRE, cyclic adenosine monophosphate response element.
Proneth et al.
Chem Biol Drug Des 2006; 67: 215–229
Page 3
tant mixture layered on a Bio-Gel P2 (Bio-Rad Labs) column (1.0 · 30 cm Econocolumn,
Bio-Rad Labs) for NDP-MSH or on a Bio-Gel P6 (Bio-Rad Labs) column (1.0 · 50 cm
Econocolumn, Bio-Rad Labs) for AGRP(87–132) for separation by size exclusion chroma-
tography using 50 mM sodium phosphate buffer, pH 7.4 as column eluant. Fifteen drop
fractions (approximately 500 lL) were collected into glass tubes containing 500 lL of
1% BSA. Each fraction was then counted on the Apex Automatic Gamma Counter (ICN
Micromedic Systems Model 28023, with RIA AID software; Robert Maciel Associates,
Inc.) to determine peak125I incorporation fractions.
Receptor-binding studies
Human embryonic kidney-293 cells stably expressing the WT and mutant receptors
were maintained as described above. One day preceding the experiment, (0.6–
0.8) · 106cells per well were plated onto 12-well plates. The peptides NDP-MSH and
hAGRP(87–132) were used to competitively displace the
(150 000 c.p.m. per well) NDP-MSH and hAGRP(87–132), respectively. Dose–response
curves (10)6to 10)12M) and IC50values were generated and analyzed by non-linear
least-squares analysis (55) and the PRISM program (version 4.0, GraphPad Inc.). The per-
centage total specific binding was determined based upon the non-specific values
obtained using 10)6M NDP-MSH or hAGRP(87–132) for the respective radiolabeled
peptide. Each experiment was performed using duplicate data points and repeated in
at least two independent experiments. The standard deviation errors of the mean were
derived from the average percentage specific binding values from at least two inde-
pendent experiments and using the PRISM program (v4.0, GraphPad Inc.). Statistical ana-
lysis was performed using a Student's t-test.
125I-radiolabeled peptides
FACS analysis of wild-type and mutant Flag-
tagged hMC4 receptors
Fluorescence-activated cell sorting (FACS) analysis of N-terminally Flag-tagged WT
and mutant hMC4R was performed as described previously for the detection of the
intracellular protein cyclo-oxygenase/prostaglandin synthase 2 (COX2/PGS2) (56). The
protocol was modified by using an allophycocyanin (APC)-conjugated anti-Flag mono-
clonal antibody (Prozyme, San Leandro, CA, USA) for both, cell surface and intracellu-
lar detection of the Flag-hMC4Rs. To detect cell surface expression of WT and
mutant Flag-hMC4Rs, cells were incubated 45 min with APC-conjugated anti-Flag
monoclonal antibody at a concentration of 1 lg/million cells. To detect the total (sur-
face and intracellular) receptor expression cells were subsequently permeabilized with
saponin buffer and stained an additional hour with the APC-conjugated anti-Flag
monoclonal antibody. Unlabeled cells were used to set the background fluorescence
staining for these analyses. BD Biosciences (San Jose, CA, USA) FACS Calibur flow
cytometers were used to collect both stained cell percentages (surface and total) and
mean fluorescence data were measured from a minimum of 10 000 collected events
per sample. Experiments were repeated three independent times. The data are pre-
sented as the mean of the three experiments (€SEM). Statistical analysis was per-
formed using a Student's t-test.
Three-dimensional hMC4R homology molecular
modeling studies
Homology modeling of the hMC4R in the 'inactive' state in complex with AGRP (25),
and in the 'active' state in complex with NDP-MSH (57), were derived based upon
the crystal structure of rhodopsin (PDB-ID: 1 gzm) (58). The modeling of the active
receptor conformation satisfied distance constraints resulting from modifications (disul-
fides, metal-binding clusters, hydrogen bonds between neighboring polar residues)
that facilitate activation, which were applied in distance geometry calculations, as
was described for modeling of the active conformation of the l-opioid receptor (59).
Based upon receptor mutagenesis and modeling results it has been previously pro-
posed that for the MCRs, a b-hairpin is formed in the extracellular loop 1 (EL1; as in
bacterial rhodopsins), and an additional short b-strand in the N-terminal segment is
attached to EL3 by a disulfide bond between Cys40and Cys279, and another important
disulfide bond Cys271–Cys277connects the EL3 to TM6 (25). Sequence alignment of
MCRs and rhodopsin (25) assumes the disappearance of an a-aneurism [an insertion
of an extra-residue(s) in an a-helix] in TM2 that is present in rhodopsin (60), but con-
servation of the a-aneurism in TM5 found in the rhodopsin crystal structure. In order
to understand the results of the L250 mutations to Ala, Glu, Phe, Lys, Asn, Arg, and
Gln the L250 side chain was substituted in the active and the inactive MC4R models
to the corresponding side chains and the side chain rotamers were adjusted to avoid
steric overlap and to provide favorable interactions (hydrogen bonds and van der Wa-
als) with neighboring residues. Final energy minimization of all hMC4R models was
performed using the CHARM (61) potential with e ¼ 10, and utilizing the adopted basic
Newton-Raphson method (50-iterations). The models of the hMC4R in the 'active' and
'inactive'conformationcanbe found
on thewebsite: http://mosberglab.
hMC4R residue numbering
Highly conserved GPCR residues in the TM domain of the hMC4R and other GPCRs are
given two numbering schemes. First, residues are numbered according to their posi-
tions in the receptor amino acid sequence. Secondly, residues are numbered relative to
the most conserved residue in the TM in which it is located (N1, N2). N1 refers to the
TM number, and N2 refers to the position of the residue relative to the most con-
served one with numbers decreasing towards the N-terminus and increasing toward
the C-terminus. The most conserved residue is assigned the position number 50, e.g.
P299 (7.50) and therefore D298 (7.49) and Y302 (7.53). This numbering scheme simpli-
fies the identification of aligned residues in different GPCRs (62).
Seven adult male Sprague–Dawley rats (250–300 g, Charles Rivers) were utilized in
these studies. All animals were treated humanely and protocols have been approved
by the University of Florida Animal Care and Use Committee (IACUC). For the immunoh-
istochemical study of hypothalamic arcuate neurons and their terminals, three rats
were injected ICV with 120 mg colchicine in 10 lL 0.9% saline 24 h prior to cardiac
perfusion. To increase the expression of AGRP, four rats were food deprived for 48 h
and ICV injected with colchicine 24 h prior to kill to block axonal transport and
increase the quantity of peptide in the neuronal cell bodies. Rodents were anesthetized
i.p. with sodium pentobarbital and perfused via the ascending aorta with 0.9% sodium
chloride solution (37 ?C), followed by a mixture of formalin and picric acid (4% parafor-
maldehyde and 0.4% picric acid in 0.16 M phosphate buffer, pH 6.9, 37 ?C). The brains
were rapidly dissected out, immersed in the same fixative for 12 h, and subsequently
rinsed in 0.1 M phosphate buffer, pH 7.4 and stored at )20 ?C until utilized.
For double labeling of the a-MSH and AGRP proteins, floating sections were incubated
sequentially in: (i) 1% BSA in PBS for 1 h; (ii) a mixture of two primary antibodies, sheep
anti-a-MSH (diluted 1:2000; Chemicon, Temecula, CA, USA) and rabbit anti-AGRP (dilu-
ted 1:1000; Phoenix Pharmaceuticals, Belmont, CA, USA), which were diluted with 0.1%
BSA and 5% Triton in PBS for 48 h; and (iii) two secondary antibodies, Cy3-conjugated
donkey anti-sheep immunoglobulin G (IgG; diluted 1:100) and Cy2-conjugated donkey
anti-rabbit IgG (diluted 1:100, Jackson Immunoresearch Laboratories, West Grove, PA,
USA) in PBS for 24 h. The sections were rinsed in PBS for 30 min at room temperature
between each incubation step. Sections were mounted on gelatin-coated slides and cov-
erslipped. After processing, the sections were examined in a Zeiss (Germany) fluores-
cence microscope equipped by digital camera (Sony, Japan) using appropriate filters. For
confocal microscopy, samples were recorded using a Zeiss LSM410 instrument. Approxi-
mately 30 optical slices of 0.5 lm thickness were sampled and combined to produce
composite confocal images. All manipulations with contrast and illumination on color
images are made using ADOBE PHOTOSHOP 6.0 software on a Windows PC.
The primary sequences of the endogenous melanocortin agonists a-
MSH, b-MSH, c2-MSH, and ACTH(1–24) (10,11), the synthetic mel-
anocortin agonists NDP-MSH (63), MTII (19), and JRH887–9 (48),
and the endogenous MC4R antagonist hAGRP(87–132) (8) used in
this study are summarized in Table 2. Endogenous and synthetic ag-
onists as well as the hAGRP(87–132) antagonist were included in
this study to identify potential differences in ligand–receptor inter-
actions between these structurally different compounds. To deter-
mine the molecular mechanism of constitutive activity of the L250Q
Chem Biol Drug Des 2006; 67: 215–229
L250X hMC4R
Page 4
hMC4R, in vitro mutagenesis was performed by mutating residue
Leu250 (6.40) of the hMC4R to Ala, Glu, Phe, Lys, Asn, Arg, and
Gln to determine requirements at this position for constitutive acti-
vation. Amino acids for receptor mutagenesis were systematically
selected based upon differences in charge, hydrophobicity, aromatic-
ity or size of the side chains of the amino acids. Figure 1 illustrates
a two-dimensional (2D) model of the hMC4R and emphasizes the
putative receptor residues implicated in 'activation' and maintaining
the 'active' or 'inactive' receptor conformation in WT and mutant
Mutation of L250 does not affect ligand-binding
affinity of the hMC4R
Table 3 summarizes the binding affinity (IC50) values of NDP-MSH
and hAGRP(87–132) at each of the seven modified hMC4Rs pre-
pared in this study. For each radiolabeled peptide [125I-NDP-MSH
125I-AGRP(87–132)] the respective non-iodinated peptide was
used to competitively displace the labeled compound resulting in
the binding affinity IC50values. Mutation of the L250 (6.40) residue
had no significant effect on NDP-MSH or hAGRP(87–132)-binding
affinity, with the exception of hAGRP(87–132)-binding affinity at the
L250N hMC4R. Ligand-binding affinity values of NDP-MSH and
hAGRP(87–132) at the mutant receptors were essentially equipotent
to the WT hMC4R, indicating that the L250 residue is not involved
in ligand binding of either of these agonist or antagonist peptide
Agonist pharmacology observed at the wild-
type and mutant hMC4Rs
Table 4 summarizes the mean agonist EC50values (€SEM) of the
peptides examined at the WT and mutant hMC4Rs. Figure 2 illus-
trates the functional agonist pharmacology of the WT control and
the seven modified hMC4Rs generated in this study. The ligands
Table 2: Primary sequences of
agonists ACTH(1–24), a-MSH, b-MSH,
c2-MSH (endogenous), MTII, NDP-MSH,
JRH887–9 (synthetic) and the
endogenous antagonist/inverse agonist
hAGRP(87–132) used in this study
PeptidePrimary sequence
Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val -NH2
MSH, melanocyte-stimulating hormone; NDP, 4-norleucine-7-D-phenylalanine; ACTH, adrenocorticotropic hormone;
AGRP, agouti-related protein.
Figure 1: Two-dimensional model of the human Melanocortin-4 Receptor (hMC4R). Residues, depicted in black circles with white
letters are involved in maintaining the active or inactive receptor conformation in L250 wild-type and mutant L250A, L250E, L250F, L250K, L250N, L250R, and L250Q
hMC4Rs. The Flag-tag (highlighted in a square) was inserted downstream of the methionine start codon for immunocytochemical detection of the receptor.
Proneth et al.
Chem Biol Drug Des 2006; 67: 215–229
Page 5
used in this experiment (Table 2) were selected to test and differ-
entiate ligand–receptor interactions between the endogenous linear
agonist peptides a-MSH, b-MSH, c2-MSH, and ACTH(1–24), the
synthetic linear analog of a-MSH [NDP-MSH (63)], the cyclic syn-
thetic agonist MTII (19), and the synthetic tetrapeptide JRH887–9
(48). At the WT hMC4R, the endogenous agonists a-MSH, b-MSH,
and ACTH(1–24) and the synthetic tetrapeptide JRH887–9 all pos-
sessed essentially equipotent activity. The synthetic superpotent
NDP-MSH and MTII agonist ligands possessed sub-nM potencies
while the endogenous agonist c2-MSH possessed approximately
200-fold decreased potency when compared with the other endog-
enous agonists at the hMC4R. The a-MSH ligand was equipotent
at the L250N hMC4Rs, slightly reduced potency at the L250F,
L250K, L250E, and L250Q hMC4Rs, and significantly decreased
potency at the L250A (39-fold) and L250R (65-fold) hMC4Rs, when
compared with the WT control. When compared with the control
hMC4R, the b-MSH agonist ligand possessed 15- to 33-fold
decreased potency at the L250F, L250Q, and L250N hMC4Rs and
significantly decreased potency at the L250A (43-fold), L250R (165-
fold), L250K (70-fold), and L250E (41-fold). The endogenous c2-MSH
agonist essentially possessed equipotent pharmacology at all the
mutant hMC4Rs examined in this study. Relative to the WT hMC4R,
ACTH(1–24) possessed 14- to 27-fold reduced potency at the L250F,
L250E, L250Q, and L250N hMC4Rs, 55- to 58-fold decreased
potency at the L250A and L250K hMC4Rs, respectively, and 220-fold
reduced agonist potency at the L250R. The synthetic cyclic MTII
peptide resulted in nearly equipotent WT activity at the L250F,
L250E, L250Q, and L250N hMC4Rs, 14- to 18-fold decreased
potency at the L250A and L250K hMC4Rs and 39-fold decreased
potency at the L250R hMC4R. NDP-MSH possessed five- to seven-
fold reduced potency at the L250F and L250Q hMC4Rs, 21- to 33-
fold decreased potency at the L250A, L250E, and L250N hMC4Rs
and 44- to 78-fold decreased potency at the L250K and L250R
hMC4Rs, respectively. Interestingly, the tetrapeptide JRH887–9 (Ac-
His-D-Phe-Arg-Trp-NH2) possessed equipotent activity at the L250A,
L250Q, and L250N hMC4Rs, and slightly reduced potency at the
L250F, L250R, L250K, and L250E hMC4Rs. Overall, the structurally
distinct endogenous and synthetic melanocortin agonist ligands
Table 4: Functional activity of melanocortin ligands at the wild-type and mutant hMC4 receptors
Potency EC50(nM)
0.88 € 0.11
0.24 € 0.07
170 € 4.80
0.86 € 0.04
0.017 € 0.007
0.022 € 0.006
0.72 € 0.12
12.0 € 2.58*
6.66 € 1.88*
725 € 88*
16.6 € 2.74*
0.051 € 0.003*
0.15 € 0.003*
11.1 € 3.57*
34.5 € 12.7*
10.3 € 3.44*
970 € 207*
46.9 € 12.0*
0.23 € 0.06
0.52 € 0.16*
6.25 € 3.26
57.6 € 16.3*
39.5 € 5.32*
1480 € 370
190 € 18*
0.67 € 0.007*
1.72 € 0.30*
10.3 € 0.95*
11.9 € 6.94
16.7 € 2.90*
530 € 20*
49.9 € 24.3
0.30 € 0.05*
0.96 € 0.20*
6.93 € 0.98*
11.8 € 3.17*
9.84 € 0.99*
913 € 560
23.5 € 11.9
0.14 € 0.035
0.73 € 0.09*
3.76 € 0.98*
4.32 € 1.10*
3.55 € 1.67*
442 € 24*
3.63 € 0.82
0.034 € 0.020
0.10 € 0.02*
1.09 € 0.29
1.55 € 1.92
8.00 € 0.91*
193 € 63
13.2 € 3.48*
0.054 € 0.052
0.46 € 0.16*
0.92 € 0.31
EC50values (nM; €SEM) are derived from at least three independent experiments. The fold difference is calculated relative to the wild-type hMC4R.
*p < 0.05 when compared with the wild-type hMC4R.
MSH, melanocyte-stimulating hormone; hMC4, human Melanocortin-4 Receptor; NDP, 4-norleucine-7-D-phenylalanine; ACTH, adrenocorticotropic hormone.
Table 3: Competitive binding affinity IC50 (nM) results at the wild-type
and mutant hMC4Rs. Non-iodinated NDP-MSH and hAGRP(87–132) were
used to competitively displace the corresponding125I-radiolabeled peptide
Binding IC50(nM)
5.20 € 0.59
5.79 € 2.14
4.85 € 0.87
5.80 € 0.52
4.49 € 0.87
7.13 € 8.30
4.17 € 0.83
5.38 € 0.34
24.7 € 12.6
4.56 € 0.75
5.68 € 0.96
34.0 € 32.0
14.3 € 6.91
14.0 € 12.4
5.98 € 0.53
42.8 € 15.5*
The indicated errors represent the standard deviation of the mean from at
least two independent experiments performed in duplicate.
*p < 0.05 when compared with the wild-type hMC4R.
MSH, melanocyte-stimulating hormone; hMC4R, human Melanocortin-4
Receptor; NDP, 4-norleucine-7-D-phenylalanine; AGRP, agouti-related protein.
L250X hMC4R
Chem Biol Drug Des 2006; 67: 215–229
Page 6
examined in this study possess distinct pharmacologic profiles at
the WT and mutant hMC4Rs.
L250X hMC4R constitutive activation
It has been previously demonstrated that the L250Q hMC4R poly-
morphism results in a constitutively active receptor that possesses
increased cellular basal activity in the absence of agonist ligand
(17,35,40). Figure 3 illustrates the basal activities observed for the
stably expressed hMC4Rs examined in this study. Relative to the
WT hMC4R, the L250F hMC4R possesses reduced basal activity
(p < 0.05), the L250A hMC4R has equipotent basal activity, and the
L250R, L250K, L250E, and L250N possess increased basal activity
(p < 0.01). Transient transfection assays have been used to
Figure 2: Agonist pharmacology of melanocortin ligands at the wild-type and mutant human Melanocortin-4
Receptor (hMC4Rs) stably expressed in human embryonic kidney-293 (HEK-293) cells.
Proneth et al.
Chem Biol Drug Des 2006; 67: 215–229
Page 7
ascertain if a receptor is a candidate as a constitutively active
receptor versus possessing an artifact related to cell surface
expression levels (49,53,64). Figure 4 summarizes the transient
transfection of the hMC4Rs examined in this study and supports
the hypothesis that the L250E, L250Q, and L250N hMC4Rs are con-
stitutively active GPCRs. The L250K hMC4R possesses increased
basal activity with increasing DNA concentrations when compared
with the WT, L250F, L250A, and L250R hMC4Rs and thus may also
be considered as constitutively active.
Cell surface expression of wild-type and mutant
hMC4 receptors
A decrease in relative cell surface expression of the receptor using
FACS has previously been proposed as a mechanism of impaired
receptor function of the constitutively active mutant L250Q hMC4R
(35,40). Therefore, we determined the cell surface expression of the
WT and mutant hMC4Rs using the N-terminal Flag-hMC4Rs and
FACS analysis quantification. Figure 5 summarizes the percentage
of cell surface expression of the WT and mutant hMC4Rs stably
expressed in HEK-293, relative to the WT hMC4R. Cell surface and
total receptor expression of the WT hMC4R were defined as 100%.
In Table 5, the receptor cell surface expression of WT and mutant
hMC4Rs are compared with the functional activity of a-MSH at
those receptors and their status as constitutively active receptors.
The mutant hMC4Rs L250A, L250F, and L250R possessed receptor
cell surface expression levels of 57%, 46%, and 52%, relative to
the WT hMC4R. The L250K mutant hMC4R which shows intermedi-
ate and constitutive activation and possessed cell surface expres-
sion levels of 58% relative to the WT hMC4R. The constitutively
active L250E, L250N, and L250Q mutant hMC4Rs (Figure 3) pos-
sessed cell surface expression levels of 66%, 54% and 72%,
respectively, relative to the WT hMC4R (Figure 5, Table 5). Thus,
there does not appear to be a correlation between the endogenous
agonist a-MSH potency and receptor cell surface expression levels.
hAGRP(87–132) antagonist and inverse agonist
pharmacology at the wild-type and mutant
Table 6 summarizes the pA2values (€SD) of hAGRP(87–132) at the
WT and mutant hMC4Rs. Figure 6 illustrates the inverse agonist
and antagonist curves of the hAGRP(87–132) ligand at the hMC4Rs
examined in this study. The pharmacologic profile of the hAGRP
(87–132) ligand at the WT hMC4R presented herein is consistent
with previously published results that this ligand possesses inverse
agonist activity at both mouse and human MC4Rs (26,27). Varying
degrees of hAGRP(87–132) inverse agonist activity is observed at
the L250K, L250E, L250Q, and L250N hMC4Rs, with the most pro-
nounced effect observed at the L250N hMC4R. These data support
the hypothesis that these latter hMC4Rs are constitutively active
receptors (Figures 3 and 4). Interestingly, the hAGRP(87–132) antag-
onist is equipotent at all the receptors examined in this study
within independent experiments and the error, with the exception
Figure 3: Basal levels of the wild-type and mutant
human Melanocortin-4Receptor
expressed in human embryonic kidney-293 (HEK-293)
cells. Statistical analysis was performed using a Student's t-test where
*p < 0.05, **p < 0.01, and ***p < 0.001.
(hMC4R) stably
Figure 4: Transient transfection bioassay: increasing amounts of wild-type and mutant human Melanocortin-4 Receptor (hMC4R) DNA were
transiently transfected into human embryonic kidney-293 (HEK-293) cells as described under Materials and Methods. The L250E, L250Q, and L250N mutants
result in increased basal activity with increasing amount of DNA and are therefore indicative of possessing constitutive activity in the absence of an agonist lig-
and. The wild-type hMC4R, the L250R, L250A, and L250F hMC4R mutants do not result in significantly increased basal activity and either lack or show very
low levels of constitutive activation. The mutant L250K hMC4R shows moderate constitutive activity.
L250X hMC4R
Chem Biol Drug Des 2006; 67: 215–229
Page 8
of the L250F (p < 0.05). However, at the L250N hMC4R it possess a
ninefold enhanced potency based on comparison of Ki values,
where Ki¼ )log pA2(52) (Table 6).
Dual label rat hypothalamic
The endogenous melanocortin agonist a-MSH and antagonist AGRP
have been reported to be expressed at the mRNA level in the arcu-
ate nucleus (ARC) of the hypothalamus in rodents and primates
(65,66). Additionally, both the a-MSH and AGRP proteins are
expressed in distinct neuronal populations within the ARC and pro-
ject to brain nuclei that express melanocortin receptor (MC3R and
MC4R) mRNA. Because AGRP has been identified to function as an
inverse agonist of the MC4R using in vitro heterologous cell lines,
we utilized a fasted rat model in attempts to identify any physiologic
data that might support the hypothesis that AGRP might function as
an inverse agonist in the hypothalamus as well. Confocal microsco-
py of dual-labeled immunohistochemical hypothalamic coronal sec-
tions of fasted and colchicines-treated rats demonstrates the
expression of a-MSH and AGRP in different neuronal populations of
the ARC (Figure 7A), consistent with previous results observed for
singly labeled samples (65,66). Furthermore, in agreement with pre-
vious studies (65,66), we observed a-MSH and AGRP high-density
networks of labeled neuronal fibers in various hypothalamic nuclei
of the brain and within the macrostructure of the paraventricular
nucleus (PVN; Figure 7). Interestingly, as illustrated in Figure 7B,
distinct subnuclei neuronal populations of the PVN appear to
express only hAGRP (green) or a-MSH (red). These data support the
hypothesis that hAGRP may possess additional physiologic roles
such as inverse agonism, in addition to its well recognized role as
an MC3R and MC4R competitive antagonist of the endogenous
a-MSH ligand. Furthermore, within the ARC it appears that AGRP-
expressing fibers (green) interact with the POMC-expressing neu-
rons (red; Figure 7C,D), to function in an autoregulatory feedback
mechanism, whereas the AGRP-expressing neurons are devoid of
POMC-expressing fiber contacts (Figure 7E). These data are
consistent with previous reports demonstrating neuropeptide Y
(NPY)-expressing neurons that co-express AGRP synapse on POMC-
expression neurons (67). These experiments provide physiologic data
Figure 5: Comparison of the wild-type and mutant human Melanocortin-4 Receptor (hMC4R) cell surface and
total receptor expression levels using fluorescent-activated cell sorting (FACS). Cell surface and total receptor expression of
the wild-type hMC4R were defined as 100% for comparative purposes. The values listed indicate the average of the mean from three independent experiments
and the error bars represent the standard error of the mean. Statistical analysis was performed using a Student's t-test with statistical significance indicated
as *p < 0.05 and **p < 0.01.
Table 5: Comparison of receptor cell surface expression with the func-
tional activity of the endogenous ligand a-MSH and the level of constitutive
activation of mutant and wild type hMC4 receptors
Receptor cell
expression (%)
a-MSH potency
(EC50in nM)
46 € 9.6*
57 € 9.6*
52 € 6.1*
58 € 6.8*
66 € 11.6*
72 € 5.8*
54 € 10.9*
0.88 € 0.11
12.0 € 2.58*
34.5 € 12.7*
57.6 € 16.3*
11.9 € 6.94
11.8 € 3.17*
4.32 € 1.10*
1.55 € 1.92
1 No
Receptor cell surface expression of the wild-type hMC4R was set to 100%.
EC50values (nM; €SEM) are derived from at least three experiments per-
formed in duplicate. The fold difference in EC50values is calculated relative
to the wild-type hMC4R.
*p < 0.05 when compared with the wild-type hMC4R.
MSH, melanocyte-stimulating hormone; hMC4, human Melanocortin-4 Recep-
Table 6: Functional activity of the antagonist hAGRP(87–132) at the wild-
type and mutant hMC4Rs
Antagonist pA2value
8.20 € 0.007
8.64 € 0.04*
8.12 € 0.22
7.64 € 0.35
8.24 € 0.01
8.27 € 0.50
8.53 € 0.64
9.13 € 0.97
pA2values (€SD) are derived from at least two independent experiments
performed in duplicate.
*p < 0.05 when compared with the wild-type human Melanocortin-4 Recep-
tor (hMC4R).
hMC4R, human Melanocortin-4 Receptor; AGRP, agouti-related protein.
Proneth et al.
Chem Biol Drug Des 2006; 67: 215–229
Page 9
supporting the hypothesis that the endogenous AGRP ligand
appears to possess additional physiologic functions than simply act-
ing as a competitive antagonist of a-MSH at the centrally
expressed MCRs. These additional physiologic functions may include
autoregulation of POMC-expressing neuronal homeostasis and per-
haps as an inverse agonist in subnuclei that are not innervated by
the a-MSH agonist.
Herein, we investigated not only the mechanism by which a natur-
ally occurring L250Q mutant hMC4R (found in a severely obese
woman) might contribute to the obesity development in this patient,
but also the molecular mechanism and requirements responsible for
constitutive receptor activation of the hMC4R. The melanocortin
system, in particular the MC4R, plays a major physiologic role in
obesity, satiety, and energy homeostasis in both humans and
rodents (12–16,28,29). Since the first discovery of naturally occur-
ring mutations in the hMC4R in severely obese patients in 1998
(28,29), a large number of polymorphisms in the MC4R have been
discovered in morbidly obese children and adults (17,18,28–47).
However, the molecular mechanisms by which these mutations alter
receptor function and subsequently may cause obesity are currently
being unraveled by several research groups (17,18,35,42,47). Mul-
tiple studies have been undertaken to characterize these mutant re-
ceptors functionally in vitro using heterologous cell lines transiently
or stably expressing polymorphic hMC4Rs. Current hypotheses for
hMC4R polymorphic receptor malfunction associated with an obese
human phenotype include modified potency of the endogenous mel-
anocortin agonists, truncated or structurally non-functional recep-
tors, and decreased cell surface expression and intracellular
Figure 6: Antagonist and inverse agonist pharmacology of hAGRP(87–132) at the wild-type and mutant human
Melanocortin-4 Receptor (hMC4Rs) stably expressed in human embryonic kidney-293 (HEK-293) cells. The correspond-
ing pA2values were generated using the Schild analysis method.
L250X hMC4R
Chem Biol Drug Des 2006; 67: 215–229
Page 10
Figure 7: Confocal images of double-labeled anti-a-
melanocyte-stimulating hormone (MSH; red) and anti-
agouti-related protein (AGRP; green) antibody sections
through the paraventricular nucleus (PVN) and arcuate
nucleus (ARC) of the hypothalamus in the brain of
food-deprived and colchicine-treated rats (A, C–E). (A)
Confocal image of the ARC adjacent to the third ventricle (3V) demonstrating
a-MSH (red) and AGRP (green) protein expressions in distinct neuronal popula-
tions. (B) Confocal image through the PVN with a schematic diagram showing
PVN neuronal subdivisions. LM and MM, lateral and medial magnocellular
regions of PVN; MM and Pe, medial and periventricular parvocellular region of
the PVN; DC, dorsomedial cap of PVN; V, ventral region of PVN. (D) Three-
dimensional reconstruction of (C) sections indicating a large number of AGRP-
immunoreactive fibers (green) interacting with a-MSH-immunoreactive (red)
neurons. Scale bars: 100 lm (A and B) and 25 lm (E).
Proneth et al.
Chem Biol Drug Des 2006; 67: 215–229
Page 11
retention in the endoplasmic reticulum (17,18,28–47). However,
none of the above proposed mechanisms singly could explain the
mechanism by which the constitutively active L250Q mutant MC4R
may be involved in obesity development in humans. Binding affinit-
ies of the melanocortin agonist NDP-MSH and the antagonist/
inverse agonist hAGRP(87–132) ligands to the L250Q polymorphic
receptor hMC4R are equipotent to the WT hMC4R (Table 3), which
indicates that this L250 (6.40) residue is not important for ligand
binding or the molecular recognition aspects of ligand–receptor
interactions. A decrease in relative cell surface expression using
FACS has been proposed as a mechanism of impaired receptor
function of the mutant L250Q hMC4R (35,40), but this alone
(Figure 5,Table 5) in the context of a heterozygote genotype of a
morbidly obese female patient with a body mass index (BMI) of 59
(17), does not correlated well with a constitutively active hMC4R,
which based upon current research would be hypothesized to result
in a lean phenotype. This rationale led us to the hypothesis that
AGRP may act as inverse agonist at this constitutively active poly-
morphic L250Q hMC4R, thus decreasing the high basal cAMP lev-
els. Herein, we have demonstrated that hAGRP(87–132) is able to
dose-dependently suppress basal activity of the L250Q hMC4R to
levels similar to the basal activity of the WT hMC4R (Figure 6). The
identification of AGRP as inverse agonist (25–27) provided evidence
that AGRP may play an alternative role in energy homeostasis, in
addition to competitive antagonism of a-MSH (26,27). Furthermore,
AGRP could have a physiologic function in and of itself, thus acting
both independently and in concert with the POMC-derived melano-
cortin agonist a-MSH (26,27). The endogenous AGRP ligand is
expressed in neurons of the ARC (Figure 7) in the hypothalamus of
the brain and is implicated in the regulation of energy homeostasis
(8,21). These AGRP-containing neurons are near the POMC-contain-
ing ARC neurons that produce the MCR agonist a-MSH (68–72). It
has been shown that during chronic food restriction, mRNA levels
encoding the orexigenic peptide AGRP are increased (8,21) and
mRNA levels encoding the prohormone POMC of the anorexigenic
peptide a-MSH are decreased in the hypothalamus (73,74). It has
also been suggested that regulation of MC4R signaling in the hypo-
thalamus could be mediated primarily by changes in AGRP expres-
sion rather than POMC, the precursor of a-MSH (69). Based upon
these data, we hypothesize that increased basal cAMP levels, as
observed at the constitutively active L250Q hMC4R mutant receptor
could mimic chronic food restriction and subsequently trigger
increased AGRP release from nearby situated AGRP neurons
(Figure 7). Thus, AGRP could decrease the basal activity of the
receptor by acting as inverse agonist and modify the POMC-derived
agonist homeostasis in adjoining ARC neurons (Figure 7), resulting
in a counterregulatory action of increased appetite and hyperphagia
which in turn may lead to the development of obesity in this
patient. Additionally, it has been demonstrated that AGRP can
cause a long-term orexigenic effect, which completely blocks the
anorectic effect of the synthetic MTII agonist (23). This observation
would support the hyperphagia and obesity observed in the L250Q
patient. However, the extent and the physiologic mechanism by
which AGRP and the constitutively active L250Q mutant hMC4R
contribute to the obesity development in the 'L250Q phenotype'
remain to be experimentally verified using in vivo physiologic stud-
ies. An additional physiologic mechanism may be that the constitu-
tive activation of L250Q is accompanied by the constitutive
desensitization of the L250Q hMC4R and its sequestration into the
internal vesicles. This would decrease the number of functional re-
ceptors on the neuronal membrane. Similar effects have been previ-
ously observed for a number of constitutively active mutants of
GPCRs, such as mutant b-adrenoreceptors (75) and rhodopsin
mutants causing congenital night blindness (76). Again, further stud-
ies need to be performed to examine this possible alternative
Hypothesized molecular mechanism of the
constitutively active L250Q hMC4R
To understand the putative molecular mechanisms by which the
L250Q hMC4R human polymorphism results in a constitutively active
GPCR, we systematically mutated the L250 residue to Ala, Glu, Phe,
Lys, Asn, Arg, and Gln in order to determine the requirements at
this position that are necessary for hMC4R constitutive activation.
Amino acids for receptor mutagenesis were selected due to differ-
ences in hydrophobicity, charge, size, length of the side chain, and
aromaticity to test our hypothesis that this L250Q hMC4R modifies
the different 'active' and 'inactive' receptor populations of the GPCR
ternary complex model, specifically via intramolecular interactions
with TM3 and TM7 residues. We observed that L250N, L250Q, and
L250E hMC4Rs possessed significantly increased basal activity
(p < 0.001) whencompared
L250N > L250Q > L250E (Figure 3). Based upon the transient trans-
fection experiment (Figure 4), the L250K hMC4R possessed moder-
ate constitutive activation and the L250F, L250A, and L250R
hMC4Rs were not constitutively active. Competitive antagonists can
act as inverse agonists at constitutively active GPCRs (77,78) and
indeed, we have observed inverse agonist activity of the hAGRP(87–
132) at the constitutively active L250E, L250Q, and L250N hMC4Rs
(Figure 6). At the non-constitutively active L250F, L250A, and L250R
mutant hMC4Rs, hAGRP(87–132) possessed normal competitive ant-
agonist activity with little observable inverse agonist activity.
with theWT control with
The hMC4R mutagenesis results observed in the study presented
herein, might be explained using theoretical three-dimensional (3D)
homology molecular models of an active (agonist-bound) and an
inactive (antagonist-bound) conformation of the hMC4R. The models
used herein have been developed based on a number of experimen-
tal constraints collected for different GPCRs and MC4R ligand and
receptor mutagenesis studies (25,57). Numerous experimental
studies provide evidence for the conformational changes during
activation of the receptors, such as side chain rearrangement lead-
ing to the disruption of inactivating intermolecular constraints, rota-
tion, and shift of TM6 and some adjustment of all other helices
(79–81). The major difference between models of the 'active' and
'inactive' hMC4R state is the putative large movement of the TMs
creating a water-filled cavity between the intracellular segments of
TMs 6, 3, and 7, and the rearrangement of hydrogen bond networks
between the TMs (79,81,82). For example, the hydrogen bond net-
work between residues D146 (3.49), R147 (3.50), and N240 (6.30) in
the inactive state model of the hMC4R is replaced by a hydrogen
bond system between residues R147 (3.50), Y212 (5.58), and Y302
(7.53) in the model of the active state (Figure 8) (25,57). In fact, the
essential role of the side chain interactions of Asp/Glu (3.49), Arg
(3.50), Tyr (5.58), and Tyr (7.53) during GPCR activation have been
experimentally supported (79,81,83,84).
L250X hMC4R
Chem Biol Drug Des 2006; 67: 215–229
Page 12
Our theoretical modeling studies reveal a putative significant
change in the environment of the L250 residue during activation of
hMC4R. In the model of the 'inactive' receptor state L250 (6.40)
occupies a hydrophobic site between TMs 3, 6, and 7 formed by
residues L140 (3.43), I143 (3.46), L247 (6.37), F254 (6.44), I301
(7.52), Y302 (7.53). In the 'active' state model the space between
TM3 and TM6 is enlarged and the L250 side chain faces the
water-filled cavity, while retaining weakened hydrophobic contacts
with TM7. The substitution of L250 by the bulky aromatic side
chain of Phe may increase the hydrophobic interactions between
TMs 3, 6, and 7 and therefore may be advantageous for keeping
the receptor in the 'inactive' conformation (Figure 8A). In contrast,
the L250 substitution by polar side chains of Asn, Gln, and Glu (but
not Lys and Arg) would not only destabilize the hydrophobic interac-
tions suitable for the 'inactive' state, but also may be involved in
the formation of interhelical hydrogen bonds with R147 (3.49) and
Y212 (5.58) appropriate for the 'active' receptor state (Figure 8B).
Such hydrogen bond interactions that stabilize the 'active' receptor
state would explain the increased constitutive activity of the
L250N, L250Q, and L250E mutants of hMC4R. A similar mechanism
may account for the observed constitutive activity of M257N,
M257Y, and M257S mutants of rhodopsin (86). Therefore, L250N,
L250Q, and L250E hMC4R mutations represent the rare case of con-
stitutively active mutants that are caused by the stabilization of the
'active' conformation rather than by destabilization of the 'inactive'
receptor state. Another example of a mutation that may lock the
receptor in the active conformation has been recently observed for
the L457R mutant of lutropin receptor (85) in which the active con-
formation is stabilized by formation of a salt bridge between resi-
dues R457 in TM3 (3.43) and D578 in TM6 (6.44).
Figure 8: Comparison of three-dimensional homology human Melanocortin-4 Receptor (hMC4R) models of the
wild-type hMC4R (A, gray) and L250Q hMC4R (B, yellow) mutants in the putative 'inactive' and 'active' receptor
conformations, respectively. Homology modeling of the hMC4R in the 'inactive' state in complex with agouti-related protein (AGRP) and in the 'act-
ive' state in complex with 4-norleucine-7-D-phenylalanine melanocyte-stimulating hormone (NDP-MSH) was based on the crystal structure of rhodopsin and was
carried out as described under Materials and Methods. The fragments of transmembrane (TMs) 3, 5, 6, and 7 are shown as ribbon, the mutated L250 residue
(purple) and several residues surrounding the mutated residue and forming hydrogen-bonding interactions are shown by licorice. Schematic representations of
four TM helices with mutated and some interacting residues are shown in the bottom part of the picture.
Proneth et al.
Chem Biol Drug Des 2006; 67: 215–229
Page 13
Changes in the potency of different agonist ligands at the different
L250X hMC4R mutants may be related to the continuum of receptor
substates that has been hypothesized to be formed in the presence
of different ligands (86–88). Some of the activated receptor sub-
states are appropriate for the interaction with specific G-proteins,
kinases, b-arrestins or other proteins interacting with activated
receptors (89). The results of this study demonstrating increased
basal activity of L250N, L250Q, and L250E (6.40) mutants of the
hMC4R, in addition to previous reports of constitutive activity of
corresponding M257N and M257Y (6.40) mutants in rhodopsin (90)
indicate that residue 6.40 may have an important structural role for
GPCR activation, regulating the position of TM6 relative to TM3
and TM7. The different lengths of the Glu and Asn side chains and
the putative different strength of Glu and Gln interactions with
R147 (3.50) may favor formation of several active substates charac-
terized by dissimilar positions of TM6 relative to the rest of the
receptor (59). Indeed, the L250N, L250Q, and L250E hMC4R mutants
demonstrated different levels of increased basal activity (Figures 3
and 4) and of agonist potency changes (Table 4). The L250N
mutant, with the highest constitutive activity, probably has a side
chain length more suitable for the formation of a hydrogen bond
with R147 (3.50) and for the placement of TM6 that provides most
effective activation of G-protein. The L250N, L250Q, and L250E
mutants may also be constitutively phosphorylated at different lev-
els and to a certain extent may lead to long-term desensitization
and downregulation of the receptor number at the cell surface.
However, this latter speculation remains to be experimentally veri-
fied. The presence of the polar, basic side chains of K250 or R250
putatively decrease the hydrophobic interactions placing TMs 3, 6,
and 7 in the 'inactive' receptor, but do not provide the hydrogen
bonding interactions necessary for stabilizing the 'active' state.
Therefore, the basal activity of the corresponding L250K and L250R
mutants were relatively low. Moreover, the R250 side chain may
form hydrogen bonds with S136 (3.39), D298 (7.49), and Y302
(7.53), appropriate for the 'inactive-like' rather then for the 'active-
like' receptor conformation. This could explain the minimal level of
constitutive activation of the L250R mutant and the decreased
agonist potency in this case (Table 4). The small side chain of the
L250A mutant probably destabilizes both 'active' and 'inactive' states,
thus causing decreased potency of agonists. The bulky L250F side
chain can reinforce TM6–7 interactions and impede separation of
these helices upon receptor activation, which would decrease the
amount of activated receptors. This would explain the reduced
potency of agonists at the mutant L250F hMC4R. On the other hand,
the smaller hydrophobic side chain of L250 in the WT hMC4R may
have smaller stabilization effect on the inactive conformation, leading
to a higher population of activated receptors upon agonist binding.
These mechanistic speculations remain to be experimentally verified;
however, based upon the data generated in this study, these specula-
tions provide specific molecular hypotheses to be further examined.
Conclusions and Future Directions
In conclusion, by performing receptor mutagenesis and creating
seven hMC4R mutations at position 250 (6.40), we have identified
requirements for receptor stimulation of the hMC4R and residues
putatively involved in hMC4R activation. We propose that the
hMC4R residue L250 is playing a key role in switching the
hMC4R from the 'active' to the 'inactive' receptor conformation.
These results can be used to gain further insight into the receptor
activation mechanism of GPCRs and can be applied to other GPCR
systems in order to stabilize the active receptor state for further
crystallographic studies. We have generated experimental evidence
that AGRP functions as an in vitro inverse agonist at the constitu-
tively active L250Q hMC4R, and physiologic data supporting the
hypothesis for additional functional roles for AGRP besides a com-
petitive antagonist in the brain. These data taken together support
the hypothesis that in addition to reduced cell surface expression,
inverse agonist activity of the hAGRP antagonist may provide the
molecular basis to explain the unanticipated phenotype of the
obese phenotype associated with the constitutively active L250Q
The authors are grateful to Dr Andrei Lomize for helpful discussions. This study has
been supported by NIH grants RO1DK063974, RO1DK57080, RO1DK064250, and an
American Diabetes Association Research Award (CHL). Grant DA03910 from the
National Institute on Drug Abuse (H.I.M) and the University of Michigan Upjohn
Research Award G003878 (I.P.).
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text:
Latest update stated that the roll out of Android 4.4.3 Kitkat OS update for the Nexus 5 handset will be delayed.
Last week, US carrier Sprint announced that it will roll out the Android 4.4.3 Kitkat OS update to its Nexus 5 handsets on Apr. 21. Specifically, the KTU84F, also known as Android Kitkat 4.4.3 was expected to roll out gradually from Apr. 14 to 21 starting with the Nexus 5. However, latest update claimed that will not be the case.
A note was posted on the carriers' website on the aforementioned date that reads, "Important Note: Due to an issue with the software, this update has been put on hold. Will update this post as soon as the rollout resumes. Google Nexus 5 Software Update - KTU84F".
Furthermore, a customer service representative then posted on the Nexus 5 forum saying, "An issue was found with software and hence the reason for pulling out this update. My understanding is a new software update will come out very soon. I will update this space with that information as soon as I can. I apologize for the delay."
Sprint has not disclosed the details on the software issue that was found. However, it was assumed Google encountered some problems along the way and needed some time to fix them.
Google has not revealed the official change log yet of the Android 4.4.3 Kitkat OS update. However, it is assumed that the update will fix more than three dozen bugs that was brought along by the 4.4.2 version of the Kitkat OS.
Moreover, the update was claimed to address Bluetooth and Wi-Fi connectivity issuses, random reboots, USB debugging and VPN problems, among others. The latest update will bring bug fixes mostly and will not bring any new features to the devices.
The Android 4.4.3 Kitkat will be the last update before Google introduce the next version of Android OS in June at the I/O Conference dubbed as Android 4.5 Lollipop.

subset: commoncrawl


text:
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Walt Disney Studios released a sneak peek of the new "Frozen" short that will air before Disney's live-action "Cinderella," which hits theaters March 13.
Tip of the WeekWhen the acceptance letter arrives from a college or university, it�s cause for celebration. But it�s also time to do some serious number crunching and take steps to mitigate potential education-related debt.
Five ways you can have fun this weekend:
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Dear Dave,
A Taiwanese flight with 58 people aboard clipped a bridge shortly after takeoff and careened into a shallow river Wednesday in the island's capital of Taipei, killing at least eight people, state media said.
The Immaculata Raiders are familiar with players getting in the zone.Nearly a week after senior Connor Goetz zoned in to the tune of 39 points, the Raiders experienced the feeling from the other side of the court.Needless to...
Come morn, take a much-needed break from your usual ho-hum cereal or yogurt and try one of these seven unique avocado breakfast ideas.
[Warning: The following contains spoiler's from Tuesday's episode of Forever.Read at your own risk!]Adam returned to
Professor Anthony E. Dixon, Ph.D., will be the guest speaker at the Leavenworth Community Service Organization's Annual Black History Month Celebration at 7 p.m. Saturday at the Riverfront Community Center, 123 S. Esplanade
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KPC students are back in classes today and enrollment across the KPC system remains stable. At this year's spring convocation, held on Jan. 5, KPC Director Gary Turner reported to staff and faculty that the college is fortunate to see spring semester's current enrollment essentially flat compared to the same period last year.
As has been the trend for the past year, many institutions around the state are seeing decreasing enrollments.
With unusual weather this winter including freezing rain and hardly any snow, some elementary schools around the Kenai Peninsula have had to occasionally adjust their normal recess routines to ensure safety and, at the same time, keep kids active.
Karl Kircher, principal of Mountain View Elementary, said he doesn't recall such an unusual winter. He said that when the playground area gets slick due to freezing rain, the school is forced to have indoor recess.
"Our number one job is safety," Kircher said.
The Kenai Peninsula Borough School District Board of Education meets at 7 p.m. in the borough building at 148 N. Binkley Street in Soldotna (unless otherwise noted). For more information, call 907-714-8888 or visit kpbsd.k12.ak.us. The agenda and packet items are posted on Wednesday afternoon prior to the date of the Board meeting. Persons with disabilities who need accommodations to participate at the School Board meetings should contact Debbie Tressler at 907-714-8836 or email firstname.lastname@example.com no later than three business days before the meeting date. The board will meet:
School board to meet
KPC has always striven to keep pace with technology. In recent years, the advent of electronic devices such as cell phones and tablets has necessitated a new, responsive design website to provide optimal viewing experiences for all devices.
Whereas the previous static site translated poorly to small screens, requiring resizing, panning and scrolling, the new site adapts the layout to the viewing environment by using fluid, proportion-based grids that allows page features to resize in order to fit the screen.
Soldotna High School students have had greater access to a variety of academic and extracurricular services since the start of the 2014-2015 school year.
The increase in scholastic programs is just one result of the reconfiguration of three central peninsula schools, said Principal Todd Syverson. The high school received more staffing and supply dollars when the number of students increased at the beginning of the year.
KPC's enrollment for next semester is tracking about the same as last spring. Classes that meet general education requirements, program prerequisites and requirements, as well as popular electives are generally the first to fill. Classes that are offered online are very popular as Alaskans juggle jobs, family and college.
Search for courses from the KPC website and then register at UAOnline at https://uaonline.alaska.edu/.
The first day of classes will be Jan. 12 at all KPC locations. For more information, contact Student Services at 262-0330.
In the past week, I discovered that I enjoy looking forward to Thanksgiving more than the holiday itself.
Before, I was thinking about how much pie I was going to eat.
After, I was regretting eating so much yummy pie.
Looking forward to Thanksgiving during that week was what got me out of bed with a smile. It's what kept me on track and productive. Looking forward to something shaped my entire week.
Anticipating a fun event can keep anyone happy.
It could be a big thing, like a vacation, or a small thing, like what's for lunch.
KPC's spring 2015 television commercial has been released!
The commercial was written, shot and produced totally in-house by KPC Advancement, with the voice-over provided by Rob Lewis, KPC's IT HelpDesk Supervisor.
Check out the commercial on NBC, ABC, FOX, KACN, CW and online on KPC's YouTube channel (https://www.youtube.com/user/kpcuaa).
Early bird high school advising, registration set
Peninsula Clarion ©2015. All Rights Reserved.


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The Spring Paris CPH sale will be held Friday, April 22 at the Paris Stockyards. The sale will carry the requirements of all CPH sales including age verification of all animals.
With extremely high beef prices most CPH sales have realized extra benefits which are rewarded to those who have done the requirements of the program. These requirements include a vaccination program, de-worming and adequate mineral program and livestock knowing how to eat and drink from feeders and waterers.
For additional details on the sale stop by the Extension Office or call 234-5510. If you plan on selling in a CPH Sale, call as soon as possible. Your cattle must be PVP Certified to participate in the sale.
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You can now invite anyone to meet at your favorite Starbucks.
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61 year old woman | Fort Lauderdale, FL, USA | Seeking men 61-69 within 300 miles
Active within 24 hours
Seeking college educated, successful gentleman for LTR. Prefer the all-American guy! Please no winks, likes, etc. Just a short note.
Greetings! ..this profile is somewhat tongue in cheek, and was written to amuse myself at the time, don't take it too seriously. Seeking a peer, ( in Ft. Lauderdale or Cape Cod, Colorado or?) I am not naive enough to think that my only match is in Ft. Lauderdale. I am open to a long distance relationship. Prefer a conservative man on the outside and although successful, a midwest type of personality. It is important that we be age and income compatible! Please look like your main photo! I am accomplished, and highly educated, but very down to earth, being raised on ten acres! Unlike many internet females....I bring to the table everything I request in a man...(well...almost everything)....I am first and foremost a fun person,an optimist, and expect the same, never needy or clingy, and absolutely no drama!...I have free time and am not tied to my business. I love to travel, like the theatre, movies, exercise,and men who are easy-going and make me laugh! I belong to a gym, a beach club, love finding new restaurants, seaching out antiques at old shops and garage sales, snowshoeing up Bellyache Ridge in Vail, hiking Bryce Canyon in Utah, Brewster in Cape Cod in the summer. Not much old baggage.....certainly no ex mates lurking under the hedges, (the old husband still likes me!!)..Please have no minor children that live with you full time, or other sundry relatives!...... I am natural blonde, and the photos look just like me!!!!.......(An internet first)...... PLEASE READ WHAT I AM SEEKING....if you feel we are on the same page, I would welcome a note of introduction from you.......I will respond to profiles without a photo, but I shall wish to see a photo at a later date.......(preferably one of you!)...and taken within the past year. Not seeking Adonis....personality much more important to me.....but must be in decent shape! Must be open-minded and willing to experiment ..prefer a college education with a cute imagination! (but be the epitome of propriety in public!)...Sense of humor is paramount!... .Must be financially and emotionally secure. I love children, but prefer yours be grown.....no heavy drinkers and no smokers! Please, no foreign men! (or foreign accents, this includes N.Y. and N.J. (smile) (Foreign cars, O.K.!) Must have a love of travel, love to laugh, have the respect of your peers. !Please be OVER your old loves before contacting me! Now...some of these rigid requirements are not written in stone, but you should have a good idea if you are a match or not! (Hint..If you are driving a subcompact with roll-up windows, and it's smoking,..you might assume we are not a match!, If you are in your 30's and the Corvette is more attractive to you than I am, you might assume we are not a match, if your job requires you to wear an orange neon vest, you may assume we are not a match, likewise if your favorite t-shirt says "worlds best grampa". I seek a man who reads the verbage and smiles!, a man who has a great sense of humor,.....this is, afterall, my ad! PS....Absolutely NO MARRIED MEN, unless you are currently in a divorce or have been legally separated for some time, and please, no men with minor chidren living with them full time , ( I have already done my time selling hot dogs at the high school games to raise money for the team! I am not the least bit pretentious....I don't intend to waste your time or mine, if we are not on the same flight. Lastly, my photos are exactly what I look like.. The only man in the photos is my brother, and he won't be any competition! Please don't be intimidated by this quirky ad, if you are a really nice guy, have a great sense of humor, are age and income compatible, we could click!! BTW, the men I have met have said, "gee, your much sweeter in real life than your profile!". I just like to have fun, so you must be savvy enough to understand my verbage!
Alumni connections , Book club , Coffee and conversation , Cooking , Dining out , Gardening/Landscaping , Hobbies and crafts , Movies/Videos , Museums and art , Music and concerts , Exploring new areas , Performing arts , Religion/Spiritual , Shopping/Antiques , Travel/Sightseeing , Volunteering , Wine tasting
Dancing, Golf, Other types of exercise, Running, Skiing, Swimming, Walking / Hiking, Weights / Machines, Yoga
I exercise 1-2 times per week.
I have Cats
I like Dogs
I have a couple of rescued cats. My Basset Hound passed last April. I would like to have another dog, but right now it is so much easier to take off for the weekend without having to worry about a dog sitter. You must like pets!
In Florida, travel destinations include the Fl. Keys, Sanibel/Captiva Islands, Mt. Dora, and Fernandina Beach. Summers in Brewster on Cape Cod, and Denver, Co. Looking forward to Ca. wine country with my new significant other, (when I find him) :)
Florida State University, Tallahassee, FL
Spending part of the summer in Brewster on Cape Cod. Short weekend trips, love art shows, galleries, the theatre, and men that make me laugh. Love dogs, but currently have two little boy cats whom I rescued from my yard. Also like car shows.
I love to travel, even short weekend trips. I belong to a beach club, and we always have events going on there. I try to work out at the club gym a few days a week/or walk on the beach. I have one very unusual hobby....ask me about it if we speak.
I read a book within a few days once I get into it. Browse at Barnes and Nobles when time permits.Prefer non-fiction. Just read Waiter Rant, story of a professional waiter which was eye-opening, Subscribe to the Sunday paper. Receive prof. journals.
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|What she is looking for
|5'7" (170cms)
|5'9" (175cms) to 6'2" (187cms)
|Body type:
|Slender, About average, Athletic and toned, A few extra pounds
|Blue, Brown, Grey, Hazel
|Light brown, Dark brown, Blonde, Salt and pepper, Silver, Dark blonde, Grey, Platinum, Bald
|No Way
|No Way, Cigar aficionado
|Social Drinker
|Never, Social Drinker
|Self-Employed / Entrepreneur
|Executive / Management, Legal, Medical / Dental / Veterinary / Fitness, Retired, Self-Employed / Entrepreneur, Other profession
I love what I do! My time is basically my own, and I only have to put in about 20 hours per week. I am free to take off on a trip if something interesting arises! I frequent Cape Cod, (Brewster), and also travel frequently to the Denver area.
|$75,001 to $100,000
|$75,001 to $100,000, $100,001 to $150,000, $150,001+
|Never Married, Widow / Widower, Currently Separated, Divorced
|Have kids:
|Yes, and they live away from home (1)
|Yes, and they sometimes live at home, No, Yes, and they live away from home
|Wants kids:
|White / Caucasian
|White / Caucasian
Prefer the all-American type guy. No foreign men, no foreign accents, including N.Y. and N. J......LOL....Light southern accents.....o.k. A somewhat reserved guy with a great sense of humor would be perfect. Over 55, over 5'8. A few extra lbs. o.k
|Christian / Protestant
|Christian / Catholic, Christian / LDS, Christian / Protestant, Jewish, Spiritual but not religious, Other, Christian / Other
I am a Christian. I respect others faiths, but do best matched with a Christian or person of the Jewish faith.... I do not wish to date an atheist. My two great loves...a Presbyterian and a Mormon! Still friends today....I dislike drama.
|Graduate degree
|Some college, Associates degree, Bachelors degree, Graduate degree, PhD / Post Doctoral
2 degrees, alum of FSU. I prefer someone with at least an undergrad degree....however....if you are bright and sucessful without a degree, that is o.k. too. .I prefer that you not be divorced more than once. No commitmentphobics, no 12 steppers.


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Clarion Inn and Conference Center
6625 Dean Memorial Parkway
Hudson, OH 44236
Phone: (330) 653-9191
Fax: (330) 656-0048
The Arcade
401 Euclid Avenue
Cleveland, OH 44114
+1 216 696 1408
Mo to Sa from 10:00 AM to 06:00 PMThe Arcade opened as early as in 1890 and was the first indoor shopping center in America. It still sports a Victorian facade and consists of three buildings with a glass skylight spanning over four balconies. Restaurants include Chocolate Bar, Greek Express and Presto Sandwiches among numerous others. Stores include flower shops, clinics, clothing boutiques and more. The space is available for events, too.
Earn valuable points toward free nights.


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, Cambodia Official Info - US State Department Travel Considerations for Cambodia
, Cambodia Official Info - U.S. Department Of State information for The Kingdom of Cambodia.
More Cambodia Vacations - Travel guide to More Cambodia accommodations and vacation packages. Visit More Cambodia for getaways and adventure travel. Plan your More Cambodia vacation today.


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Locally sourced ingredients form the flavor foundations of these craft beers, including Michigan-grown hops and grain
$16 $10
$146.99 for Pedal-Pub Cruise for Eight on March 21–April 28, 2015 ($280 Value). 33 Options Available.
Great Lakes Pub Cruiser
Pedal pubs are equipped with stereos and an iPod hookup; Groupon is valid for eight people, but can seat 16
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Wine Tasting and Architectural Tour for Two or Four at the Henderson Castle Winery (Up to 55% Off)
French Restaurant at the Henderson Castle
Sample six of Henderson Castle's estate-label wines, such as meritage and riesling, and tour the castle, which was established in 1895
Wine Tasting for Two or Four at Baroda Founders Wine Cellar (Up to 47% Off)
Baroda Founders Wine Cellar
Founders Wine Cellar
Each guest samples seven wines, five classic varieties and two speciality wines, during the winery visit
Private Wine Tasting for Two or Four at Chateau Aeronautique Winery (Half Off)
Chateau Aeronautique Winery
After a winery tour, guests sample wines fermented from Michigan grapes paired with snacks alongside an airplane hangar and grass runway
Craft Brewery Bus Tours Presents Midwest Craft Beer Fest for One or Two with VIP Option on May 9 (Up to 56% Off)
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Four Winds Field
Celebrate craft beer at a festival; VIP entry includes lunch, workshops, and unlimited drinks
Distillery Tour with Fine Spirits Tasting for Two, Four, Six, or Eight at Virtuoso Distillery (Up to 56% Off)
Virtuoso Distillery
Tour and tasting of a distillery that makes fine spirits using American rye that's distilled 18 times
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The following is an abstract for the selected article. A PDF download of the full text of this article is available here. Members may download full texts at no charge. Non-members may be charged a small fee for certain articles.
A Rapid Spectrographic Method for the Determination of Beryllium in Air Dust
Volume 8, Number 2 (May 1954) Page 71-75
Landis, Frank P.; Coons, Mary C.
A method has been developed for the determination of beryllium in air dust samples at the rate of 11 man-minutes per sample. The method involves the use of a small filter paper sample which is inserted into a cupped graphite electrode and arced in a conventional direct current arc. Certain treatments are necessary to ensure optimum excitation of the sample, including the addition of a barium chloride carrier and internal standard. The method is sensitive to .002 microgram of beryllium on the filter paper, and the range of analysis extends to .1 microgram. The accuracy of the method is about ±30% of the amount of beryllium present.