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14. Raeburn, S. P., Ilton, E. S. & Veblen, D. R. Quantitative determination of the oxidation state of iron in
biotite using X-ray photelectron spectroscopy: II. In situ analyses. Geochim. Cosmochim. Acta 61,4531±4537 (1997).
15. Harris, J., Hutchison, M. T., Hursthouse, M., Light, M. & Harte, B. A new tetragonal silicate mineraloccurring as inclusions in lower-mantle diamonds. Nature 387, 486±488 (1997).
16. Mao, H.-K., Shen, G. & Hemley, R. J. Multivariable dependence of Fe-Mg partitioning in the lower
mantle. Science 278, 2098±2100 (1997).
17. Wood, B. J. & Rubie, D. C. The effect of alumina on phase transformations at the 660-kilometerdiscontinuity from Fe-Mg partitioning experiments. Science 273, 1522±1524 (1996).
18. Disko, M. M., Ahn, C. C. & Fultz, B. Transmission Electron Energy Loss Spectrometry in Materials
Science (Minerals, Metals & Materials Soc., Warrendale, IL, 1992).
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1996).
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York, 1996).
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5, 323±327 (1998).

regulator of feeding and energy balance which acts downstream of
leptin and the melanocortin system, and that deletion of a geneencoding a single orexigenic peptide can result in leanness.
The MCH gene was disrupted in embryonic stem cells byhomologous recombination using a targeting vector in whichexons 1±3 of the MCH gene were replaced by a construct consistingof phosphoglycerate kinase (PGK) promoter and the neomycinresistance (neor) gene (PGK-neor) (Fig. 1a). Two embryonic stem
cell lines with correctly targeted recombination were detected bySouthern blot analysis, and a line of mice with a disrupted MCH
gene (MCH-/-) was established (Fig. 1b). Reverse transcription withpolymerase chain reactions (RT-PCR; Fig. 1c) and in situ hybridization (Fig. 1d) con®rmed disruption of the MCH gene in MCH-/mice. In addition, immunocytochemistry showed that MCH was
not immunostained in the brain of MCH-/- mice (data not shown).
MCH-/- mice were born at the expected mendelian frequency,
viable into adulthood, and fertile, and they appeared phenotypicallyaWild typeBN

Exon
I II IIIA

Probe

Targeted vector

Mutant allele

P

B

N

X

BN

Acknowledgements. We thank P. Rez and J. Bradley for discussions, and K. Righter, D. Canil and
G. Gudmundsson for providing us with samples. This work was supported by the Earth Sciences Divisionof the US NSF.
Correspondence and requests for materials should be addressed to L.A.J.G. (e-mail: Igarvie@asu.edu).

X

X

(C)

P

(C)

P

PGK-neo
B

B

B

PGK-neo

WT 11 Kb
HR 7.5 Kbb-/- +/+ +/- -/- -/- +/- +/-Mice lacking melaninconcentrating hormone
are hypophagic and lean

c

+/+

+/-

-/-

M r (K) M 200 200 0.2 200 20 NC

1.6
1.0
0.5
MCH
Leptin receptor(long form)

Masako Shimada*, Nicholas A. Tritos², Bradford B. Lowell*,
Jeffrey S. Flier* & Eleftheria Maratos-Flier³+/+ +/- ND -/- NDNCd* Division of Endocrinology, Beth Israel Deaconess Medical Center,
330 Brookline Avenue, Boston, Massachusetts 02115, USA² Research Division, Joslin Diabetes Center, 1 Joslin Place, Boston,
Massachusetts 02215, USA.........................................................................................................................

Feeding is in¯uenced by hypothalamic neuropeptides that promote(orexigenic peptides) or inhibit feeding1. Of these, neuropeptide Y(NPY) in the arcuate nucleus2 and melanin-concentrating hormone(MCH)3 and orexins/hypocretins4,5 in the lateral hypothalamus
have received attention because their expression is increased
during fasting and because they promote feeding when administered centrally. Surprisingly, absence of the orexigenic neuropeptide NPY fails to alter feeding or body weight in normal mice6. As
de®ciency of a single component of the pathway that limits foodintake (such as leptin or receptors for melanocortin-4)7,8 causes
obesity, it has been suggested that orexigenic signals are more
redundant than those limiting food intake7,8. To de®ne further thephysiological role of MCH and to test the redundancy of orexigenic signals, we generated mice carrying a targeted deletion of
the MCH gene. MCH-de®cient mice have reduced body weight
and leanness due to hypophagia (reduced feeding) and an inappropriately increased metabolic rate, despite their reduced
amounts of both leptin and arcuate nucleus pro-opiomelanocortin messenger RNA. Our results show that MCH is a critical
670

Figure 1 Targeted disruption of the MCH gene in mice. a, Top, the targeted regionof the MCH gene locus. Middle, the PGK±neor cassette was used to disrupt the
MCH gene coding region. Bottom, the expected targeted allele. Restriction
enzyme sites: B, BglII; N, NcoI; X, XbaI; A, A¯; P, PstI; (C), ClaI in polylinker. The
thick black bar indicates the genomic 320-bp BglII±NcoI probe. WT, wild-type; HR,
the homologous recombinant allele. b, Southern blot analysis of mouse tailgenomic DNA. Genotypes are shown at the top. c, Top, detection of MCH mRNAin the hypothalamus using RT-PCR. The mouse genotype and total amount of
RNA (in ng) used for RT-PCR in each group is shown at the top. Bottom, RT-PCR
analysis, using primers for the long form of the leptin receptor and the same RNA
as in the top panel. M, marker; NC, negative control; ND, not done. d, In situ
hybridization with a digoxigenin-labelled riboprobe was used to analyse MCH
expression in the hypothalamus of wild-type (left) and MCH-/- (right) mice. 3V,
third ventricle.

Nature © Macmillan Publishers Ltd 1998

NATURE | VOL 396 | 17 DECEMBER 1998 | www.nature.com

8letters to naturenormal by gross inspection except for their reduced size. The mean
body weight of both male and female MCH-/- mice was reduced,
and this was ®rst apparent between 4 and 5 weeks of age. At 17 weeks
of age, male MCH-/- mice weighed 28% less (Fig. 1a) and female
MCH-/- mice weighed 24% less (Fig. 2b) than control mice; weightsof heterozygous MCH+/- mice tended to be slightly but notsigni®cantly lower than weights of control mice (Fig. 2a, b). Carcass
analysis showed that MCH-/- mice had a reduced triglyceride
content; they were leaner than control littermates (Fig. 2c). We
measured nose±anus length of male mice at 16 weeks of age;
this measurement did not vary between groups (MCH+/+,
9:2 6 0:1; MCH-/-, 9:1 6 0:1 cm; n  6 in each group).
The reduced body weight and lean phenotype were associatedwith hypophagia, as 6-week-old MCH-/- mice ingested 12% fewer
calories than control mice over a 24-hour period (Fig. 2d). The
entire reduction in food intake occurred during the dark cycle, andthe amount of food consumed during the light period, although asmall fraction of total daily intake, was signi®cantly increased
compared with control animals, indicating that MCH may exert
its primary effect on feeding during the dark period (Fig. 2d).
However, as MCH-/- mice consumed most of their calories during
the dark cycle, like normal mice, it seems that MCH is not necessary
for the normal diurnal feeding pattern (Fig. 2d). Consistent with
their lean phenotype, the MCH-/- mice showed decreased leptin
levels, being 51%, 59% and 42% of control levels at 12:00, 18:00 and
c 18

30
25

†† † † †††† ††† ††† ††† † † †
†
**

20
15
10
5
0

0

4

8

12

16

16
14
12
10

6
4

a

*

2
0
25

20

††

8

26

27

28

29

30

31

Body weight (g)

d

b 40
35

5

Food intake (g)30Body weight (g)6Female

25
20

†† †
* **

15

††

†† † † † †

*

10

*
4

*

4

8

12

16

20

5

*
**

b

2

*

0

Day

Night

Whole dayFigure 2 Body weight, fat composition and food intake in MCH-/- and control mice
fed on a chow diet. a, b, Growth curves. Three to four mice were housed per cage.

7

5
4
3
2

00

mice. Open circles, wild-type mice; open triangles,d6MCH+/-, n  28; MCH-/-, n  12. c, Percentage of triglycerides relative to bodyand MCH

-5
-10
-15

Wild type8

1

weight in MCH

0

-20

0

Male: MCH+/+, n  8; MCH+/-, n  19; MCH-/-, n  13. Female: MCH+/+, n  6;
+/+

5

*
MCH (-/-)

Time (h)Time (week)

-/-

**

10

6:00 12:00 18:00 24:00

1

0

10

3

5
0

c

15

Body weight reduction
after 24 h starvation (%)

Body weight (g)35Body weight during food shift (g)40Plasma leptin level (ng ml–1)

Fat in 12-week-old mice (%)

Male

8

Food intake after 24 h
starvation (g)a 45

24:00, respectively (P , 0:05 versus control) (Fig. 3a).
As MCH has been proposed to play a role in motivatedbehaviours, we studied the behaviour of MCH-/- mice using an
open-®eld locomotion test9, in which MCH-/- mice were as active
as wild-type controls. Control male mice (n  6) crossed 41 6 6squares over 5 minutes whereas MCH-/- male mice (n  6) crossed
43 6 6 squares over the same interval.
We studied potential effects of MCH de®ciency on neuroendocrine function. Thyroxine levels (MCH+/+ mice, 4:7 6 0:6 mg dl 2 1 ;MCH-/- mice, 5:2 6 1:2 mg dl 2 1 ; mean 6 s:e:m:) and corticosterone levels (MCH+/+ mice, 16:9 6 1:5 ng ml 2 1 ; MCH-/- mice,
14:7 6 1:3 ng ml 2 1 ) measured at 06:00 were not signi®cantly different in 15-week-old male MCH-/- mice and controls. Levels ofblood glucose and insulin measured in 15-week-old males werealso not signi®cantly different (glucose: MCH+/+ mice,
107:7 6 5:3 mg dl 2 1 ; MCH-/- mice, 102:6 6 5:9 mg dl 2 1 ; insulin;
MCH+/+ mice, 0:99 6 0:06 ng ml 2 1 ; MCH-/- mice, 0:72 6 0:07 ng
ml-1; n  7 each group).
The reduction in body weight in MCH-/- mice exceeded thedegree to which food intake was reduced, indicating that increased
metabolic rate might contribute to the lower weight. Rectal temperature, an imperfect indicator of metabolic rate, was unchanged,
being 37:9 6 0:03 8C in control animals (n  6) and 37:6 6 0:04 8C
in MCH-/- animals (n  6). We therefore measured the metabolic
rate of these mice in the fed state. MCH-/- mice show rates of oxygen
consumption that are slightly but not signi®cantly higher thancontrol rates when expressed on a per mouse basis (MCH+/+ mice,
1:08 6 0:06 ml O2 per min per mouse; MCH-/- mice, 1:14 6 0:06 ml
O2 per min per mouse). However, the rate of oxygen consumption isincreased by 20% in MCH-/- mice when normalized to body mass
(MCH-/- mice, 34:1 6 0:4 ml O2 per min per kg body weight;
MCH+/+ mice, 27:8 6 0:4 ml O2 per min per kg body weight).
Whether expressed per mouse or per kg body weight, this rate of

4

8

12

16

20

Hours after refeeding

24

28
26
24
22
20

** *

*

18
16
0 2 4

6

8 10 12 14 16 18

Days after starting food shift

MCH+/- mice; shaded circles, MCH-/- mice. d, Food intake by 6-week-old wildtype (n  6; white bars) and MCH-/- (n  9; shaded bars) male mice during the

Figure 3 Leptin levels and feeding response in MCH-/- mice. a, Leptin levels in 8-

light and dark cycles. Mice were housed individually 4 days before the experimentweek-old male MCH-/- (n  6; ®lled circles) and MCH+/+ (n  8; open circles)

and the values were determined over a 5-day period. Data are mean intake per

mice. Data are means 6 s.e.m. b, Cumulative food intake in response to starvationday 6 s.e.m. Asterisk indicates P , 0:05; double asterisk indicates P , 0:01;

in MCH-/- (®lled circles) and MCH+/+ (open circles) mice. c, Body-weight reduc-dagger indicates P , 0:001; double dagger indicates P , 0:0001. Statisticaltion after 24-h starvation. d, Response of wild-type (open circles) and MCH-/-signi®cance was determined by Fischer's PLSD (post-hoc least signi®cant(®lled circles) mice to food shift. Asterisk indicates P , 0:05; double asterisk

difference).

indicates P , 0:01. Statistical signi®cance was determined by Fischer's PLSD.

NATURE | VOL 396 | 17 DECEMBER 1998 | www.nature.com

Nature © Macmillan Publishers Ltd 1998671

letters to nature
oxygen consumption may re¯ect an impaired ability of MCH-/mice to appropriately regulate their metabolic rate in response to
their reduced feeding and lower amounts of leptin (see below).
Thus, MCH de®ciency promotes weight loss by reducing feeding
and may also limit the normal suppression of metabolic rateduring food restriction. This combination of results was predicted
for NPY-/- mice on the basis of the results of intracerebral injectionsof this neuropeptide, but mice de®cient in NPY showed no such
defects6.
We studied the response of MCH-/- mice to food deprivation.
MCH-/- mice responded to 24 hours of starvation with compensatory hyperphagia equal to that of wild-type mice (Fig. 3b).
Thus, like NPY6, MCH is not essential for the hyperphagic
response to starvation. However, two lines of evidence indicatethat MCH de®ciency may create susceptibility to the adverse effectsof starvation. First, when age-matched mice were starved for
24 hours, weight loss was greater in MCH-/- mice than in controls(17:0 6 1:0% vs 12:8 6 0:9% weight loss, respectively; n  6,
P , 0:05) (Fig. 3c). Second, when age-matched mice were starved
for 48 hours, 3 of 4 MCH-/- but 0 of 4 MCH+/+ mice died during thelast 4 hours of the fast. The precise basis for these adverse effects of
starvation is not yet known, but inappropriate levels of thermogenesis, coupled with reduced initial fat stores, would be expected tocause greater weight loss, and earlier death, in the absence of foodintake.
To further explore the response to altered food availability, weused a restricted-feeding paradigm10. Food was offered only during
the light cycle, between 11:00 and 15:00, to 7-week-old mice. Over a
9-day period, wild-type controls responded to this stress with a 16%
reduction in body weight, whereas MCH-/- mice showed 23%
weight loss (P , 0:05) (Fig. 3d). In contrast, food intake fell tothe same degree in wild-type and MCH-/- mice (data not shown),
consistent with the possibility that MCH de®ciency causes increased
thermogenesis under these circumstances.
The hypophagia and leanness of MCH-/- mice led to the production of lower leptin levels, which should cause hyperphagia. However, in the context of MCH de®ciency, low leptin levels resulted indecreased rather than increased food intake, indicating that MCH
may be essential for the hyperphagic response to leptin de®ciency
under these conditions. We also examined the effect of exogenous

0

-1

-2

-3

-4

*

-5

-6

20

10

0

-20

Day 1

Day 2

*

-30

-40

-50

Beforeinjection

140

-10

Beforeinjection

Day 1

Day 2

Figure 4 Response of six MCH+/+ (open circles) and six MCH-/- mice (shaded
circles) to recombinant mouse leptin, administered at a dose of 1 mg per g bodyExpression of hypothalamic mRNA (%)

b

1

Change in food intake after leptin injection (%)

Change in body weight after leptin injection (%)

aleptin on MCH-/- mice. Leptin was injected at a dose of 1 mg per g
body weight twice a day for 2 days. Leptin was able to reduce bothfood intake and body weight11 within the initial 24-hour period(Fig. 4). The initial response of MCH-/- mice to leptin was twice thatof wild-type mice (P , 0:05). This exaggerated response to exogenous leptin, which was also seen in NPY-/- mice, was attenuated by
day 2. As leptin inhibits food intake in MCH-/- mice, suppression of
MCH is unlikely to be the mechanism by which leptin inhibits foodintake in normal mice. Furthermore, MCH may, either directly or
indirectly, oppose the effect of leptin in diminishing food intake.
Finally, to assess the state of hypothalamic compensation in
response to both MCH de®ciency and lower leptin levels, we studied
hypothalamic expression of mRNAs encoding four other appetiteregulating neuropeptides in MCH-/- mice, namely NPY, orexin4,
Agouti-related protein (AGRP)12,13 and pro-opiomelanocortin(POMC)14,15. Expression of NPY and AGRP in the arcuate nucleus
and of orexin in the lateral hypothalamus remained unchanged in
MCH-/- mice in the fed state. However, expression of POMC in thearcuate nucleus was markedly suppressed (decreased by 63%
compared with controls; P , 0:006) (Fig. 5). As leptin is a positive
regulator of arcuate POMC expression16,17 the reduced POMC
expression could result from lower leptin levels, which, in turn,result from a lean body composition. Lower leptin amounts did notincrease NPY expression in MCH-/- mice but did in wild-type
mice18. These results might indicate that POMC expression ismore sensitive to the reduction in leptin levels than is NPY
expression. Alternatively, MCH de®ciency could reduce arcuate
POMC expression directly, through the loss of direct innervationof arcuate POMC-expressing neurons by MCH-expressing neurons;
MCH immunoreactivity is seen in the arcuate nucleus of wild-type
mice19, consistent with bidirectional communication between
POMC- and MCH-expressing systems. There are bidirectional
connections between arcuate neurons expressing NPY, AGRP and
POMC and lateral hypothalamic neurons expressing MCH andorexins20. The lower expression of arcuate POMC, which in othercontexts promotes hyperphagia and obesity21, might be expected tolimit the consequences of MCH de®ciency. However, our results
show that the ability of low arcuate POMC expression (and/or a low
POMC:AGRP ratio) to cause hyperphagia and obesity is limited in
the absence of an intact MCH gene.
Our MCH-/- mice have a deletion of the entire coding region of
the MCH gene, which also encodes the neuropeptides EI and GE22.
There is little information available on the biological role of thesetwo peptides; direct administration of either peptide alone or in
combination in rats has no effect on eating behaviour (E.M.-F.,
unpublished observations). In theory, the phenotypes of MCH-/mice could result from de®ciency of any or all of these peptides.

120
100
80
60

*
40
20
0

NPY

OrexinAGRP

POMC

weight twice daily (09:00 and 18:00), vs saline control (n  3; triangles). Values fora, the change in body weight and b, the change in food intake are the per centFigure 5 Expression of neuropeptide mRNAs in hypothalami of 24-week-old wild-change from a 7-day baseline period. Asterisk indicates P , 0:05. Statistical

type (n  6; white bars) and MCH-/- (n  6; shaded bars) mice. Asterisk indicatessigni®cance was determined by Fischer's PLSD.

P , 0:006. Statistical signi®cance was determined with the Mann±Whitney test.

672

Nature © Macmillan Publishers Ltd 1998

NATURE | VOL 396 | 17 DECEMBER 1998 | www.nature.com

8letters to nature
However, given the ability of MCH to promote feeding3,23 and theabsence of evidence that NEI and NGE promote such an effect, it isreasonable to assume that MCH de®ciency is the primary, or evensole, cause of the phenotype reported here.
MCH is a cyclic 19-amino-acid polypeptide whose expression islimited to the lateral hypothalamus and zona incerta22,24. It has been
implicated as an important regulator of eating behaviour, because
central administration of MCH promotes feeding, and MCH
mRNA amounts rise as a result of starvation and leptin de®ciency3.
MCH-expressing neurons are well placed anatomically to participate in feeding behaviour, because they make monosynaptic connections with several areas in the brain involved in integrating
inputs related to taste, olfaction and visceral sensations, including
the nucleus of the solitary tract, the parabrachial nucleus and theinsular and medial prefrontal cortex24. MCH-expressing neurons
have therefore been suspected to participate in complex integrative
behaviours, and the acute feeding response to MCH3,23 indicatedthat feeding was among these behaviours. The phenotype of the
MCH-/- mouse reported here strongly supports this hypothesis. As
the leanness of MCH-/- mice occurs despite reduced expression of
leptin and arcuate POMC, which produce obesity when MCH ispresent7,21,25, MCH appears to be a critical effector of energy balance
downstream of leptin and the POMC/melanocortin system. Further
genetic crosses between MCH-/- mice and mice with different causesof obesity will be required to test this hypothesis directly. Finally, as
MCH-/- mice are lean, antagonists of MCH action may be effective
treatments for obesity.
.........................................................................................................................

Methods

Creation of MCH-/- mice. We screened a mouse C129 SvJ P1 genomic library(Genome Systems) using a 250-base-pair (bp) PCR fragment, generated on thebasis of the published sequence of the murine MCH gene26, as a probe. One ofthree clones was mapped; it consisted of the 16-kilobase (kb) P1 vector and a
72-kb genomic insert containing the MCH coding region between ,25 kb of 59
and 45 kb of 39 ¯anking sequence. We replaced a ,1.8 kb-fragment extending
from 340 bp upstream of the translation initiation codon (XbaI site) and 200 bp
downstream of the stop codon (A¯ III site)) of this clone with a PGK±neor
cassette. This targeting vector in pGEM5 (Promega) was linearized at the SalI
site and electroporated into the J1 embryonic stem line27 (cells provided by E. Li
and A. Sharp); selection of G418-resistant clones was done as described27. We
identi®ed targeted clones by Southern blot analysis, using a 320-bp BgII±NcoI
fragment located 59 of the targeting vector as a probe; 2 of 68 clones werepositive. These positive clones were injected into C57B1/6 embryos at theblastocyst stage. Chimaeric offspring were mated with C57B1/6 mice. Germline
transmission of the mutant allele was determined by Southern analysis ofmouse tail genomic DNA. One line of mice carrying the disrupted MCH gene
was generated, and F3 hybrids were used in all experiments. We reversetranscribed 1 mg of total RNA from the hypothalamus of a mouse from eachgroup and ampli®ed aliquots equivalent to 200, 20 and 0.2 ng total RNA by
PCR, using the following primers: sense, 59-ATGGCAAAGATGACTCTCTCT39; and antisense, 59-GACTTGCCAACATGGTCGGTA-39, as described26. In
situ hybridization was done using a digoxigenin-labelled MHC complementary
RNA probe28.
Studies of MCH-/- mice. Mice were maintained in a daily cycle of 12 h light(06:00±18:00) and 12 h darkness (18:00±06:00) and were allowed free access tochow (Purina Formulab 5008) and water. Total body lipid content was assessedusing alcoholic potassium hydroxide digestion with saponi®cation of all fats,neutralization and then enzymatic determination of glycerol (Sigma) as
described29. Leptin and insulin concentrations were measured by RIA (Linco
Research), as were corticosterone and thyroxine levels (ICN). Glucose wasmeasured using a One Touch meter (Lifescan). Rectal temperature wasmeasured with a rectal probe (Yellow Springs Instrument). Oxygen consumption was measured in 20-week-old male wild-type (n  4) and MCH (n  4)
mice using an Oxymax 4.93 (Columbus Instruments).
Response to starvation in MCH-/- and MCH+/+ mice. We deprived 10-weekold mice (n  6) of food for 24 h, beginning at 09:00; the mice were thenallowed free access to chow. To measure cumulative food intake after refeeding,
NATURE | VOL 396 | 17 DECEMBER 1998 | www.nature.com

we measured the weight of consumed food at 2, 4, 6, 12 and 24 h after refeeding.
Body weights were measured after the 24-h starvation period and after
refeeding.
-/Food-shift experiment. To determine the response of wild-type and MCH
mice to food shift, body-weight-matched animals (wild-type male, n  7; MCH-/male, n  4) were individually housed and allowed free access to chow for only
4 h during the light cycle (11:00±15:00) for 16 days10. Body weights were measured
at 11:00. Animals were allowed free access to water during the period.
Response to leptin. To determine the response to leptin, mice were injected
intraperitoneally with recombinant mouse leptin (Eli Lilly) at a dose of 1 mg per
g body weight twice daily (09:00 and 18:00). Three control mice were injectedwith saline.
Expression of hypothalamic mRNAs. Mice were killed at 09:00 byadministration of pentobarbital and were perfused transcardially ®rst withsaline and then with 10% buffered formalin. In situ hybridization was donewith probes for NPY, POMC, AGRP and orexin mRNAs. The images captured
on ®lm were quanti®ed with a computing densitometer (Molecular Dynamics)
and ImageQuant Software (Molecular Dynamics). We integrated the absorbance in rectangular areas encompassing each nucleus of interest (arcuate
nucleus for NPY, POMC and AGRP, and lateral hypothalamus for orexin) overeach set of brain sections, and subtracted background density from an adjacentarea without speci®c hybridization signal. We determined statistical signi®cance with the Mann±Whitney test.

8

Received 15 July; accepted 5 October 1998.
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for the treatment of obesity? Eur. J. Clin. Invest. 24, 293±308 (1994).
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Nature 380, 243±247 (1996).
4. Sakurai, T. et al. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G proteincoupled receptors that regulate feeding behavior. Cell 92, 573±585 (1998).
5. de Lecea, L. et al. The hypocretins: hypothalamus-speci®c peptides with neuroexcitatory activity. Proc.
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letters to nature
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Acknowledgements. We thank J. Mastaitis, C. Behn and C. Lee for technical assistance, J. Elmquist forhelp in analysing and interpreting brain anatomy, and D. S. Ludwig for the P1 clone used in making theconstruct for the knockout mice. This work was supported in part by grants from NIH to J.S.F.
and E.M.-F., from the American Diabetes Association to E.M.-F., and from Eli Lilly to J.S.F. andE.M.-F., and by the Transgenic Core of the Boston Obesity Nutrition Research Center. M.S. was supportedby the Banyu Fellowship in Lipid Metabolism and Atherosclerosis which is sponsored by Banyu
Pharmaceutical Co Ltd and the Merck Foundation.
Correspondence and requests for materials should be addressed to E.M.-F. (e-mail: emarat@joslab.
harvard.edu).

GABAB receptors function
as a heteromeric assemblyof the subunits GABABR1and GABABR2
Kenneth A. Jones, Beth Borowsky, Joe A. Tamm,
Douglas A. Craig, Margaret M. Durkin, Meng Dai,
Wen-Jeng Yao, Mary Johnson, Caryn Gunwaldsen,
Ling-Yan Huang, Cheng Tang, Quanrong Shen,John A. Salon, Kelley Morse, Thomas Laz, Kelli E. Smith,
Dhanapalan Nagarathnam, Stewart A. Noble,
Theresa A. Branchek & Christophe Gerald
Synaptic Pharmaceutical Corporation, 215 College Road, Paramus,
New Jersey 07652, USA.........................................................................................................................

The principal inhibitory neurotransmitter GABA (g-aminobutyric acid) exerts its effects through two ligand-gated channels,
GABAA and GABAC receptors, and a third receptor, GABAB (ref.
1), which acts through G proteins to regulate potassium andcalcium channels. Cells heterologously expressing the cloned
DNA encoding the GABABR1 protein exhibit high-af®nity antagonist-binding sites2, but they produce little of the functional
activity expected from studies of endogenous GABAB receptors
in the brain. Here we describe a new member of the GABAB
polypeptide family, GABABR2, that shows sequence homology to
GABABR1. Neither GABABR1 nor GABABR2, when expressedindividually, activates GIRK-type potassium channels; however,the combination of GABABR1 and GABABR2 confers robust
stimulation of channel activity. Both genes are co-expressed inindividual neurons, and both proteins co-localize in transfected
cells. Moreover, immunoprecipitation experiments indicate thatthe two polypeptides associate with each other, probably asheterodimers. Several G-protein-coupled receptors (GPCRs)
exist as high-molecular-weight species, consistent with the formation of dimers by these receptors3±7, but the relevance of these
species for the functioning of GPCRs has not been established. We
have now shown that co-expression of two GPCR structures,
GABABR1 and GABABR2, belonging to the same subfamily isessential for signal transduction by GABAB receptors.
To ®nd other genes related to the GABABR1 gene, we searched theexpressed sequence tags (ESTs) of GenBank using the GABABR1
sequence. (Throughout the text, `GABABR1' refers to the
GABABR1b splice variant.) Two entries had scores that suggested
signi®cant homology to GABABR1. We used oligonucleotide probes
from these sequences to isolate a full-length clone from a rat
hypothalamic complementary DNA library. Sequence analysis of
this new clone showed that it has a 2.8-kilobase open reading framethat is predicted to encode a protein of 940 amino acids. A BLAST
search of the GenEMBL database indicated that this amino-acidsequence was most closely related to that of GABABR1, exhibiting
35% and 41% identity overall and within the predicted transmembrane domains, respectively (Fig. 1a). The structural similarity to
674

GABABR1 indicated that this sequence might encode a new GABAB
polypeptide, which we refer to as GABABR2. The next most relatedsequences were other members of the metabotropic glutamate
receptor (mGluR) family, with 21±24% overall amino-acid identity.
Like GABABR1 and other members of the mGluR family8,
GABABR2 contains a large amino-terminal extracellular domain
with regions of homology to bacterial amino-acid-binding proteins.
We studied the distribution of GABABR2 messenger RNA within
the central nervous system by in situ hybridization. Strong hybridization signals were observed in regions of the rat brain that havehigh densities of GABAB-receptor-binding sites9, such as the hippocampus, medial habenula, thalamus and cerebellum (Fig. 1b±e).
There was a high degree of similarity in the distribution andintensity of GABABR1 and GABABR2 hybridization signals.
Postsynaptic inhibition of neurons by GABAB-receptor activation
is caused by the opening of inwardly rectifying K+ channels(GIRKs)10±13. We assessed the ability of either GABABR1 or
GABABR2 to regulate K+ currents in cells transfected with GIRK1
and GIRK4 subunits14±16. Xenopus oocytes injected with either
GABABR1 or GABABR2 mRNAs failed to generate GABA-evoked
GIRK currents (Fig. 2b). The longer splice variant of GABABR1,GABABR1a, did not stimulate GIRK activity either, as reported
elsewhere2,17. In a mammalian host, HEK293 cells, small agonistevoked currents (10±50 pA) were observed in 5 of 26 cells expressing GABABR1; similar weak currents were evoked in 1 of 23 cells
expressing GABABR2. In contrast, large currents were producedin 100% of cells expressing the galanin receptor GalR1 (ref. 18)(Fig. 2i).
The overlapping expression patterns of GABABR1 and GABABR2transcripts in the brain indicated that the corresponding proteinsmight be co-expressed in individual neurons and that both might berequired for functional activity. When GABABR1 and GABABR2
were co-injected into oocytes together with GIRK subunits, theapplication of GABA produced robust K+ currents (Fig. 2a, b).
Responses to GABA were abolished in oocytes pretreated with
pertussis toxin, showing that receptor stimulation leads to activation of a heterotrimeric G protein of the Gao/Gai class. The GABAinduced currents showed the following properties, which suggestthat the currents were mediated by GIRK channels: ®rst, a dependency on a raised external K+ concentration; second, strong inward
recti®cation; third, a reversal potential (-23 mV) near the predictedequilibrium potential for K+; and fourth, sensitivity to block by
100 mM Ba2+ (Fig. 2c).
The pharmacology of agonists at the GABABR1/GABABR2 combination was comparable to that reported for native receptors19,20.
GABA, baclofen and 3-aminopropylmethyl phosphinic acid (3APMPA) exhibited half-maximally effective concentrations (EC50values) of 1:3 6 0:1 (n  20), 3:3 6 0:4 (n  8), and 0:051 6 0:003(n  6) mM, respectively (Fig. 2d, g). Concentration±effect curves
for GABA were shifted to the right, in an apparently competitive
manner, by well-characterized GABAB-selective antagonists (Fig.
2h). Estimates of af®nity of the antagonists CGP54626(33:1 6 6:3 nM; n  4) and CGP55845 (2:5 6 0:2 nM; n  6) for
GABABR1/GABABR2 were similar to values reported in previous
electrophysiological studies using brain tissue19,20, as well as to thoseobtained by measuring displacement of radioligand from cells
expressing GABABR1 alone2.
The appearance of robust functional responses after GABABR1/GABABR2 co-expression was also observed in HEK293 cells. Upon coexpression of GABABR1 and GABABR2 together with GIRKs, GABA
evoked currents in 70 of 81 recorded cells (Fig. 2e, i). These currents
were blocked by low concentrations of the competitive GABABreceptor antagonist CGP55845 (ref. 21) and by the GIRK-channel
blocker Ba2+ (Fig. 2f), indicating that they resulted from the stimulation of GABAB receptors and the subsequent activation of GIRKs.
Large-amplitude currents were also observed when GABABR2 waspaired with the GABABR1a splice variant (1;046 6 247 pA; n  9). To

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