Molecular Biology of the Cell
Vol. 19, 1540–1547, April 2008
Identification of Ciliary Localization Sequences within the
Third Intracellular Loop of G Protein-coupled Receptors
Nicolas F. Berbari,* Andrew D. Johnson,* Jacqueline S. Lewis,*
Candice C. Askwith,†and Kirk Mykytyn*‡
Departments of *Pharmacology,†Neuroscience, and‡Internal Medicine, Division of Human Genetics,
and College of Medicine, The Ohio State University, Columbus, OH 43210
Submitted September 18, 2007; Revised January 2, 2008; Accepted January 29, 2008
Monitoring Editor: Francis Barr
Primary cilia are sensory organelles present on most mammalian cells. The functions of cilia are defined by the signaling
proteins localized to the ciliary membrane. Certain G protein–coupled receptors (GPCRs), including somatostatin receptor
3 (Sstr3) and serotonin receptor 6 (Htr6), localize to cilia. As Sstr3 and Htr6 are the only somatostatin and serotonin
receptor subtypes that localize to cilia, we hypothesized they contain ciliary localization sequences. To test this hypothesis
we expressed chimeric receptors containing fragments of Sstr3 and Htr6 in the nonciliary receptors Sstr5 and Htr7,
respectively, in ciliated cells. We found the third intracellular loop of Sstr3 or Htr6 is sufficient for ciliary localization.
Comparison of these loops revealed a loose consensus sequence. To determine whether this consensus sequence predicts
ciliary localization of other GPCRs, we compared it with the third intracellular loop of all human GPCRs. We identified
the consensus sequence in melanin-concentrating hormone receptor 1 (Mchr1) and confirmed Mchr1 localizes to primary
cilia in vitro and in vivo. Thus, we have identified a putative GPCR ciliary localization sequence and used this sequence
to identify a novel ciliary GPCR. As Mchr1 mediates feeding behavior and metabolism, our results implicate ciliary
signaling in the regulation of body weight.
Primary cilia are appendages that project from almost all
human cell types (Wheatley et al., 1996). It is generally
accepted that primary cilia serve important specialized sig-
naling functions (Pazour and Witman, 2003; Marshall and
Nonaka, 2006; Singla and Reiter, 2006). In the eye, photore-
ceptors, which are modified primary cilia, sense and re-
spond to light. In the nose, specialized olfactory cilia detect
odors and initiate signaling cascades in olfactory neurons. In
the kidney, it is proposed that bending of cilia on epithelial
cells by fluid flow triggers an increase in intracellular cal-
cium mediated by an ion channel located on the cilium
(Praetorius and Spring, 2001; Nauli et al., 2003). In each case,
the function of the cilium is defined by the signaling proteins
that are enriched in the ciliary membrane (i.e., light recep-
tors, odorant receptors, and mechanoreceptors). Importantly,
disruption of the signaling mediated by these receptors
can cause disease and altered development (Davenport
and Yoder, 2005; Hildebrandt and Otto, 2005; Pan et al.,
2005; Bisgrove and Yost, 2006). Yet, the specific signaling
proteins that localize to the vast majority of cilia in the
mammalian body are unknown. Thus, the functions of pri-
mary cilia on most cell types in the body are unknown.
Neuronal primary cilia are abundant throughout the ro-
dent brain (Bishop et al., 2007). The functional importance of
these cilia is suggested by the fact that several human ciliary
disorders, including Bardet-Biedl syndrome (BBS), Joubert
syndrome (JS), and Meckel syndrome (MKS), have promi-
nent functional and structural CNS phenotypes (Badano et
al., 2006). Although the specific functions of neuronal cilia
are unknown, certain G protein–coupled receptors (GPCRs),
including somatostatin receptor 3 (Sstr3; Handel et al., 1999;
Schulz et al., 2000) and serotonin receptor 6 (Htr6; Hamon et
al., 1999; Brailov et al., 2000), localize to the ciliary membrane
on neurons. This observation suggests that cilia may sense
levels of neuromodulators in the local environment of neu-
rons. Given that the minority of the total ciliated neurons are
positive for Sstr3 (Berbari et al., 2007) and the distribution of
Htr6 ciliary localization is restricted to a few regions of the
brain (Hamon et al., 1999; Brailov et al., 2000), it is likely that
additional GPCRs localize to neuronal cilia.
Although the ciliary membrane is continuous with the
plasma membrane, only certain GPCRs localize to cilia. This
is clearly demonstrated by the somatostatin receptors. There
are five somatostatin receptor genes present in humans and
rodents (Sstr1-5; Patel, 1999). In addition, the carboxy (C)-
tail of Sstr2 can undergo alternative splicing to yield Sstr2a
and Sstr2b. They are all members of the GPCR superfamily
of cell-surface receptors that couple to heterotrimeric G pro-
teins and regulate numerous downstream effectors. There
are ?950 GPCRs in the human genome, with 500 of those
coding for odorant or taste receptors and the remaining 450
coding for receptors with endogenous ligands (Takeda et al.,
2002). All GPCRs share a common molecular topology of
seven transmembrane-spanning domains, three intracellular
loops, three extracellular loops, an amino (N)-terminus out-
side the cell, and a C-terminus inside the cell (Dong et al.,
2007). The six somatostatin receptor subtypes are expressed
in the rat CNS and show overlapping regional distributions
(Schulz et al., 2000). Yet, Sstr3 is selectively targeted to the
ciliary membrane (Handel et al., 1999; Schulz et al., 2000).
This article was published online ahead of print in MBC in Press
on February 6, 2007.
Address correspondence to: Kirk Mykytyn (email@example.com).
1540© 2008 by The American Society for Cell Biology
Similarly, of the 13 mammalian G protein–coupled seroto-
nin receptor subtypes only Htr6 has been shown to localize
to cilia (Hamon et al., 1999; Brailov et al., 2000). These obser-
vations suggest that Sstr3 and Htr6 contain unique se-
quences that mediate their localization to cilia.
Very little is known about the underlying mechanisms of
sorting and trafficking of GPCRs to the cilium. One GPCR
ciliary localization sequence that has been described is an
adjacent hydrophobic and basic residue motif immediately
C-terminal to the seventh transmembrane segment. In Cae-
norhabditis elegans, this sequence is required for ciliary local-
ization of olfactory GPCRs (Dwyer et al., 2001). In mamma-
lian cells, this motif is required for the ciliary localization of
the GPCR Smoothened (Corbit et al., 2005). Although this
motif is present in Sstr3 and Htr6, it is also present and
conserved in all of the somatostatin and serotonin receptor
subtypes. Yet, only Sstr3 and Htr6 localize to cilia. Thus, this
motif may be necessary but it is not sufficient to specify
We hypothesized that ciliary GPCRs contain unique se-
quences that mediate localization to cilia. Here, we report
that the third intracellular loop of Sstr3 and Htr6 is sufficient
to localize nonciliary GPCRs to cilia, suggesting that Sstr3
and Htr6 are targeted to cilia through similar mechanisms.
Comparison of the sequences within these loops reveals a
putative consensus sequence. This consensus sequence is
present in the third intracellular loop of numerous known
ciliary GPCRs and also identifies a number of candidate
ciliary GPCRs. We subsequently show that one of these
(Mchr1), localizes to cilia in vitro and in vivo. These findings
identify a new role for cilia in MCH signaling and, because
Mchr1 is involved in the regulation of energy homeostasis,
implicate ciliary signaling in the regulation of body weight.
MATERIALS AND METHODS
Animals and Tissue Processing
The mice used in this study were on a 129:BL6 background. All procedures
were approved by the Institutional Animal Care and Use Committee at The
Ohio State University. Animals were anesthetized by a 0.1 ml/10 g intraperi-
toneal injection of 2.5% tribromoethanol (Sigma-Aldrich, St. Louis, MO),
killed by cardiac puncture, and perfused with phosphate-buffered saline (PBS)
followed by a mixture of 2% paraformaldehyde and HistoChoice (Amresco,
Solon, OH). The brains were then further fixed in 2% PFA/HistoChoice for
16–24 h at 4°C followed by cryoprotection in 30% sucrose in PBS for 16–24 h.
Cryoprotected brains were embedded in Optimal Cutting Temperature com-
pound (VWR, West Chester, PA), and sectioned in a cryostat at a thickness of
30 ?m. Labelings were performed in three different animals.
The coding sequences of the somatostatin receptors were amplified from
mouse genomic DNA. The unspliced version of Sstr2 that was amplified
corresponds to Sstr2a. The coding sequences of serotonin receptors 6 and 7
and melanin-concentrating hormone receptor 1 were amplified from reverse-
transcribed mouse whole brain RNA using the Superscript First-Strand Syn-
thesis RT-PCR Kit (Invitrogen, Carlsbad, CA). All coding sequences were
cloned into a TA cloning vector (pSTBlue-1; Novagen, San Diego, CA).
Receptor open reading frames were utilized as templates for overlap exten-
sion PCR to generate chimeric receptors, as previously described (Horton et
al., 1990). Briefly, oligonucleotide primers (36mers; IDT Technologies, Cor-
alville, IA) corresponding to the nucleotide sequence of the junction regions
were generated with 5? nucleotide sequence from one receptor and 3? se-
quence from the other. The junctions were designed at conserved residues,
the coding frames were maintained, and no sequences were inserted or
deleted. Primers corresponding to the N-terminal and C-terminal regions of
both receptors were designed with 5? restriction sites for directional cloning.
The initial PCR generated fragments of the two receptors to be combined with
complementary overhanging sequence. The products from the first reaction
were gel-purified and combined as template for the fusion PCR. All amplifi-
cations were performed with HiFi Platinum Taq Polymerase (Invitrogen). The
final PCR products were cloned into the pEGFP-N vector (Clontech, Moun-
tain View, CA) using the added restriction enzyme sites. All DNA constructs
were sequence verified. Primer sequences are available upon request. Chi-
meric receptor sequences were mutated using the QuikChange Site-directed
Mutagenesis Kit (Stratagene, La Jolla, CA).
Cell Culture and Transient Transfections
IMCD-3 cells (ATCC, Manassas, VA) were maintained in DMEM:F12 media
supplemented with 10% FBS, 1.2 g/l of sodium bicarbonate, and 0.5 mM
sodium pyruvate (Invitrogen). Cells (n ? 5 ? 106) were electroporated with 10
?g DNA and plated at high density on glass coverslips. Cells were harvested
24 h after transfection for immunocytochemistry.
Cells were fixed in 4% paraformaldehyde and permeabilized with 0.3% Triton
X-100 in PBS with 2% goat serum, 0.02% sodium azide, and 10 mg/ml bovine
serum albumin (BSA). The IMCD cells were then labeled with anti-acetylated
?-tubulin (T-6793; Sigma-Aldrich). Brain sections were permeabilized with
0.3% Triton X-100 in PBS with 2% donkey serum, 0.02% sodium azide, and 10
mg/ml BSA and simultaneously labeled with anti-adenylyl cyclase III (ACIII)
rabbit polyclonal antibody (sc-588; Santa Cruz Biotechnology, Santa Cruz,
CA), used at 1:500, and anti-melanin–concentrating hormone receptor 1 goat
polyclonal antibody (sc-5534; Santa Cruz Biotechnology), used at 1:250. All
incubations and washes were carried out in PBS with 2% serum, 0.02%
sodium azide, and 10 mg/ml BSA. Primary antibody incubations were carried
out for 16–24 h at 4°C, and secondary antibody incubations were carried out
for 1 h at room temperature. Secondary antibodies included the following:
Alexa Fluor 546–conjugated goat anti-mouse IgG (Invitrogen), Alexa Fluor
488–conjugated donkey anti-goat IgG (Invitrogen), or Cy3-conjugated don-
key anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA). Nuclei were
visualized by DRAQ5 (Axxora, San Diego, CA). Slides were mounted using
Immu-Mount (Thermo Scientific, Pittsburgh, PA).
All samples were imaged on a Zeiss 510 META laser scanning confocal
microscope (Thornwood, NY) at the Ohio State University Central Micros-
copy Imaging Facility. Multiple consecutive focal planes (Z-stack), spaced at
0.5–1-?m intervals, were captured. For all collected images, the brightness
and contrast of each channel was adjusted using the Zeiss LSM Image
Browser program. For quantitative analysis, coverslips were centered on the
microscope objective, and 3–5 consecutive fields were imaged. The number of
ciliated transfected cells and the number of transfected cells expressing cilia
localization in each image were counted by individuals blinded to the exper-
imental conditions. The results are expressed as the percent of transfected
cells showing ciliary localization. A total of ?100 cells were counted from
three distinct transfections (n ? 3) for each construct. Data are expressed as
mean ? SEM.
Prediction of Human GPCR Third Intracellular Loop
Human GPCR protein sequences were downloaded from the GPCRDB infor-
mation system (http://www.gpcr.org/7tm/; Horn et al., 2003). The protein
sequences were input into the transmembrane hidden Markov model
(TMHMM) program (version 2.0) to generate predictions of transmembrane,
intracellular, and extracellular sequences (Krogh et al., 2001). A Perl program was
then applied to the TMHMM output to extract the predicted third intracellular
loop sequence for all human GPCRDB proteins. The resulting database was
searched using the ambiguous regular expression “AX[S/A]XQ” to identify
GPCRs predicted to contain this motif within their third intracellular loop.
To determine whether heterologous expression could be
used to identify GPCR ciliary localization sequences, we
generated constructs encoding mouse somatostatin recep-
tors one through five (Sstr1–5) fused at the C-terminus to
enhanced green fluorescent protein (EGFP). These con-
structs were then expressed in inner medullary collecting
duct (IMCD) cells, which are derived from mouse kidney
and develop cilia in culture. Visualization of the subcellular
localization of the fluorescently labeled receptors 24–48 h
after transfection showed that of the somatostatin receptor
subtypes, only Sstr3 selectively localized to cilia (Figure 1,
A–E). The remaining somatostatin receptor subtypes failed
to localize to cilia, as indicated by a lack of colocalization
with the ciliary marker acetylated ?-tubulin, and instead
localized to the plasma membrane or within intracellular
compartments. Similarly, transfection of IMCD cells with Htr6
GPCR Ciliary Localization Sequences
Vol. 19, April 20081541
and the closely related Htr7 fused at the C-terminus to EGFP
revealed that Htr6 preferentially localized to cilia, whereas
Htr7 localized primarily to the plasma membrane (Figure 1, F
and G). Interestingly, cilia on cells expressing Sstr3 or Htr6
consistently appeared longer than cilia on untransfected cells
or on cells expressing nonciliary GPCRs (Figure 1, A–G). The
underlying mechanism for this difference is unknown. Overall,
these results suggest that Sstr3 and Htr6 contain sequences that
specify ciliary localization, and IMCD cells possess the neces-
sary machinery to traffic specific receptors to cilia.
To identify the region of Sstr3 containing ciliary localiza-
tion sequences, we utilized a fusion PCR approach to con-
struct EGFP-fused chimeric receptors. Chimeras were gen-
erated containing segments of Sstr3 and the structurally and
pharmacologically similar Sstr5. To minimize the likelihood
of protein misfolding, the chimeric receptor fusion sites
were engineered at conserved residues located within the
transmembrane domains. Initially, we generated chimeric
receptors of Sstr5 in which the sequence from the N-termi-
nus to the second, fourth, or sixth transmembrane (TM)
domains was substituted with the corresponding sequence
from Sstr3 (Figure 2A). Expression of these chimeric recep-
subtype 6 selectively localize to cilia when heterologously expressed
in IMCD cells. (A–G) Representative confocal microscopy images of
transiently transfected IMCD cells expressing somatostatin recep-
tors 1 through 5 (Sstr1–5) and serotonin receptors 6 (Htr6) and 7
(Htr7) fused at the C-terminus to EGFP. Left, EGFP fluorescence
(green) shows expression of the receptors; middle, acetylated ?-tu-
bulin (red) marks the cilia; right, merged images. Below each panel
is a confocal image in the xz plane to show cilia projecting vertically
from the apical surface of cells. Note that only Sstr3-EGFP (C) and
Htr6-EGFP (F) show localization that overlaps with acetylated ?-tu-
bulin ciliary labeling. Sstr2 corresponds to Sstr2a. Nuclei are labeled
with the DNA stain DRAQ5 (blue). All scale bars, 10 ?m.
Somatostatin receptor subtype 3 and serotonin receptor
domains of Sstr3 are important for ciliary localization. (A) Schematic
of chimeric receptors containing portions of Sstr3 (indicated by
black lines) and Sstr5 (indicated by white lines) fused at the C-
terminus to EGFP. Transmembrane domains (TM) are depicted as
boxes. (B–D) Representative images of transiently transfected IMCD
cells expressing the indicated chimeric receptors. Left, EGFP fluo-
rescence (green); middle, acetylated ?-tubulin (red); right, merged
images. Chimeric receptors Sstr5[N-TM2Sstr3] (B) and Sstr5[N-
TM4Sstr3] (C) do not localize to cilia. Chimeric receptor Sstr5[N-
TM6Sstr3] does localize to cilia, suggesting that ciliary localization
of Sstr3 is mediated by sequences between TM4 and TM6. Nuclei
are labeled with DRAQ5 (blue). All scale bars, 10 ?m.
Sequences between the fourth and sixth transmembrane
N. F. Berbari et al.
Molecular Biology of the Cell1542
tors in IMCD cells revealed that only chimeric receptor
Sstr5[N-TM6Sstr3] selectively localized to cilia (Figure 2,
B–D), suggesting that sequences between the TM4 and TM6
domains in Sstr3 mediate ciliary localization. To test this
hypothesis, we generated a chimeric receptor of Sstr5 in
which the sequence between the TM4 and TM6 domains was
replaced with the corresponding sequence from Sstr3 (Fig-
ure 3A). This chimera also localized to cilia (Figure 3B),
suggesting that ciliary localization sequences are located
within the third extracellular (e3) loop or the third intracel-
lular (i3) loop of Sstr3. To distinguish between these two
possibilities, we generated chimeric receptors of Sstr5 con-
taining only the e3 or i3 loop of Sstr3 (Figure 3A). Notably,
chimeric receptor Sstr5[TM4-5Sstr3] did not localize to cilia
(Figure 3C), but chimeric receptor Sstr5[TM5-6Sstr3] did
localize to cilia (Figure 3D), indicating that sequences within
the i3 loop of Sstr3 are sufficient to localize Sstr5 to cilia.
Immunoblotting of proteins isolated from cells transiently
transfected with chimeric receptors Sstr5[TM4-5Sstr3] and
Sstr5[TM5-6Sstr3] revealed similar expression patterns (Sup-
plementary Figure 1), indicating that absence of cilia local-
ization was not due to the lack of receptor expression or
To determine whether the i3 loop of Htr6 also mediates
ciliary localization, we generated a chimeric receptor in
which the i3 loop of Htr7 had been substituted with the i3
loop of Htr6 (Figure 4A). Notably, this chimeric receptor
selectively localized to cilia (Figure 4B). This result indicates
that Sstr3 and Htr6 contain ciliary localization sequences
within the same domain and suggest that they may be
targeted to cilia through similar mechanisms. Because the
predicted i3 loop of Htr6 (63 residues) is significantly larger
than the i3 loop of Sstr3 (36 residues), we further narrowed
the region of Htr6 sufficient to traffic Htr7 to the cilium by
generating chimeric receptors containing only the N- or
C-portion of the i3 loop (Figure 4A). V241 in Htr6, which
corresponds to V295 in Htr7, was used as the fusion site.
Chimeric receptor Htr7[TM5-V241Htr6] selectively localized
to cilia (Figure 4C) but chimeric receptor Htr7[V241-
TM6Htr6] did not (Figure 4D), indicating that sequences
within the N-portion of the i3 loop of Htr6 are sufficient to
localize Htr7 to cilia.
localize Sstr5 to cilia. (A) Schematic of chimeric receptors containing
portions of Sstr3 (black lines) and Sstr5 (white lines) fused at the
C-terminus to EGFP. TM domains are depicted as boxes. (B–D)
Representative images of transiently transfected IMCD cells ex-
pressing the indicated chimeric receptors. Left, EGFP fluorescence
(green); middle, acetylated ?-tubulin (red); right, merged images.
Chimeric receptors Sstr5[TM4-6Sstr3] (B) and Sstr5[TM5-6Sstr3] (D)
localize to cilia, whereas chimeric receptor Sstr5[TM4-5Sstr3] (C)
does not localize to cilia, suggesting that the i3 loop of Sstr3 contains
ciliary localization sequences. Nuclei are labeled with DRAQ5
(blue). All scale bars, 10 ?m.
The third intracellular (i3) loop of Sstr3 is sufficient to
localize Htr7 to cilia. (A) Schematic of chimeric receptors containing
portions of Htr6 (black lines) and Htr7 (white lines) fused at the
C-terminus to EGFP. TM domains are depicted as boxes. (B–D)
Representative images of transiently transfected IMCD cells ex-
pressing the indicated chimeric receptors. Left, EGFP fluorescence
(green); middle, acetylated ?-tubulin (red); right, merged images.
Chimeric receptors Htr7[TM5-6Htr6] (B) and Htr7[TM5-V241Htr6]
(C) selectively localize to cilia, whereas chimeric receptor Htr7
[V241-TM6Htr6] (D) does not, suggesting that the N-portion of the
i3 loop of Htr6 contains ciliary localization sequences. Nuclei are
labeled with DRAQ5 (blue). All scale bars, 10 ?m.
The amino portion of the i3 loop of Htr6 is sufficient to
GPCR Ciliary Localization Sequences
Vol. 19, April 20081543
We hypothesized that ciliary localization sequences would
be unique to the receptor subtype that localizes to cilia,
would be conserved in species in which the receptor is
ciliary, and would be similar between Sstr3 and Htr6. Com-
parison of the i3 loop sequences of the mouse somatostatin
receptors reveals similarity between Sstr3 and the other
subtypes, except for several residues in the N-portion and an
insertion of 11 amino acids that is unique to Sstr3 (Figure
5A). To further narrow the sequences of interest we com-
pared the i3 loop sequences of Sstr3 across species. Com-
parison of the mouse and human sequences reveals that
the unique residues in the N-portion of the loop are not
conserved, but 5 of the 11 amino acids inserted in mouse
Sstr3 (APSCQ) are completely conserved in human SSTR3
(Figure 5B). Given that human SSTR3 also localizes to cilia
when expressed in IMCD cells (data not shown), this
suggested that residues within “APSCQ” might confer
We then examined the N-portion of the i3 loop of Htr6
and identified a sequence (ATAGQ) with modest similarity
that is not present in Htr7 (Figure 5C). Because the A and Q
at the ends of the sequence are identical between Sstr3 and
Htr6, we performed site-directed mutagenesis on these res-
idues to verify they are important for ciliary localization. The
A and Q residues in chimeric receptor Sstr5[TM5-6Sstr3] were
mutated to phenylalanine, and ciliary localization was then
quantified in IMCD cells expressing Sstr3, Sstr5, Sstr5[TM5-
6Sstr3], or Sstr5[TM5-6Sstr3mut]. Sstr3 localized to cilia in
?91% of transfected cells, whereas Sstr5 never localized to
cilia (Figure 5D). Chimeric receptor Sstr5[TM5-6Sstr3]
showed a similar percentage of cilia localization (?93%) as
Sstr3 (Figure 5D and Supplementary Figure 2A). The mu-
tated version of this chimera (Sstr5[TM5-6Sstr3mut1]) showed
a dramatic reduction in the percentage of ciliary localization
but still localized to cilia in ?50% of cells (Figure 5D).
However, the i3 loop of mouse Sstr3 contains a second
ciliary localization consensus sequence (APACQ; Figure 5B).
To test whether the second site was contributing to ciliary
localization, we mutated the A and Q residues to F in
Sstr5[TM5-6Sstr3mut1] and quantified ciliary localization.
Notably, when all four residues were mutated in this chi-
mera (Sstr5[TM5-6Sstr3mut2]) ciliary localization was re-
duced to ?6% of cells (Figure 5D and Supplementary Figure
2B). Immunoblotting of proteins isolated from transiently
transfected cells expressing chimera Sstr5[TM5-6Sstr3mut2]
confirmed that the mutant chimeric receptor was expressed
(Supplementary Figure 1).
Similarly, the A and Q residues in chimeric receptor Htr7
[TM5-V241Htr6] were mutated to phenylalanine and ciliary
localization was then quantified in IMCD cells expressing
Htr6, Htr7, Htr7[TM5-V241Htr6], or Htr7[TM5-V241Htr6mut].
Htr6 localized to cilia in ?93% of transfected cells, whereas
Htr7 localized to cilia in ?20% of transfected cells (Figure
5E). Of note, ciliary localization of Htr7 was almost exclu-
sively seen in cells that were expressing the receptor at a
very high level. Chimeric receptor Htr7[TM5-V241Htr6] lo-
fies unique residues in Sstr3 and Htr6 that are important for ciliary
localization of GPCRs. (A) Alignment of the predicted i3 loop se-
quences of the mouse somatostatin receptors reveals an insertion of
11 unique residues in Sstr3. The corresponding positions of the
residues are indicated. Red signifies identical residues and blue
signifies conserved residues. (B) Alignment of the predicted i3 loop
sequence of somatostatin receptor subtype 3 from mouse (Sstr3) and
human (SSTR3). Five of the unique 11 residues (APSCQ; boxed) are
completely conserved between mouse and human. (C) Alignment of
the predicted i3 loop sequences of mouse serotonin receptors 6 and
7 shows the presence of a unique sequence in Htr6 (ATAGQ; boxed)
with modest similarity to the Sstr3 sequence. (D) Percentage of
transiently transfected IMCD cells that show ciliary localization
when expressing Sstr3, Sstr5, Sstr5[TM5-6Sstr3], Sstr5[TM5-6Sstr3mut1],
or Sstr5[TM5-6Sstr3mut2]. Sstr3 localizes to cilia in ?91% of trans-
fected cells. Sstr5 never localizes to cilia. Chimeric receptor
Sstr5[TM5-6Sstr3] localizes to cilia in ?93% of transfected cells.
Chimeric receptor Sstr5[TM5-6Sstr3mut1], in which the A and Q
in the conserved consensus sequence have been mutated to F,
localizes to cilia in ?50% of transfected cells. Chimeric receptor
Sstr5[TM5–6Sstr3mut2], in which the A and Q in the second consen-
sus sequence have also been mutated to F, localizes to cilia in ?6% of
transfected cells. Values are expressed as mean ? SEM *Significantly
different from Sstr3 and Sstr5[TM5–6Sstr3] percentages. (E) Percentage
Comparative genomics and mutational analysis identi-
of transiently transfected IMCD cells that show ciliary localization
when expressing Htr6, Htr7, Htr7[TM5-V241Htr6], or Htr7[TM5-
V241Htr6mut]. Htr6 localizes to cilia in ?93% of transfected cells.
Htr7 localizes to cilia in ?20% of transfected cells. Chimeric receptor
Htr7[TM5-V241Htr6] localizes to cilia in ?70% of transfected cells.
Chimeric receptor Htr7[TM5-V241Htr6mut], in which the A and Q
have been mutated to F, localizes to cilia in ?4% of transfected cells.
Values are expressed as mean ? SEM. *Significantly different from
Htr6 and Htr7[TM5-V241Htr6] percentages.
N. F. Berbari et al.
Molecular Biology of the Cell 1544
calized to cilia in ?70% of transfected cells (Figure 5E and
Supplementary Figure 2C). Remarkably, the mutated ver-
sion of this chimera (Htr7[TM5-V241Htr6mut]) almost never
localized to cilia (?4%; Figure 5E and Supplementary Figure
2D). Together, these results indicate that the A and Q resi-
dues in the i3 loop of Sstr3 and Htr6 are important for ciliary
To test whether these sequences could be used to predict
novel ciliary GPCRs, we formulated a consensus sequence
based on Sstr3 and Htr6. Because serine and alanine belong
to a “strong” Gonnet Pam250 matrix conservation group
(Gonnet et al., 1992), we formulated the following loose
consensus sequence (AX[S/A]XQ), where position 1 is an A,
position 5 is a Q, position 3 is an S or A, and positions 2 and
4 are any amino acid. As the ciliary localization sequences
were identified within the i3 loop of both Sstr3 and Htr6, we
searched for the consensus sequence in a database contain-
ing the sequences of the predicted i3 loop of all human
GPCRs. The consensus sequence is present in the i3 loop of
11 additional GPCRs (Table 1). Remarkably, these 11 GPCRs
include 3 odorant receptors, 3 cone opsins, and rhodopsin,
all of which are ciliary proteins. The remaining four GPCRs
(?-2A adrenergic receptor, chemokine orphan receptor 1,
melanin-concentrating hormone receptor 1, and muscarinic
acetylcholine receptor M5) have not been shown to localize
to cilia and were classified as candidate ciliary GPCRs. Be-
cause melanin-concentrating hormone receptor 1 (Mchr1) is
involved in the regulation of feeding behavior and energy
balance (Pissios et al., 2006) and multiple human ciliary
disorders are associated with obesity (Badano et al., 2006),
Mchr1 was considered a particularly good candidate ciliary
To test whether Mchr1 localizes to cilia, we cloned the
coding sequence of Mchr1 from mouse cDNA, fused it to
EGFP, expressed it in IMCD cells, and assessed subcellular
localization. Notably, Mchr1 localized to cilia when ex-
pressed in IMCD cells (Figure 6A). We then tested whether
Mchr1 localizes to cilia in tissue by labeling mouse brain
sections with an antibody to Mchr1. We detected ciliary
localization of Mchr1 in several brain regions (Figure 6B),
including the hippocampus, nucleus accumbens, olfactory
bulb, and hypothalamus. Ciliary localization was confirmed
by colabeling with ACIII, which is a marker of neuronal cilia
(Berbari et al., 2007; Bishop et al., 2007). These results indi-
cate, for the first time, that Mchr1 localizes to cilia in vitro
and in vivo.
It is presumed that ciliary membrane proteins are synthe-
sized in the endoplasmic reticulum, trafficked through the
Golgi, and then transported in post-Golgi vesicles to the
base of the cilium (Bloodgood, 2000). As access to the cilium
is restricted and only certain proteins localize to the ciliary
membrane (Bloodgood, 2000), it is thought that ciliary pro-
teins contain targeting signals that direct them to the cilial
compartment (Rosenbaum and Witman, 2002). Few mam-
malian ciliary localization sequences have been described
(Deretic et al., 1998; Corbit et al., 2005; Geng et al., 2006; Hu
et al., 2006; Jenkins et al., 2006), and only one sequence has
been shown to be sufficient for ciliary targeting in mamma-
lian cells. The N-terminal 15 amino acids of the ciliary cation
channel polycystin-2 are sufficient to localize heterologous
proteins to cilia (Geng et al., 2006). Within this domain is a
conserved motif, RVXP, that is required for ciliary localiza-
tion (Geng et al., 2006). This motif is also found at the
C-terminus of olfactory cyclic nucleotide–gated channel 1b
and is required for trafficking of the channel into cilia (Jen-
kins et al., 2006). This motif is not found in Sstr3 or Htr6, but
is found in the i3 loop of Htr7, which does not localize to
cilia, suggesting this motif is not functional in Htr7 or is not
involved in the targeting of GPCRs to cilia.
In this work, we show that sequences within the i3 loop of
Sstr3 and Htr6 are sufficient to localize nonciliary GPCRs to
cilia on IMCD cells. Notably, we see the same subcellular
localization results when we express the constructs in cul-
tured primary hippocampal neurons (Berbari and Mykytyn,
unpublished results). This suggests that the trafficking ma-
chinery for ciliary receptors is conserved between neurons
and IMCD cells and supports the utility of this system for
identifying the mechanisms of ciliary protein sorting and
trafficking. Interestingly, replacing the i3 loop in Sstr3 and
Htr6 with the i3 loop from Sstr5 and Htr7, respectively, does
Table 1. G protein–coupled receptors identified by the ciliary lo-
calization consensus sequence
G protein–coupled receptor
Olfactory receptor 52N1
Olfactory receptor 52N4
Olfactory receptor 6V1
?-2A adrenergic receptor
hormone receptor 1
Chemokine orphan receptor 1
calizes to cilia in vitro and in vivo. (A) Representative image of
transiently transfected IMCD cells expressing Mchr1 fused at the
C-terminus to EGFP. Left, EGFP fluorescence (green); middle, acety-
lated ?-tubulin (red); right, merged image. (B) Representative image
of the nucleus accumbens from an adult mouse colabeled with
antibodies to Mchr1 (green; left), ACIII (red; middle), and merged
(right). The majority of cilia are positive for both Mchr1 and ACIII.
Cilia that are positive for ACIII but negative for Mchr1 are indicated
with arrows. Nuclei are labeled with DRAQ5 (blue). All scale bars,
Melanin-concentrating hormone receptor 1 (Mchr1) lo-
GPCR Ciliary Localization Sequences
Vol. 19, April 20081545
not prevent ciliary localization (data not shown). Thus, al-
though the i3 loops are sufficient to localize nonciliary
GPCRs to cilia, they are not necessary for ciliary localization
of Sstr3 and Htr6. This is an intriguing result that suggests
there are additional ciliary localization sequences within
Sstr3 and Htr6.
The i3 loops of GPCRs, including somatostatin and sero-
tonin receptors, are normally associated with G protein cou-
pling and desensitization (Oksenberg et al., 1995; Gelber et
al., 1999; Hipkin et al., 2000). This is the first time the i3 loop
has been implicated in GPCR ciliary localization. Most
known GPCR trafficking motifs are located within the C-
terminus (Dong et al., 2007). However, the i3 loop of mouse
vasopressin V2 receptor contains two RXR ER retention
motifs that are thought to be masked under normal condi-
tions but can be unmasked in mutant receptors and block
trafficking to the plasma membrane (Hermosilla and Schul-
ein, 2001). An RXR motif is present in the i3 loop of Sstr3 and
Sstr5, but neither Htr6 nor Htr7 contain an RXR motif in the
i3 loop. Thus, it is unlikely that ER retention is a mechanism
for affecting ciliary localization in our system. The i3 loop of
many GPCRs also contains a loose consensus sequence
(BBXXB), where B is a basic residue and X is a nonbasic
residue, at the junction of the i3 loop and the TM6 domain
that is important for GPCR structure and activity (Kjelsberg
et al., 1992; Ren et al., 1993; Egan et al., 1998). This consensus
sequence is present in the C-terminal end of the i3 loop of
Sstr3, Sstr5, Htr6, and Htr7 and is required for Htr6 activity
(Kohen et al., 2001). However, it is likely unrelated to ciliary
localization given the ciliary localization domain in Htr6
maps to the N-portion of the i3 loop.
Comparison of the sequences within the i3 loops of Sstr3
and Htr6 reveals a consensus sequence (AX[S/A]XQ) that
may comprise a ciliary localization sequence. In support of
this idea, the consensus sequence is also present in the i3
loop of Mchr1, which we have shown localizes to cilia.
Further, mutating the A and Q residues in chimeric receptor
Htr7[TM5-V241Htr6] decreases ciliary localization from
?70% of cells to ?4%. Mutating the A and Q residues in
both consensus sequences in chimeric receptor Sstr5[TM5-
6Sstr3] decreases ciliary localization from ?93% of cells to
?6%. It is interesting that Htr7 localizes to cilia in ?20% of
cells, whereas Sstr5 never localizes to cilia. This may be due
to the fact that Sstr5 appears to be associated primarily with
intracellular structures (Figure 1E), as opposed to Htr7 that
appears to localize mainly to the plasma membrane (Figure
1G). It is likely that high levels of heterologously expressed
proteins can overcome restrictions on ciliary localization
when they are on the plasma membrane. Indeed, cotrans-
fecting Htr7 with an empty expression vector (pcDNA3.1) at
a ratio of 1:4, to lower the level of Htr7 expression, reduces
ciliary localization of Htr7 to ?6% of cells (data not shown).
Comparison of our consensus ciliary localization sequence
against the i3 loop sequence of all human GPCRs identified
known ciliary GPCRs and four candidate ciliary GPCRs. We
have confirmed that one of these candidates, Mchr1, local-
izes to cilia. The consensus sequences of the remaining three
candidates are present in the human protein but are not
completely conserved in lower organisms. An exciting pos-
sibility is ciliary localization of some GPCRs may have
evolved only in higher organisms. The development of cil-
iary localization would potentially be a mechanism for or-
ganisms to create additional complexity and more special-
ized functions in existing signaling pathways. Another
possibility is our consensus sequence is too stringent. In
support of this possibility, the ciliary localization sequence in
human HTR6 contains a G rather than an A at position 1,
suggesting there is flexibility to the consensus sequence. We
also hypothesized that localization of the sequence within the
i3 loop was a requirement for ciliary localization and limited
our search to the i3 loop. The amount of flexibility in the
consensus sequence and its location within receptors could
potentially increase the number of candidate ciliary GPCRs
dramatically. It will be necessary to experimentally determine
what residues are required and permissive and whether the
consensus sequence can be present in other domains in order
to accurately predict all ciliary GPCRs.
Although the ciliary localization sequence we identified is
common to Sstr3, Htr6, and Mchr1, it is not present in Smo,
despite the fact that Smo localizes to cilia in vitro and in vivo
(Corbit et al., 2005). One possibility is that Smo is targeted to
the cilium through a different mechanism. Indeed, substitu-
tion of the hydrophobic-basic residues in the C-terminal tail
of Smo with alanines prevents ciliary localization (Corbit et
al., 2005), but mutation of this motif in Sstr3 or Htr6 does not
prevent ciliary localization (Berbari and Mykytyn, unpub-
lished results). Perhaps there are divergent mechanisms of
ciliary trafficking between the different GPCR families. Sstr3,
Htr6, and Mchr1 are members of the Rhodopsin family of
GPCRs, whereas Smo is a member of the Frizzled/Smooth-
ened family of GPCRs. Several studies have found that defects
in ciliary protein transport can be associated with mislocaliza-
tion of some membrane proteins but not others (Marszalek et
suggesting there are multiple pathways for targeting mem-
brane proteins to the cilium.
An important outcome from these studies is the demon-
stration that Mchr1 localizes to neuronal cilia in regions of
the mouse brain, including the hypothalamus. A role for
MCH and its receptor in the regulation of feeding and
metabolism is well established. Specifically, injection of
MCH into the brains of mice induces a rapid increase in
feeding behavior (Qu et al., 1996), whereas injection of
Mchr1 antagonists reduces feeding behavior (Borowsky et
al., 2002). Further, transgenic mice overexpressing MCH
(Ludwig et al., 2001) are obese, and mice lacking expression
of either MCH (Shimada et al., 1998) or Mchr1 (Chen et al.,
2002) are lean. The fact that Mchr1 localizes to cilia in re-
gions of the brain known to regulate feeding behavior sug-
gests that localization of Mchr1 to cilia may be important for
signaling through the receptor and proper regulation of
these processes. Interestingly, mice lacking cilia in the brain
or specifically on pro-opiomelanocortin–expressing cells in
the hypothalamus are hyperphagic and become obese (Dav-
enport et al., 2007). Further, obesity is a hallmark of some
human ciliary disorders. The role of cilia in this phenotype
in particular has been a long-standing mystery. The identi-
fication of Mchr1 as a ciliary GPCR provides, for the first
time, a potential molecular mechanism to link defects in cilia
We thank W. Sade ´e, D. Lautenbach, L. Wallace, E. Fink, and M. Vest for
technical assistance. This work was supported in part by research grant
5-FY05–39 from the March of Dimes Birth Defects Foundation (K.M.)
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