may
Uppa
GPCR
Serotonin1A receptor
Specific interaction
rs (
pres
ster
tain the CRAC motif in their sequence. We report here the presence of CRAC motifs in three representative
t in hig
rane o
The G-protein coupled receptor (GPCR) superfamily is the larg-
est and most diverse protein family in mammals, involved in signal
transduction across membranes [8]. They represent major targets
for the development of novel drug candidates in all clinical areas
[9]. GPCRs are integral membrane proteins with a considerable
GPCRs [18]. Serotonergic signaling plays a key role in the genera-
tion and modulation of various cognitive, behavioral and develop-
mental functions. Work from other laboratories has shown that the
function of GPCRs such as rhodopsin [19] and the b2-adrenergic
receptor [20] is sensitive to membrane cholesterol.
Two possible mechanisms have been suggested by which mem-
brane cholesterol could influence the structure and function of
GPCRs [21]: (i) through a direct/specific interaction with GPCRs,
or (ii) through an indirect way by altering membrane physical
properties in which the receptor is embedded, or due to a combi-
nation of both. Interestingly, recently reported crystal structures
Abbreviations: 5-HT1A receptor, 5-hydroxytryptamine-1A receptor; CRAC, cho-
lesterol recognition/interaction amino acid consensus; GPCR, G-protein coupled
receptor; TMD, transmembrane domain.
⇑ Corresponding author. Fax: +91 40 2716 0311.
Biochemical and Biophysical Research Communications 404 (2011) 569–573
Contents lists availab
Biochemical and Biophysical
.e lE-mail address: amit@ccmb.res.in (A. Chattopadhyay).function, and sorting. It is often found distributed non-randomly
in domains in biological andmodel membranes [1,2]. Many of these
domains (sometimes termed as ‘lipid rafts’ [3]) are thought to be
important for the maintenance of membrane structure and func-
tion, although characterizing the spatiotemporal resolution of these
domains has proven to be challenging [4]. The concept of such spe-
cialized membrane domains gains relevance in cell biology since
important functions such as signal transduction [5], and the entry
of pathogens [6,7] have been implicated to these putative domains.
dynamics simulation that the lipid–protein interface corresponds
to 38% of the total surface area of the receptor [10]. Interestingly,
membrane cholesterol has been shown to modulate the function of
a number of GPCRs (reviewed in [11–13]). Previous work from our
laboratory has shown the crucial requirement of membrane cho-
lesterol in the organization and function of the serotonin1A (5-
HT1A) receptor, an important neurotransmitter receptor [12–17].
Serotonin1A receptors represent one of the largest, evolutionarily
ancient, and highly conserved families of seven transmembrane1. Introduction
Cholesterol is a major constituen
membranes and is crucial in memb0006-291X/$ - see front matter  2010 Elsevier Inc. A
doi:10.1016/j.bbrc.2010.12.031GPCRs, namely, rhodopsin, the b2-adrenergic receptor, and the serotonin1A receptor. Interestingly, the
function of these GPCRs has been previously shown to be dependent on membrane cholesterol. The pres-
ence of CRAC motifs in GPCRs indicates that interaction of cholesterol with GPCRs could be specific in nat-
ure. Further analysis shows that CRAC motifs are inherent characteristic features of the serotonin1A
receptor and are conserved over natural evolution. These results constitute the first report of the presence
of CRAC motifs in GPCRs and provide novel insight in the molecular nature of GPCR–cholesterol
interaction.
 2010 Elsevier Inc. All rights reserved.
her eukaryotic cellular
rganization, dynamics,
portion of the protein embedded in the membrane. This raises
the obvious possibility that the membrane lipid environment could
be an important modulator of receptor structure and function. In
case of rhodopsin, for example, it is estimated from molecularKeywords:
CRAC
Cholesterol
of GPCRs. Several structural features of proteins, believed to result in preferential association with cho-
lesterol, have been recognized. Cholesterol recognition/interaction amino acid consensus (CRAC)
sequence represents such a motif. Many proteins that interact with cholesterol have been shown to con-Identification of cholesterol recognition a
motif in G-protein coupled receptors
Md. Jafurulla, Shrish Tiwari, Amitabha Chattopadhy
Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research,
a r t i c l e i n f o
Article history:
Received 30 November 2010
Available online 10 December 2010
a b s t r a c t
G-protein coupled recepto
across membranes, and re
ical areas. Membrane chole
journal homepage: wwwll rights reserved.ino acid consensus (CRAC)
⇑
l Road, Hyderabad 500 007, India
GPCRs) are the largest class of molecules involved in signal transduction
ent major targets in the development of novel drug candidates in all clin-
ol has been reported to have an important role in the function of a number
le at ScienceDirect
Research Communications
sevier .com/locate /ybbrc

of GPCRs have shown structural evidence of cholesterol binding
sites [22,23]. Several structural features of proteins that are be-
lieved to result in preferential association with cholesterol have
been recognized [24,25]. In many cases, proteins that interact with
cholesterol have a characteristic amino acid sequence, termed the
cholesterol recognition/interaction amino acid consensus (CRAC)
motif. The CRAC sequence is defined by the presence of the pattern
-L/V-(X) -Y-(X) -R/K-, in which (X) represents between one
acid sequences used for the analysis belong to diverse taxa that in-
clude insects, fish and other marine species, amphibians and
extending up to mammals. The sequences for Trichoplax adhaerens,
Manduca sexta and Anopheles gambiae are putative serotonin1A
receptors whereas those of Strongylocentrotus purpuratus, Bos tau-
rus, Ornithorhynchus anatinus, Danio rerio, Monodelphis domestica,
Macaca mulatta and Taeniopygia guttata are predicted by homology.
The sequence for Caenorhabditis elegans belongs to the serotonin
570 Md. Jafurulla et al. / Biochemical and Biophysical Research Communications 404 (2011) 569–5731–5 1–5 1–5
and five residues of any amino acid [24,26]. This motif has been
shown to be present in caveolin-1 [27], the peripheral-type benzo-
diazepine receptor [26,28], the HIV-1 transmembrane protein gp41
[29], and the mammalian seminal plasma protein PDC-109 [30].
In spite of the importance of membrane cholesterol in GPCR
function [11–13], specific motifs for binding of cholesterol to
GPCRs have not been identified yet. We report here the presence
of CRAC motifs in three representative GPCRs, namely, rhodopsin,
the b2-adrenergic receptor, and the serotonin1A receptor. As men-
tioned above, all three receptors have been shown to have choles-
terol dependence for their function. Interestingly, we show that
CRAC motifs are inherent characteristic features of the serotonin1A
receptor and are conserved over natural evolution. Our results con-
stitute the first report of the presence of CRAC motifs in GPCRs.
2. Materials and methods
2.1. Identification of CRAC motif in representative human GPCRs:
rhodopsin, the b2-adrenergic receptor and serotonin1A receptor
The putative CRAC motifs in these GPCRs (with the conserved
tyrosine (Y) along with leucine (L)/valine (V) toward its amino ter-
minus and lysine (K)/arginine (R) toward carboxy terminus, within
a distance of 5 residues on either side), were manually identified
(see Fig. 1). The amino acid sequences of GPCRs were from NCBI
database. The transmembrane helices of the serotonin1A receptor
were predicted using the program TMHMM2 (see Fig. 2). These
predictions were confirmed by other programs such as MEMSTAT,
SPLIT4 and HMMTOP2 (it has been earlier reported that these pro-
grams were among the best and perform equally well in predicting
transmembrane helices [31]). Since the structure of human rho-
dopsin is not available, we aligned it to bovine rhodopsin [32]
using ClustalW and mapped the transmembrane regions based
on their similarity.
2.2. Sequence alignment of the serotonin1A receptor and identification
of CRAC motif
The evolution of CRAC motif(s) in the serotonin1A receptor over
various phyla was analyzed by examining amino acid sequences of
the serotonin1A receptor obtained from NCBI and Expasy databases
(see Figs 2 and 3). Partial, duplicate and other non-specific se-
quences were removed from the set of sequences obtained. Initial
alignment of sequences was carried out using ClustalW. After elim-
inating the relatively divergent parts of the receptor, the sequence
was realigned using the same program. The putative CRAC motifs
in the serotonin1A receptor were manually identified. The aminoFig. 1. Putative cholesterol recognition/interaction amino acid consensus (CRAC) mo
transmembrane domains (TMD) of rhodopsin, the b2-adrenergic receptor, and serotonin1A
in the respective sequences are mentioned in parentheses.receptor family.
3. Results and discussion
In the overall context of cholesterol sensitivity of GPCR function
[11–13], we examined whether the sequence of these GPCRs con-
tains any CRAC motif(s). Interestingly, we identified the presence
of CRAC motifs in all three GPCRs, namely, rhodopsin, the b2-adren-
ergic receptor and the serotonin1A receptor (see Fig. 1). Our analy-
sis shows that while the sequences of rhodopsin and the
serotonin1A receptor contain three CRAC motifs, the b2-adrenergic
receptor sequence shows two CRAC motifs. Rhodopsin sequence
contains CRAC motifs in putative transmembrane helices I (resi-
dues 57–66), III (residues 131–141) and VII (residues 304–311),
while the serotonin1A receptor sequence is characterized by CRAC
motifs in putative transmembrane helices II (residues 90–101), V
(residues 208–219) and VII (residues 394–405). The b2-adrenergic
receptor sequence, on the other hand, exhibits CRAC motifs in
putative transmembrane helices V (residues 213–221) and VII (res-
idues 324–328).
The serotonin1A receptor is an important member of the GPCR
superfamily and is estimated to have differentiated 650 million
years ago from the serotonin1 receptor subfamily in the time per-
iod during which vertebrates diverged from invertebrates [33]. In
the context of the presence of CRAC motifs in the human seroto-
nin1A receptor, we further analyzed whether the motif(s) is con-
served during the natural evolution of the receptor. In order to
examine the evolution of CRAC motif(s) in the serotonin1A receptor
over various phyla, we analyzed amino acid sequences of the sero-
tonin1A receptor from available databases (see Fig. 3). Partial,
duplicate and other non-specific sequences were removed from
the set of sequences obtained. The amino acid sequences used for
the analysis belong to diverse taxa that include insects, fish and
other marine species, amphibians and extending up to mammals.
Initial alignment carried out using ClustalW showed that CRAC
motif(s) are conserved in most species. Realignment with ClustalW
(after eliminating the relatively divergent parts of the receptor)
exhibited conservation of CRAC motif(s) across all phyla analyzed
(see Fig. 3). It therefore appears that CRAC motif(s) represents an
inherent characteristic feature of the serotonin1A receptor and is
conserved during the course of natural evolution. It appears from
Fig. 3 that the CRAC motif(s) is present even in organisms that
are not capable of biosynthesis of cholesterol. Organisms which
lack cholesterol biosynthesis could, however, acquire cholesterol
through diet [34]. Organisms such as insects possess sterols that
are different from cholesterol which have diverged from choles-
terol during the sterol evolution pathway [35]. The presence oftifs in representative human G-protein coupled receptors. The CRAC motifs in
receptor are shown. The numbers corresponding to the starting amino acid position