![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Karolinska Institute, Department of Biosciences at NOVUM, Huddinge, Sweden (H.A.); Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut (A.M.D., D.A.K.); and Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden (G.v.H, C.-N.C.)
Received November 5, 2002; accepted May 20, 2003.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
).
Despite the central importance of GPCRs, little is known about their folding
and membrane assembly. With respect to the mechanism of endoplasmic reticulum
(ER) targeting and insertion, members of the GPCR family can be divided in two
groups: proteins that have a cleavable signal sequence at the N terminus and
proteins that do not (Wallin and von
Heijne, 1995). The presence of an N-terminal cleavable signal
sequence is predominantly found among GPCRs whose extracellular N-termini form
stably folded, ligand-binding domains
(Kochl et al., 2002). The
majority (about 90%) of GPCRs do not contain a signal sequence. In this case,
the first TM domain is thought to function as a targeting sequence (reverse
signal anchor sequence) for the protein to initiate the assembly process in
the ER membrane via the signal recognition particle (SRP)-Sec61 pathway
(Friedlander and Blobel,
1985). Translocation of the extracellular N-terminal tail (N-tail)
across the ER membrane may occur after its synthesis, possibly in a
C-to-N-terminal direction (Nilsson et al.,
2000). It has been suggested that the ER targeting and
translocation of opsin can occur either co- or post-translationally, although
in both instances, it requires that the nascent peptide chain be attached to
the ribosome (Kanner et al.,
2002).
The efficiency of N-tail translocation is dependent on several factors. The
presence of positively charged residues and rapid folding of the N-tail
prevent its translocation (von Heijne and
Gavel, 1988; Denzer et al.,
1995). Charged residues flanking the first TM segment
(von Heijne and Gavel, 1988;
Hartmann et al., 1989), and
the hydrophobicity and length of this segment are also important determinants
(Wahlberg and Spiess, 1997).
In some polytopic membrane proteins, downstream TM segments facilitate N-tail
translocation, suggesting that the second TM may serve as an ER targeting
sequence in some cases (Monne et al.,
1999; Nilsson et al.,
2000).
Previous studies indicate that the length of an N-tail is not a limiting
factor for efficient translocation (Denzer
et al., 1995). However, it is conceivable that shorter N-tails may
be translocated across the ER membrane more readily than longer ones when no
signal sequence is present. Statistical analyses indicate that GPCRs without
signal sequences do have considerably shorter N-tails (40 amino acids on
average) than the GPCRs with signal sequences (200 amino acids on average)
(Wallin and von Heijne, 1995).
Nevertheless, there are exceptions to this rule. The human cannabinoid
receptor 1 (CB1) has an N-tail of 116 amino acids and yet lacks a signal
sequence. This prompts the question of whether the long N-tail of CB1 can be
successfully translocated to the luminal side of the ER. This issue must be
examined to understand the biological implication of possessing such a large
N-terminal domain, in terms of the folding mechanism and the physiological
function, of CB1.
CB1 binds to 
9-tetrahydrocannabinol, the major psycho-active
component of marijuana (Matsuda et al.,
1990; Goutopoulos and
Makriyannis, 2002). It is one of the most abundant GPCRs in the
brain and is involved in a wide range of physiological activities
(Breivogel and Childers, 1998).
At the cellular level, the action of CB1 involves coupling with heterotrimeric
G proteins of the Gi/o family. The activation of G proteins induces
a series of signaling events, including a decrease in the level of cAMP
(Howlett, 1998), activation of
inwardly rectifying K+ channels
(Pertwee, 1997), inhibition of
N- and P/Q-type Ca2+ channels
(Mackie and Hille, 1992), and
activation of mitogen-activated protein kinases
(Bouaboula et al., 1995). CB1
is distributed in many areas of the brain
(Tsou et al., 1998). In the
hippocampus, it is localized in a subset of presynaptic axon terminals and
modulates neurotransmitter release (Katona
et al., 1999; Wilson and
Nicoll, 2002). Thus, it seems that the expression and subcellular
localization of CB1 are well regulated in specific types of cells, suggesting
that the process of membrane assembly of CB1 may be an important control point
for the function of the receptor. However, this area has not been
explored.
As the first step of this investigation, we have examined the effect of the long N-tail of CB1 on its membrane assembly. We have monitored membrane insertion, N-linked glycosylation, and surface expression of the receptor using a cell-free in vitro system and cellular expression systems. We find that the long N-tail inhibits the assembly of CB1 in the ER membrane. This may cause the relatively short half-life of the receptor in the cell and leads to a low level of expression at the plasma membrane. The addition of a signal peptide at the N terminus of CB1 or shortening of the long N-tail greatly increases the stability and cell surface expression of the receptor but has no effect on ligand binding.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
DNA Techniques. BamHI and EcoRI sites were
introduced by PCR at the 5' and 3' ends of the human CB1 gene,
respectively. The PCR fragment was cloned between BamHI and
EcoRI sites of the pcDNA3.1 vector. The N-terminal shortened mutants
64CB1, 80CB1, and 89CB1 were made in the same way. The
DNA fragment encoding the signal sequence from bovine preprolactin was
inserted between the HindIII and BamHI sites, which placed
the signal sequence at the N terminus of CB1. Mutations N77A and N83A of CB1
were made using the QuikChange site-directed mutagenesis kit. The c-Myc
epitope was introduced at the N terminus of CB1 or between the signal sequence
and CB1, using complementary oligonucleotides encoding the c-Myc peptide and
inserted at the BamHI site. The BamHI site was altered after
the insertion of c-Myc. DNA constructs based on pcDNA3.1 were used for the
cell-free in vitro study and expression in human embryonic kidney (HEK) 293
cells. The coding region of these DNA constructs was transferred into the
pSFV1 vector between BamHI and SmaI sites for expression in
baby hamster kidney (BHK) cells. PCR primers for the forward and reverse
directions were designed so that the BamHI site was followed by a
ribosomal binding site (GCCACC) and the ATG start codon, and the TAG stop
codon was placed in front of the SmaI site.
Cell-Free in Vitro Expression System. The constructs in pcDNA3.1
were transcribed by T7 RNA polymerase for 1 h at 37°C with the buffer
supplied by the manufacturer, essentially following the protocol described
previously (Monne et al.,
1999). Translation in rabbit reticulocyte lysate in the presence
of dog pancreas microsomes was performed as described previously
(Liljestrom and Garoff, 1991).
Sodium carbonate extraction of the translation mixture in the microsomal
membrane was carried out as described previously
(Monne et al., 1999).
Proteinase K treatment of microsomes was also carried out as described
previously (Nilsson et al.,
2000).
Cellular Expression Systems. Protein synthesis in BHK cells using
the SFV expression system has been described in detail previously
(Liljestrom and Garoff, 1991;
Liljestrom et al., 1991).
Briefly, CB1 constructs under the SP6 promoter in the SFV vector were
linearized for in vitro transcription. The resulting RNA was used to transfect
BHK cells by electroporation. Six hours after electroporation, cells were
prestarved of methionine for 30 min and then labeled with
[35S]methionine, followed by chase in media containing 1 mM
unlabeled methionine. MG132 was used at a final concentration of 20 µM and
added 1.5 h before labeling and during labeling. After pulse chasing, cells
were solubilized in lysate buffer containing 1% nonidet P-40. Anti-c-Myc
antibodies (1 µl) were added to 100 µl of cell lysate to
immunoprecipitate the radiolabeled receptor with 30 µl of protein G
agarose, following the protocol from the manufacturer. Samples were then
resuspended in 50 µl of Endo H buffer (50 mM sodium citrate, pH 5.5, and 1%
SDS) and incubated with Endo H for 16 h at 37°C as described previously
(Andersson et al., 1997). For
PK treatment, BHK cells were suspended in homogenization buffer (10% sucrose,
w/w, 10 mM Tris-HCl pH 7.4, 1 mM EDTA) and passed through a 23-gauge needle 15
times. The homogenates were centrifuged at 5000g for 5 min to remove
nuclei, and the supernatants were divided in aliquots and treated with PK,
PMSF, and Triton X-100 at final concentrations of 8 µg/ml, 0.46 mg/ml, and
13 mg/ml, respectively, for 30 min on ice. Samples were then lysed in nonidet
P-40 at a final concentration of 1%. The lysates were immunoprecipitated and
analyzed by SDS-PAGE. For immunofluorescence microscopy, transfected cells
were fixed with 3% paraformaldehyde 6.5 hours after electroporation and then
processed for indirect immunofluorescence essentially as described previously
(Salminen et al., 1992). For
surface staining, cells were incubated with the monoclonal antibody c-Myc
(diluted 1:200) on ice before fixation, followed by permeabilization with 0.1%
Triton X-100 and then incubated with the FITC-conjugated goat anti-mouse IgG
as the secondary antibody (diluted 1:100). For internal staining, the primary
antibody was diluted 1:1000. To analyze receptor binding, HEK 293T cells were
grown in Dulbecco's modified Eagles' medium plus 10% fetal bovine serum with
high glucose at 37°C in a 5% CO2 humidified incubator.
Partially confluent plates of cultured cells were transiently transfected with
pcDNA3.1 containing the wild-type or modified CB1 using calcium phosphate
precipitation.
Ligand Binding Assay. Membrane preparations of transiently
transfected HEK 293T cells and saturation binding assays to [3H]
CP-55,940 were carried out as described previously
(Chin et al., 1999).
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
).
|
Three possible scenarios explain the lack of glycosylation of CB1: (1) residues Asn77 and Asn83 are not good substrate sites for oligosaccharide transferase; (2) CB1 fails to integrate into the ER membrane; or (3) the N-tail of CB1 is not translocated across the ER membrane. To assess the first possibility, the signal sequence from preprolactin was attached to the N terminus of CB1 (designated ssCB1). For both CB1 and ssCB1, single or double mutations were made in which residues Asn77, Asn83, or both were mutated to Ala. The N-linked glycosylation of these N-tail variants was examined using the in vitro transcription-translation system in the presence of microsomes (Fig. 1B). The migration of the protein bands on SDS-PAGE indicates that with a signal sequence, CB1 was fully glycosylated at both positions Asn77 and Asn83 (Fig. 1B), ruling out the first possibility.
By extracting the unbound, loosely associated proteins from the membrane
using 100 mM sodium carbonate, it was demonstrated that the majority of CB1
and ssCB1 remained associated with membrane fractions
(Fig. 2A), ruling out the
second possibility. To confirm membrane integration and to probe CB1 membrane
topology, microsomes containing synthesized proteins were treated with PK.
Protein portions translocated into the ER lumen are protected from digestion
by the microsomal membrane. Two previously characterized proteins derived from
Escherichia coli leader peptidase, denoted Lep and P2, were used as
the luminal and cytoplasmic markers, respectively
(Nilsson and von Heijne,
1998). A large protected fragment of about 35 kDa was observed by
SDS-PAGE in the ssCB1 sample after the PK treatment
(Fig. 2B, lane 12). The size of
this fragment corresponds to a portion of CB1 from the N terminus to the third
cytoplasmic loop (ranging approximately from amino acid residue 310 to 345) as
depicted in Fig. 2C. The first
and the second cytoplasmic loops of CB1 are relatively short (about 8 and 15
amino acids, respectively) and were presumably inaccessible to PK digestion
under our assay conditions. However, for the wild-type CB1, no protected
fragment was detectable (Fig.
2B, lane 9). This suggested that without a signal sequence, the
long N-tail of CB1, along with the first TM domain, were left on the
cytoplasmic side of the ER membrane during protein synthesis.
|
The Effect of the Long N-Tail of CB1 on Cell Surface Expression,
Stability, and Ligand Binding to the Receptor. The data above show that
the wild-type CB1 did not attain the expected 7TM topology, possibly because
of the difficulty in translocating the long N-tail across the ER membrane.
This finding prompts several questions. Does this situation reflect what
happens in the cell? Can CB1, if incorrectly folded in the ER membrane, be
transported to the cell surface? What properties (e.g., length or
glycosylation) of the N-tail affect the expression of CB1 in a cell? To
address these questions, we characterized the synthesis of CB1 in BHK cells,
transiently expressing the receptor using the Semliki Forest virus (SFV)
expression system (Liljestrom and Garoff,
1991).
To examine the process of maturation of the receptor from the ER compartment to the plasma membrane, cells expressing either the CB1 or ssCB1 genes were metabolically labeled with [35S]methionine and chased for 2 h. A c-Myc tag was added at the N terminus of the receptor for detection by immunoprecipitation and immunofluorescent staining. The sensitivity of the protein to Endo H was used as an indicator to monitor the state of glycosylation and intracellular trafficking of the receptor. Consistent with the data from the cell-free in vitro system, only a small portion of the wild-type CB1 (less than 10%) was glycosylated (Fig. 3A, lanes 1 and 2). However, with the addition of the N-terminal signal sequence, the protein (ssCB1) was 100% glycosylated and a fraction of the molecules became complex-glycosylated (Endo H-resistant) in 2 h (Fig. 3B).
|
Most interestingly, we found that the wild-type CB1 was weakly labeled and rapidly degraded in less than an hour, whereas ssCB1 seemed to be more stable, although certain degrees of protein degradation were observed in both samples (Fig. 3, A and B). For the wild-type CB1, after a 1-hour chase, only about 10% of the 35S-labeled receptor remained detectable, whereas for ssCB1, about 50% remained [data obtained from comparing densitometric measurements of total protein in lane 1 (or 2) and lane 3 (or 4) in Fig. 3, A and B]. The above result suggests that, in BHK cells, the newly synthesized CB1 did not attain the correct conformation because of difficulty in translocating the N-tail and possibly was degraded by proteasomes via the ER quality control pathway. The addition of the N-terminal c-Myc tag is unlikely to account for the rapid degradation and lack of glycosylation of the wild-type CB1, because similar results were obtained using proteins without the c-Myc tag with antibodies against the first 14 amino acids of CB1 (data not shown). To rule out the possibility that the inhibition of CB1 processing is caused by overexpression of protein in SFV system, we performed the same pulse-chase experiments at an earlier time-point after electroporation at which the protein expression level is lower. Results similar to those shown in Fig. 3, A and B, were obtained (data not shown).
To test whether CB1 was degraded by proteasomes, the specific proteasome inhibitor MG132 was added to CB1-expressing cells during protein synthesis. We found that the stability of CB1 was greatly enhanced, which resulted in a protein increase of approximately 6-fold (Fig. 4A, lane 1 and 2), whereas ssCB1 was increased by only 2-fold (Fig. 4A, lanes 3 and 4). As shown in Fig. 4A, the enhanced stability by MG132 did not increase the amount of glycosylated CB1, indicating that the N-tail remained cytosolic even when protein degradation was prevented by MG132.
|
To further confirm membrane topology, cells expressing either the
CB1 or ssCB1 gene were metabolically labeled for 30 min
followed by homogenization. Homogenates (containing inside-out vesicles
derived from the ER) were subjected to PK treatment. In good agreement with
the observation from the cell-free system
(Fig. 2B), a large protected
fragment of about 35 kDa was observed in samples containing ssCB1, but no
protected fragment was observed in samples containing the wild-type CB1
(Fig. 4B). Can the rapid
degradation of CB1 by proteasomes be alleviated by shortening the N-tail? To
answer this question, N-tail deletion mutants 64CB1, 80CB1, and
89CB1, with N-tails of 52, 36, and 27 amino acids, respectively, were
made and characterized. Two, one, and none of the N-linked glycosylation sites
remained in 64CB1, 80CB1, and 89CB1, respectively. The
N-tail modified CB1 mutants were all stable and attained their mature forms
within 2 h (Figs. 3,
C–E). The amount of glycosylation increased as the length of
the N-tail decreased from 45% glycosylation in 64CB1 to 100% in
80CB1 (Fig. 3, C and D).
These results substantiate the relation between the stability of CB1 and the
efficiency of N-tail translocation.
Simultaneously with the pulse-chase labeling experiments, immunofluorescent
staining of receptors expressed at the cell surface was performed using
antiserum against the N-terminal c-Myc epitope. The intensity of cell surface
staining was consistent with the expression patterns seen in
Fig. 3, where strong surface
expression was observed for ssCB1, 64CB1, 80CB1, and
89CB1 (Fig. 5, B, C, D, and
E, respectively). It is worth noting that 89CB1, containing
no N-linked glycosylation site, remained stably expressed at the cell surface,
suggesting that N-linked glycosylation may not be a requirement for transport
of CB1 to the plasma membrane. That little surface staining was detectable for
wild-type CB1 could be caused by its instability, low levels of the receptor
at the plasma membrane, and/or the lack of extracellularly located N-tail
(Fig. 5A). As a control,
internal staining was performed on cells expressing the wild-type CB1, ssCB1,
and 89CB1 (Fig. 6). Weak
staining was observed with cells expressing the wild-type CB1, consistent with
our earlier notion that the protein was quickly degraded in the cell.
|
|
To evaluate whether these modifications at the N-terminal domain affect interactions of CB1 with cannabinoid ligands, saturation binding analysis was performed using membrane preparations from HEK 293 cells transiently expressing CB1. No significant difference was found between CB1 and the mutants in their binding affinities to [3H]CP-55,940, a representative cannabinoid compound (Table 1). Immunofluorescent staining of HEK 293 cells showed that the levels of both surface and intracellularly expressed receptors were much higher for ssCB1 than for CB1, as seen in BHK cells (Fig. 7). The Bmax values in this particular study did not reflect the intensity of immunofluorescent staining in HEK 293 cells, because these two experiments were performed independently, and empirical factors such as transfection efficiency needed to be taken into account. At this stage, we do not know whether cell surface expression is the prerequisite for ligand binding activity, because the membrane preparation we used in this study is derived from whole cell homogenates, which includes intracellular membranes and plasma membrane.
|
|
CB1 is expressed at the cell surface in several recombinant systems, such
as AtT20 cells, Xenopus laevis oocytes and HEK 293 cells
(Hsieh et al., 1999;
Jin et al., 1999;
McAllister et al., 2002), and
is not well expressed in other systems, such as Chinese hamster ovary cells
and COS-7 cells (D. Kendall, unpublished observations). Our data suggest that
CB1, when expressed in BHK cells, is intrinsically unstable; this instability
may be caused by the difficulty in translocating the long N-tail across the ER
membrane, which in turn causes a low level of expression at the cell surface.
The poor surface expression can be improved by adding a signal sequence or
shortening the N-tail.
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
;
Petaja-Repo et al., 2000).
Here, we demonstrate that CB1 follows this general mechanism; in this case,
translocating the N-tail and acquiring the correct topology in the ER membrane
is a key control point for receptor biogenesis. The inefficient processing of
CB1 but not CB2 in the cell-free translation system shown in
Fig. 1 has provided the initial
indication that CB1 may be intrinsically unstable, but CB2 may not. Indeed,
later we found that CB1 was rapidly degraded by proteasomes when expressed in
BHK cells. This result coincides with the observation that CB1 failed to be
expressed as a fusion protein in E. coli because of severe
proteolytic degradation, whereas CB2 was expressed in a functional form at a
high level (Calandra et al.,
1997). The addition of a cleavable signal sequence at the N
terminus enhances the stability and surface expression of the receptor. This
has been also observed for other GPCRs, such as the 
2 adrenergic
receptor and the endothelin B receptor
(Guan et al., 1992;
Kochl et al., 2002). In this
study, we show that the advantage of adding a signal sequence is to facilitate
targeting of the receptor to the ER membrane and to promote N-tail
translocation. Without a signal sequence, which occurs in the 90% of GPCRs,
the insertion of the N-tail may happen after its synthesis; in this case, the
first or the second TM may serve as the ER targeting signal. The topological
model of CB1 in the ER membrane shown in
Fig. 2C suggests a possible
scenario that the second TM directly targeted the receptor to the ER membrane
and initiated partial assembly of the receptor; subsequently, the correct
topology of the first TM and translocation of the N-tail could be achieved. As
in the case of CB1, the latter step was hindered by the long N-tail. Detailed
mechanism of GPCR assembly in the ER membrane is not clear; however, a recent
cross-linking study on the assembly of opsin and neurotensin receptor showed
that the first and the second TMs associate with distinct components of the ER
translocon during the receptor biosynthesis
(Meacock et al., 2002).
The hydrophobic nature of cannabinoid ligands suggests that their binding
site is localized within the 7TM bundle of the receptor
(McAllister et al., 2002). Our
data show that the N-tail of CB1 can be deleted up to 89 amino acids without
affecting the receptor binding to the ligand CP-55,940. This suggests that the
long N-tail of CB1, which accounts for more than 25% of the protein, may not
be relevant to ligand binding or activation of the receptor. This notion is
supported by the existence of an alternatively spliced isoform, CB1A, that has
a shorter N-tail of 55 amino acid and differs from CB1 in the first 28 amino
acids, yet displays pharmacological properties similar to those of CB1
(Gerard et al., 1991;
Rinaldi-Carmona et al., 1996).
In native tissues, the expression of CB1A amounts to about 20% of CB1 at the
mRNA level (Shire et al.,
1995). According to the results presented in this study, one may
speculate that, with a shorter N-tail, CB1A would be more stable during its
synthesis and would be expressed at the cell surface more readily than
CB1.
Cellular factors may be required to facilitate surface expression of CB1.
In hippocampal neurons, strong labeling of CB1 was found in presynaptic
terminal plasma membranes but not in other plasma membranes
(Katona et al., 1999). The
presynaptic localization of CB1 at the plasma membrane is essential for its
quick response as a neuromodulator. Agonist-induced internalization of CB1 has
been found to be an important mechanism to regulate the availability of the
receptor at the plasma membrane (Jin et
al., 1999; Coutts et al.,
2001). Nonetheless, it is reasonable to speculate that the process
of biosynthesis can also be a control point to modulate the expression,
subcellular location, and thus function of CB1. Many specific GPCR-associating
proteins have been identified to play a role as molecular chaperones to
regulate the cell surface expression of GPCRs
(Brady and Limbird, 2002). In
addition, a recent study on the 
-opioid receptor has demonstrated that
biosynthesis of a GPCR can be regulated at the ER level pharmacologically,
where hydrophobic ligands act as chemical chaperones to promote the correct
folding and maturation of the receptor
(Petaja-Repo et al., 2002).
Interestingly, it has been shown that the cell surface expression of CB1 was
increased by adding a CB1-specific inverse agonist, SR141716
(Rinaldi-Carmona et al.,
1998). A chaperone-mediated mechanism may exist that regulates the
synthesis, degradation, folding, and trafficking of CB1 in which the long
N-tail may play a role.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
ABBREVIATIONS: GPCR, G protein-coupled receptor; TM, transmembrane;
ER, endoplasmic reticulum; Endo H, endoglycosidase H; PMSF,
phenylmethylsulfonyl fluoride; PK, proteinase K; MG132,
carbobenzoxy-leucyl-leucyl-leucinal; CP-55,940,
(1
,2)-(R)-5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]phenol;
FITC, fluorescein isothiocyanate; PCR, polymerase chain reaction; HEK, human
embryonic kidney; PAGE, polyacrylamide gel electrophoresis; CB1, cannabinoid
receptor 1; CB2, cannabinoid receptor 2; SFV, Semliki Forest virus; SR141716,
N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide
hydrochloride; BHK, baby hamster kidney; Lep, leader peptidase.
1 Current address: Department of Molecular Biophysics and Biochemistry, Yale
University, New Haven, Connecticut. 
Address correspondence to: Chen-Ni Chin, Yale University, Department of Molecular Biophysics and Biochemistry, P.O. Box 208114, New Haven, CT 06520-8114. E-mail: chin{at}mail.csb.yale.edu
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Bockaert J and Pin JP (1999) Molecular tinkering of G protein-coupled receptors: an evolutionary success. EMBO (Eur Mol Biol Organ) J 18: 1723–1729.[CrossRef][Medline]
Bouaboula M, Poinot-Chazel C, Bourrie B, Canat X, Calandra B, Rinaldi-Carmona M, Le Fur G, and Casellas P (1995) Activation of mitogen-activated protein kinases by stimulation of the central cannabinoid receptor CB1. Biochem J 312: 637–641.
Brady AE and Limbird LE (2002) G protein-coupled receptor interacting proteins: emerging roles in localization and signal transduction. Cell Signal 14: 297–309.[CrossRef][Medline]
Breivogel CS and Childers SR (1998) The functional neuroanatomy of brain cannabinoid receptors. Neurobiol Dis 5: 417–431.[CrossRef][Medline]
Calandra B, Tucker J, Shire D, and Grisshammer R (1997) Expression in Escherichia coli and characterisation of the human central CB1 and peripheral CB2 cannabinoid receptors. Biotech Lett 19: 425–428.[CrossRef]
Chin CN, Murphy JW, Huffman JW, and Kendall DA (1999)
The third transmembrane helix of the cannabinoid receptor plays a role in the
selectivity of aminoalkylindoles for CB2, peripheral cannabinoid receptor.
J Pharmacol Exp Ther
291:
837–844.
Coutts AA, Anavi-Goffer S, Ross RA, MacEwan DJ, Mackie K, Pertwee
RG, and Irving AJ (2001) Agonist-induced internalization and
trafficking of cannabinoid CB1 receptors in hippocampal neurons. J
Neurosci 21:
2425–2433.
Denzer AJ, Nabholz CE, and Spiess M (1995) Transmembrane orientation of signal-anchor proteins is affected by the folding state but not the size of the N-terminal domain. EMBO (Eur Mol Biol Organ) J 14: 6311–6317.[Medline]
Friedlander M and Blobel G (1985) Bovine opsin has more than one signal sequence. Nature (Lond) 318: 338–343.[CrossRef][Medline]
Gerard CM, Mollereau C, Vassart G, and Parmentier M (1991) Molecular cloning of a human cannabinoid receptor which is also expressed in testis. Biochem J 279: 129–134.
Goutopoulos A and Makriyannis A (2002) From cannabis to cannabinergics. New therapeutic opportunities. Pharmacol Ther 95: 103–117.[CrossRef][Medline]
Guan XM, Kobilka TS, and Kobilka BK (1992) Enhancement
of membrane insertion and function in a type IIIb membrane protein following
introduction of a cleavable signal peptide. J Biol
Chem 267:
21995–21998.
Hartmann E, Rapoport TA, and Lodish HF (1989)
Predicting the orientation of eukaryotic membrane-spanning proteins.
Proc Natl Acad Sci USA
86:
5786–5790.
Howlett AC (1998) The CB1 cannabinoid receptor in the brain. Neurobiol Dis 5: 405–416.[CrossRef][Medline]
Hsieh C, Brown S, Derleth C, and Mackie K (1999) Internalization and recycling of the CB1 cannabinoid receptor. J Neurochem 73: 493–501.[CrossRef][Medline]
Jin W, Brown S, Roche JP, Hsieh C, Celver JP, Kovoor A, Chavkin C,
and Mackie K (1999) Distinct domains of the CB1 cannabinoid
receptor mediate desensitization and internalization. J
Neurosci 19:
3773–3780.
Kanner EM, Klein IK, Friedlander M, and Simon SM (2002) The amino terminus of opsin translocates "posttranslationally" as efficiently as cotranslationally. Biochemistry 41: 7707–7715.[CrossRef][Medline]
Katona I, Sperlagh B, Sik A, Kafalvi A, Vizi ES, Mackie K, and
Freund TF (1999) Presynaptically located CB1 cannabinoid
receptors regulate GABA release from axon terminals of specific hippocampal
interneurons. J Neurosci
19:
4544–4558.
Kochl R, Alken M, Rutz C, Krause G, Oksche A, Rosenthal W, and
Schulein R (2002) The signal peptide of the G protein-coupled
human endothelin B receptor is necessary for translocation of the N-terminal
tail across the endoplasmic reticulum membrane. J Biol
Chem 277:
16131–16138.
Kopito RR (1999) Biosynthesis and degradation of CFTR. Physiol Rev 79: S167–S173.
Liljestrom P and Garoff H (1991) A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Biotechnology (NY) 9: 1356–1361.[CrossRef][Medline]
Liljestrom P, Lusa S, Huylebroeck D, and Garoff H
(1991) In vitro mutagenesis of a full-length cDNA clone of
Semliki Forest virus: the small 6,000-molecular-weight membrane protein
modulates virus release. J Virol
65:
4107–4113.
Mackie K and Hille B (1992) Cannabinoids inhibit
N-type calcium channels in neuroblastoma-glioma cells. Proc Natl
Acad Sci USA 89:
3825–3829.
Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, and Bonner TI (1990) Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature (Lond) 346: 561–564.[CrossRef][Medline]
McAllister SD, Tao Q, Barnett-Norris J, Buehner K, Hurst DP, Guarnieri F, Reggio PH, Nowell Harmon KW, Cabral GA, et al. (2002) A critical role for a tyrosine residue in the cannabinoid receptors for ligand recognition. Biochem Pharmacol 63: 2121–2136.[CrossRef][Medline]
Meacock SL, Lecomte FJ, Crawshaw SG, and High S (2002)
Different transmembrane domains associate with distinct endoplasmic reticulum
components during membrane integration of a polytopic protein. Mol
Biol Cell 13:
4114–4129.
Monne M, Gafvelin G, Nilsson R, and von Heijne G (1999) N-tail translocation in a eukaryotic polytopic membrane protein: synergy between neighboring transmembrane segments. Eur J Biochem 263: 264–269.[Medline]
Nilsson I and von Heijne G (1998) Breaking the camel's back: proline-induced turns in a model transmembrane helix. J Mol Biol 284: 1185–1189.[CrossRef][Medline]
Nilsson I, Witt S, Kiefer H, Mingarro I, and von Heijne G
(2000) Distant downstream sequence determinants can control
N-tail translocation during protein insertion into the endoplasmic reticulum
membrane. J Biol Chem
275:
6207–6213.
Pertwee RG (1997) Pharmacology of cannabinoid CB1 and CB2 receptors. Pharmacol Ther 74: 129–180.[CrossRef][Medline]
Petaja-Repo UE, Hogue M, Laperriere A, Walker P, and Bouvier M
(2000) Export from the endoplasmic reticulum represents the
limiting step in the maturation and cell surface expression of the human
opioid receptor. J Biol Chem
275:
13727–13736.
Petaja-Repo UE, Hogue M, Bhalla S, Laperriere A, Morello JP, and Bouvier M (2002) Ligands act as pharmacological chaperones and increase the efficiency of delta opioid receptor maturation. EMBO (Eur Mol Biol Organ) J 21: 1628–1637.[CrossRef][Medline]
Rinaldi-Carmona M, Calandra B, Shire D, Bouaboula M, Oustric D,
Barth F, Casellas P, Ferrara P, and Le Fur G (1996)
Characterization of two cloned human CB1 cannabinoid receptor isoforms.
J Pharmacol Exp Ther
278:
871–878.
Rinaldi-Carmona M, Le Duigou A, Oustric D, Barth F, Bouaboula M,
Carayon P, Casellas P, and Le Fur G (1998) Modulation of CB1
cannabinoid receptor functions after a long-term exposure to agonist or
inverse agonist in the Chinese hamster ovary cell expression system.
J Pharmacol Exp Ther
287:
1038–1047.
Salminen A, Wahlberg JM, Lobigs M, Liljestrom P, and Garoff H
(1992) Membrane fusion process of Semliki Forest virus. II:
Cleavage-dependent reorganization of the spike protein complex controls virus
entry. J Cell Biol 116:
349–357.
Shire D, Carillon C, Kaghad M, Calandra B, Rinaldi-Carmona M, Le
Fur G, Caput D, and Ferrara P (1995) An amino-terminal variant of
the central cannabinoid receptor resulting from alternative splicing.
J Biol Chem 270:
3726–3731.
Therien AG, Grant FE, and Deber CM (2001) Interhelical hydrogen bonds in the CFTR membrane domain. Nat Struct Biol 8: 597–601.[CrossRef][Medline]
Tsou K, Brown S, Sanudo-Pena MC, Mackie K, and Walker JM (1998) Immunohistochemical distribution of cannabinoid CB1 receptors in the rat central nervous system. Neuroscience 83: 393–411.[CrossRef][Medline]
von Heijne G and Gavel Y (1988) Topogenic signals in integral membrane proteins. Eur J Biochem 174: 671–678.[Medline]
Wahlberg JM and Spiess M (1997) Multiple determinants
direct the orientation of signal-anchor proteins: the topogenic role of the
hydrophobic signal domain. J Cell Biol
137:
555–562.
Wallin E and von Heijne G (1995) Properties of
N-terminal tails in G-protein coupled receptors: a statistical study.
Protein Eng 8:
693–698.
Wilson RI and Nicoll RA (2002) Endocannabinoid
signaling in the brain. Science (Wash DC)
296:
678–682
This article has been cited by other articles:
|
|
 |
|
  N. Harada, Y. Yamada, K. Tsukiyama, C. Yamada, Y. Nakamura, E. Mukai, A. Hamasaki, X. Liu, K. Toyoda, Y. Seino, et al. A novel GIP receptor splice variant influences GIP sensitivity of pancreatic {beta}-cells in obese mice Am J Physiol Endocrinol Metab, January 1, 2008; 294(1): E61 - E68. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. M. Conn, A. Ulloa-Aguirre, J. Ito, and J. A. Janovick G Protein-Coupled Receptor Trafficking in Health and Disease: Lessons Learned to Prepare for Therapeutic Mutant Rescue in Vivo Pharmacol. Rev., September 1, 2007; 59(3): 225 - 250. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Dong and G. Wu Regulation of Anterograde Transport of {alpha}2-Adrenergic Receptors by the N Termini at Multiple Intracellular Compartments J. Biol. Chem., December 15, 2006; 281(50): 38543 - 38554. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. J. Sim-Selley, N. S. Schechter, W. K. Rorrer, G. D. Dalton, J. Hernandez, B. R. Martin, and D. E. Selley Prolonged Recovery Rate of CB1 Receptor Adaptation after Cessation of Long-Term Cannabinoid Administration Mol. Pharmacol., September 1, 2006; 70(3): 986 - 996. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Rutz, A. Renner, M. Alken, K. Schulz, M. Beyermann, B. Wiesner, W. Rosenthal, and R. Schulein The Corticotropin-releasing Factor Receptor Type 2a Contains an N-terminal Pseudo Signal Peptide J. Biol. Chem., August 25, 2006; 281(34): 24910 - 24921. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. P. Picone, A. D. Khanolkar, W. Xu, L. A. Ayotte, G. A. Thakur, D. P. Hurst, M. E. Abood, P. H. Reggio, D. J. Fournier, and A. Makriyannis (-)-7'-Isothiocyanato-11-hydroxy-1',1'-dimethylheptylhexahydrocannabinol (AM841), a High-Affinity Electrophilic Ligand, Interacts Covalently with a Cysteine in Helix Six and Activates the CB1 Cannabinoid Receptor Mol. Pharmacol., December 1, 2005; 68(6): 1623 - 1635. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. M. Pietila, J. T. Tuusa, P. M. Apaja, J. T. Aatsinki, A. E. Hakalahti, H. J. Rajaniemi, and U. E. Petaja-Repo Inefficient Maturation of the Rat Luteinizing Hormone Receptor: A PUTATIVE WAY TO REGULATE RECEPTOR NUMBERS AT THE CELL SURFACE J. Biol. Chem., July 15, 2005; 280(28): 26622 - 26629. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. S. Kearn, K. Blake-Palmer, E. Daniel, K. Mackie, and M. Glass Concurrent Stimulation of Cannabinoid CB1 and Dopamine D2 Receptors Enhances Heterodimer Formation: A Mechanism for Receptor Cross-Talk? Mol. Pharmacol., May 1, 2005; 67(5): 1697 - 1704. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Ott, Y. Neldner, R. Cebe, I. Dodevski, and A. Pluckthun Engineering and functional immobilization of opioid receptors Protein Eng. Des. Sel., March 1, 2005; 18(3): 153 - 160. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Hague, M. A. Uberti, Z. Chen, R. A. Hall, and K. P. Minneman Cell Surface Expression of {alpha}1D-Adrenergic Receptors Is Controlled by Heterodimerization with {alpha}1B-Adrenergic Receptors J. Biol. Chem., April 9, 2004; 279(15): 15541 - 15549. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
, nonglycosylated form; 
, glycosylated form;
*, nonspecific band at around 55 kDa, which frequently appeared in reactions
with microsomes. Data shown in A are representative of four independent
experiments. Data shown in B are from one experiment. Mw, molecular mass.
, protein fragments protected from PK
digestion; *, a naturally occurring proteolytic fragment (lanes 11 and 12);
, Endo H-sensitive forms (derived from core-glycosylated receptors);
