Cell surface delivery and structural re-organization by pharmacological chaperones of an oligomerization-defective alpha(1b)-adrenoceptor mutant demonstrates membrane targeting of GPCR oligomers.

Abstract

Many G-protein-coupled receptors, including the alpha(1b)-adrenoceptor, form homo-dimers or oligomers. Mutation of hydrophobic residues in transmembrane domains I and IV alters the organization of the alpha(1b)-adrenoceptor oligomer, with transmembrane domain IV playing a critical role. These mutations also result in endoplasmic reticulum trapping of the receptor. Following stable expression of this alpha(1b)-adrenoceptor mutant, cell surface delivery, receptor function and structural organization were recovered by treatment with a range of alpha(1b)-adrenoceptor antagonists that acted at the level of the endoplasmic reticulum. This was accompanied by maturation of the mutant receptor to a terminally N-glycosylated form, and only this mature form was trafficked to the cell surface. Co-expression of the mutant receptor with an otherwise wild-type form of the alpha(1b)-adrenoceptor that is unable to bind ligands resulted in this wild-type variant also being retained in the endoplasmic reticulum. Ligand-induced cell surface delivery of the mutant alpha(1b)-adrenoceptor now allowed co-recovery to the plasma membrane of the ligand-binding-deficient mutant. These results demonstrate that interactions between alpha(1b)-adrenoceptor monomers occur at an early stage in protein synthesis, that ligands of the alpha(1b)-adrenoceptor can act as pharmacological chaperones to allow a structurally compromised form of the receptor to pass cellular quality control, that the mutated receptor is not inherently deficient in function and that an oligomeric assembly of ligand-binding-competent and -incompetent forms of the alpha(1b)-adrenoceptor can be trafficked to the cell surface by binding of a ligand to only one component of the receptor oligomer.

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Biochem. J. (2009) 417, 161–172 (Printed in Great Britain) doi:10.1042/BJ20081227
161
Cell surface delivery and structural re-organization by pharmacological
chaperones of an oligomerization-defective α1b-adrenoceptor mutant
demonstrates membrane targeting of GPCR oligomers
Meritxell CANALS1, Juan F. LOPEZ-GIMENEZ1and Graeme MILLIGAN2
Molecular Pharmacology Group, Neuroscience and Molecular Pharmacology, Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
Many G-protein-coupled receptors, including the α1b-adrenocep-
tor, form homo-dimers or oligomers. Mutation of hydrophobic
residues in transmembrane domains I and IV alters the organiz-
ation of the α1b-adrenoceptor oligomer, with transmembrane do-
main IV playing a critical role. These mutations also result in
endoplasmic reticulum trapping of the receptor. Following stable
expression of this α1b-adrenoceptor mutant, cell surface delivery,
receptor function and structural organization were recovered by
treatment with a range of α1b-adrenoceptor antagonists that acted
at the level of the endoplasmic reticulum. This was accompanied
by maturation of the mutant receptor to a terminally N-glycosyl-
ated form, and only this mature form was trafficked to the cell
surface. Co-expression of the mutant receptor with an otherwise
wild-type form of the α1b-adrenoceptor that is unable to bind
ligands resulted in this wild-type variant also being retained in
the endoplasmic reticulum. Ligand-induced cell surface delivery
of the mutant α1b-adrenoceptor now allowed co-recovery to the
plasma membrane of the ligand-binding-deficient mutant. These
results demonstrate that interactions between α1b-adrenoceptor
monomersoccuratanearlystageinproteinsynthesis,thatligands
of the α1b-adrenoceptor can act as pharmacological chaperones
to allow a structurally compromised form of the receptor to
pass cellular quality control, that the mutated receptor is not
inherently deficient in function and that an oligomeric assembly
of ligand-binding-competent and -incompetent forms of the α1b-
adrenoceptor can be trafficked to the cell surface by binding of a
ligand to only one component of the receptor oligomer.
Key words: endoplasmic reticulum retention, G-protein-coupled
receptor (GPCR), glycosylation, pharmacological chaperone.
INTRODUCTION
It is now widely accepted that many GPCRs (G-protein-coupled
receptors) can form dimers and/or higher order oligomers [1,2].
The importance of this for function is currently an area of intense
investigation, with suggested roles for dimerization/oligomeriz-
ation including both providing the most appropriate footprint
for G-protein binding and enhancing the effectiveness of signal
transduction [3]. A further area in which GPCR dimerization/
oligomerization may play a key role is in the processes involved
inreceptorfolding,maturationandeffectivetransfertotheplasma
membrane [4,5]. In this scenario, protein–protein interactions are
likely to be initiated early in the biosynthetic process. Evidence
in favour of this model includes the well appreciated ‘dominant-
negative’ effects of either naturally occurring or designed, trunc-
ated or otherwise mutated GPCRs in restricting cell surface
delivery of co-expressed, wild-type forms of the receptor [6,7].
Furthermore,inthecaseofthemostwidelystudiedGPCRhetero-
dimer, the functional GABA (γ-aminobutyric acid) b receptor
[8] is only generated and delivered to the cell surface when
the GABAbR1 polypeptide, that contains an ER (endoplasmic
reticulum)-retention motif within the intracellular C-terminal tail,
interacts with the GABAbR2 polypeptide which acts to mask this
retention sequence [9].
A number of human diseases are associated with mutations
in GPCRs that result in intracellular retention of the receptor
due to a lack of effective folding and receptor maturation. These
include nephrogenic diabetes insipidus, in which a wide range of
mutations in the V2 vasopressin receptor result in this condition
[10], as well as cases of retinitis pigmentosa (mutants of rhodop-
sin) [11] and hypergonadotrophic hypergonadism (mutants of the
gonadotrophin-releasing hormone receptor) [12]. In recent times,
so-called ‘pharmacological chaperones’, cell-permeant ligands
with affinity for the GPCR in question, have been used to recover
a number of such receptor point mutants to the cell surface,
with the hope and expectation that this might be sufficient to
allow the receptor to function normally and respond to endo-
genously produced agonist ligands [13–17].
Certain GPCR mutants that appear to be compromised in
structural organization or dimerization also display improper
cell surface delivery [18,19]. It is thus conceptually possible
that pharmacological chaperones could rescue the cell surface
delivery and function of dimerization/oligomerization-impaired
receptor mutants. This hypothesis has not, however, previously
been tested, but could be employed to assess if GPCRs are
delivered to the surface of cells as pre-formed dimers/oligomers.
In recent studies on transmembrane elements important for
efficient dimerization/oligomerization of the α1b-adrenoceptor,
we generated a variant containing point mutations in both TMI
(transmembrane domain I) and TMIV (transmembrane domain
IV) that had a defect in structural organization, consistent with
altered oligomerization, as monitored by the use of single
cell sequential 3-colour FRET (fluorescence resonance energy
transfer) imaging [18]. We went on to show that the mutations
Abbreviations used: α1b-adrenoceptor TMI-TMIV, a form of the hamster α1b-adrenoceptor containing point mutations (L1.52A, V1.53A) in transmembrane
domain I and (L4.46A, L4.47A) in transmembrane domain IV; BFA, brefeldin A; DMEM, Dulbecco’s modified Eagle’s medium; eCFP, enhanced cyan
fluorescent protein; EndoH, endoglycosidase H; ER, endoplasmic reticulum; eYFP, enhanced yellow fluorescent protein; FRET, fluorescence resonance
energy transfer; GABA, γ-aminobutyric acid; GPCR, G-protein-coupled receptor; [35S]GTP[S], [35S]guanosine 5?-[γ-thio]triphosphate; HEK cell, human
embryonic kidney cell; NGaseF, N-glycosidase F; TM, transmembrane domain.
1These authors contributed equally to this work.
2To whom correspondence should be addressed (email g.milligan@bio.gla.ac.uk).
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M. Canals, J. F. Lopez-Gimenez and G. Milligan
in TMIV (L166A, L167A: L4.46A, L4.47A in the Ballesteros
and Weinstein nomenclature) [20] were principally responsible
for this effect.
Following stable expression of this mutant in HEK (human
embryonic kidney)-293 cells, we now demonstrate that it is
trappedintheER/Golgiasanimmatureproteinandthattreatment
of these cells with ligands with affinity for the α1b-adrenoceptor
results in maturation of the mutant to a form that is able to pass
cellularqualitycontrolandreachthecellsurface,andwhichFRET
studies suggest has structural organization akin to the wild-type
α1b-adrenoceptor. These ligands hence function as ‘pharmaco-
logicalchaperones’[14–17]forthemutantreceptor.Furthermore,
this mutant acts as a selective ‘dominant-negative’, preventing
cell surface delivery of co-expressed wild-type forms of the
α1b-adrenoceptor. Because a pharmacological chaperone ligand
allowstheco-traffickingofthemutantthatlackscorrectstructural
organization in the absence of the ligand and a co-expressed form
ofthewild-typeα1b-adrenoceptorthatisunabletobindtheligand,
these data demonstrate that the α1b-adrenoceptor traffics to the
cell surface as a dimeric/oligomeric complex [4] and suggest
that effective dimerization/oligomerization may be integral to the
production of the functional cell surface α1b-adrenoceptor.
EXPERIMENTAL
Materials
AllmaterialsfortissueculturewerefromInvitrogen.[3H]Prazosin
was from Perkin Elmer Life Sciences. All drugs used were from
Sigma–Aldrich.
Constructs, cell culture and transfection
Wild-type and mutant (with L1.52A, V1.53A and L4.46A,
L4.47A mutations; α1b-adrenoceptor TMI-TMIV) forms of the
α1b-adrenoceptor fused to eYFP (enhanced yellow fluorescent
protein) were described previously [18]. HEK-293 cells were
transfected using Effectene (Qiagen) transfection reagent and
subsequently selected for resistance to geneticin. The Myc-
D125A (D3.32A) α1b-adrenoceptor-Y67C-eYFP mutant was
generated using the QuikChange®site-directed mutagenesis kit
from Stratagene.
Subcellular compartments: staining and immunocytochemistry
Cells on poly-D-lysine-coated coverslips were rinsed twice with
PBS. Cell nuclei were stained by incubating the cells for 20 min
at 37◦C with fresh PBS containing the nuclear DNA-binding dye
Hoechst 33342 (Molecular Probes) (10 μg/ml). Cells were then
washed 3–4 times with PBS, fixed in 4% formaldehyde/PBS for
10 min and washed three times with ice-cold PBS prior to the
blocking step performed with 3% (w/v) non-fat dried skimmed
milkpowderinPBSfor10 min.Cellswereincubatedwitharabbit
anti-FLAGpolyclonalantibody(1:100,Sigma–Aldrich)for1 hat
room temperature (22◦C) and then washed twice with PBS. Cells
were then incubated for 1 h with an Alexa594-conjugated anti-
rabbit antibody (1:400) (Molecular Probes). After washing with
PBS, coverslips mounted on to glass slides were viewed using
an epifluorescence microscope. For ER staining, after nuclear
labelling cells were rinsed twice with PBS and incubated at 37◦C
for a further 30 min with an ER-Tracker Red dye (Molecular
Probes).
Cell surface receptor measurement: ELISA
Cells were grown in 96-well poly-D-lysine-coated plates and
treated as described in the Figure legends. Cell surface receptors
were labelled with anti-FLAG antibody (1:1000) in growth
mediumfor30 minat30◦C.Thecellswerethenwashedoncewith
20 mM Hepes/DMEM (Dulbecco’s modified Eagle’s medium)
and then incubated for 30 min at 37◦C in growth medium sup-
plemented with horseradish peroxidase-conjugated anti-rabbit
IgG as secondary antibody and 1 μM Hoechst nuclear stain. The
cells were then washed twice with PBS and Hoechst fluorescence
was measured. Finally, the cells were incubated with SureBlue
(KPL, Gaithersburg, MD, U.S.A.) for 5 min in the dark at room
temperature and then the absorbance was read at 620 nm using a
Victor2plate reader (PerkinElmer).
Cell lysates
Cell lysates were obtained by harvesting the cells with ice-cold
RIPA buffer [50 mM Hepes, 150 mM NaCl, 1% Triton X-100,
and 0.5% sodium deoxycholate supplemented with 10 mM NaF,
5 mM EDTA, 10 mM NaH2PO4, 5% ethylene glycol and a
protease inhibitor cocktail (Complete; Roche Diagnostics,
Mannheim,Germany),pH 7.4].Cellularextractswerethencentri-
fuged for 30 min at 14000 g and the supernatant was recovered.
Cell surface biotinylation experiments
For cell surface biotinylation, cells were grown in 6-well plates
coated with poly-D-lysine and treated as stated. Confluent cells
were washed with ice-cold borate buffer (10 mM boric acid,
154 mM NaCl, 7.2 mM KCl and 1.8 mM CaCl2, pH 9.0) and
incubated on ice with 1 ml of 0.8 mM EZ-Link Sulfo-NHS-SS-
Biotin (Pierce) in borate buffer for 15 min. The cells were then
rinsed with a 0.192 M glycine and 25 mM Tris/HCl (pH 8.3)
solution to quench the excess of biotin and then lysed with RIPA
buffer. Lysates were centrifuged for 30 min at 14000 g and the
supernatantwasrecovered.Analiquotofthelysateswassavedfor
Western blotting. Biotinylated cell surface proteins were isolated
using 100 μl of immunopure immobilized streptavidin (Pierce).
After 1 h incubation at 4◦C with constant rotation, samples were
centrifuged and the streptavidin beads were washed 3 times with
RIPA buffer. Finally the biotinylated proteins were eluted
with 100 μl of SDS sample buffer for 1 h at 37◦C.
Cell treatments
Deglycosylation treatments were carried out using NGaseF
(N-glycosidase F) or EndoH (endoglycosidase H) (Roche
Diagnostics) for an overnight period at final concentrations of
1 unit/μl or 100 m-units/ml respectively. Tunicamycin and BFA
(brefeldin A) treatments were performed by overnight incubation
with 12 μM or 5 μg/ml respectively.
[Ca2+]iratio imaging and [Ca2+]imobilization assays
HEK-293 cells stably expressing FLAG-α1b-adrenoceptor-eYFP
or FLAG-α1b-adrenoceptor TMI-TMIV-eYFP were plated on to
coverslips where they grew for 24 h prior to experimentation.
Cells were loaded with the Ca2+-sensitive dye Fura-2 AM
(1.5 μM), by incubation (30 min; 37◦C) under reduced light
in DMEM growth medium. Calcium ratio imaging and image
analysis were then performed as described previously [18].
For FlexStation (Molecular Devices, Sunnydale, CA, U.S.A.)
experiments, cells were grown in 96-well plates and, 24 h after
seeding, cells were loaded with the calcium-sensitive dye Fura-2
AM as above.
Membrane preparations
Cells were collected by centrifugation (1700 g, 5 min, 4◦C),
frozen at −80◦C for at least 1 h and resuspended in 10 mM
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TMIV of α1bAR in dimerization and function
163
Tris/HCl and 0.1 mM EDTA, pH 7.4 buffer. Cell suspensions
were homogenized using an Ultra Turrax for 3×20 s. The
homogenate was centrifuged at 1700 g for 10 min, and the super-
natant collected and centrifuged at 48000 g for 45 min at
4◦C. The resulting pellet was resuspended in buffer and stored
at −80◦C.
[3H]Prazosin binding studies
Binding assays were initiated by the addition of 15–20 μg of
cell membranes to an assay buffer (50 mM Tris/HCl, 100 mM
NaCl and 3 mM MgCl2, pH 7.4) containing [3H]prazosin (0.02–
1 nM in saturation assays and 0.4 nM for competition assays) in
the absence or presence of increasing concentrations of phenyl-
ephrine. Non-specific binding was determined in the presence
of 100 μM phentolamine. Reactions were incubated for 60 min
at 30◦C, and bound ligand was separated from free by vacuum
filtration through GF/B filters (Semat, St. Albans, Hertfordshire,
U.K.).Thefilterswerewashedtwicewithassaybuffer,andbound
ligand was estimated by liquid scintillation spectrometry.
[35S]GTP[S] ([35S]guanosine 5?-[γ-thio]triphosphate)
binding studies
[35S]GTP[S] bindingexperiments
addition of membranes to an assay buffer {20 mM Hepes
(pH 7.4), 3 mM MgCl2, 100 mM NaCl, 1 μM GDP, 0.2 mM
ascorbic acid and 100 nCi of [35S]GTP[S]} containing the
indicated concentrations of receptor ligands. Reactions were
incubated for 15 min at 30◦C and terminated by the addition of
0.5 mlofice-coldbuffercontaining20 mMHepes(pH 7.4),3 mM
MgCl2, 100 mM NaCl and 0.2 mM ascorbic acid. The samples
were centrifuged at 16000 g for 15 min at 4◦C, and the resulting
pellets resuspended in solubilization buffer (100 mM Tris/HCl,
pH 7.4, 200 mM NaCl, 1 mM EDTA and 1.25% Nonidet P-
40) plus 0.2% SDS. Samples were pre-cleared with Pansorbin
(Calbiochem) followed by immunoprecipitation with an anti-Gq/
G11antiserum [21]. Finally, the immunocomplexes were washed
twice with solubilization buffer, and bound [35S]GTP[S] was
measured.
wereinitiated bythe
Sequential 3-colour FRET studies
Sequential3-colourFRETstudieswereperformedasdescribedby
Lopez-Gimenezetal.[18]followingtransientco-expressionofC-
terminally eCFP (enhanced cyan fluorescent protein), eYFP and
dsRed2-tagged and N-terminally FLAG-tagged forms of either
the wild-type or mutant α1b-adrenoceptors in HEK-293 cells.
RESULTS
Cellular location of an α1b-adrenoceptor TMI-TMIV mutant and its
plasma membrane delivery by a pharmacological chaperone
We have recently described a form of the α1b-adrenoceptor
containing mutations in both TMI (L1.52A, V1.53A) and TMIV
(L4.46A, L4.47A) (α1b-adrenoceptor TMI-TMIV) that, based on
FRET data, result in alteration of its quaternary structure and de-
fective receptor maturation and delivery to the plasma membrane
following transient transfection [18]. To investigate this further,
westablyexpressedN-terminallyFLAG-taggedandC-terminally
eYFP-tagged forms of wild-type α1b-adrenoceptor (Figures 1A1
and 1B1) and the TMI-TMIV mutant (Figures 1A2and 1B2) in
HEK-293 cells. Immunocytochemistry with anti-FLAG in fixed
non-permeabilized cells confirmed cell surface delivery of a pro-
portionofwild-typeFLAG-α1b-adrenoceptor-eYFP(Figure1A1).
In contrast, no cell surface FLAG-α1b-adrenoceptor TMI-TMIV-
eYFP could be detected (Figure 1A2). In the same cells, visualiz-
ation of the eYFP linked to each form of the receptor showed
that FLAG-α1b-adrenoceptor TMI-TMIV-eYFP was distributed
throughout delineated intracellular compartments (Figures 1A2
and 1B2), whereas FLAG-α1b-adrenoceptor-eYFP was located at
both the plasma membrane and in intracellular locations (Fig-
ures 1A1 and 1B1). Plasma membrane delivery of wild-type
FLAG-α1b-adrenoceptor-eYFP was confirmed further by the
strong overlap at the cell surface of anti-FLAG-immunoreactivity
and eYFP fluorescence in non-permeabilized cells (Figure 1A1).
MergingoftheeYFPsignalincellsstainedwitharedER-defining
dye suggested that FLAG-α1b-adrenoceptor TMI-TMIV-eYFP
was mainly retained in this compartment (Figure 1B2), whereas
thiswasnotthecaseforwild-typeFLAG-α1b-adrenoceptor-eYFP
(Figure 1B1).
Mutationsresultinginintracellularretention,aswellastheabil-
ity of some chemicals and/or specific ligands to recover cell sur-
faceexpressionofsuchmutants,havebeendescribedforanumber
ofGPCRs[13–17].Therefore,inordertoevaluatewhetherFLAG-
α1b-adrenoceptor TMI-TMIV-eYFP could be trafficked to the cell
surface by pharmacological means, we tested several compounds
with affinity at the α1b-adrenoceptor, using a fixed concentration
of 10−5M of each in the initial screens. Intact cell anti-FLAG
ELISAexperimentsindicatedthatantagonistswithhigheraffinity
for the α1b-adrenoceptor were the most effective in increasing cell
surface FLAG-α1b-adrenoceptor TMI-TMIV-eYFP (Figure 2A).
For example, although the two 5-hydroxytryptamine-receptor-
selective blockers tested, ketanserin and ritanserin, are known
to display some affinity for the α1b-adrenoceptor, only ketanserin
caused significant cell surface delivery of the mutant receptor,
consistent with its higher affinity at the α1b-adrenoceptor
(15 nM compared with 190 nM respectively). Because prazosin,
along with ketanserin, was the most effective ligand tested in act-
ing as a pharmacological chaperone for FLAG-α1b-adrenoceptor
TMI-TMIV-eYFP (Figure 2A), this drug was selected for more
detailed studies. Treatment of cells for different periods of time
with prazosin revealed that the maximum effect was achieved
after14–16 hincubation(resultsnotshown).Followingthisinitial
characterization, we evaluated the effect of different concen-
trations of prazosin (Figure 2B). Prazosin increased cell surface
FLAG-α1b-adrenoceptor TMI-TMIV-eYFP levels in a concen-
tration-dependent manner, with the EC50in the nanomolar range
and a maximum effect at 10−7M (Figure 2B). Although this con-
centration is more than 100-fold higher than the Kdfor prazosin
at the α1b-adrenoceptor measured in broken cell [3H]prazosin-
binding studies, prazosin is not a highly membrane-permeant
ligand, and the effective concentration reaching the receptor
within the ER is likely to be much lower. Visualization of eYFP
and anti-FLAG staining of cells, as in Figure 1, confirmed that
treatmentwithprazosinresultedincellsurfacedeliveryofFLAG-
α1b-adrenoceptor TMI-TMIV-eYFP (Figure 2C) without altering
the cellular distribution of wild-type FLAG-α1b-adrenoceptor-
eYFP (Figure 2C).
Only the mature, terminally N-glycosylated form of the
α1b-adrenoceptor is delivered to the cell surface
Many GPCRs are produced as immature forms that require
final terminal glycosylation prior to effective plasma membrane
delivery [22,23]. Given the inability of FLAG-α1b-adrenoceptor
TMI-TMIV-eYFP to reach the cell surface, we examined the
potential existence of differences in the glycosylation pattern
between the wild-type and the TMI-TMIV mutant α1b-adreno-
ceptor constructs. SDS/PAGE and anti-FLAG immunoblotting of
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M. Canals, J. F. Lopez-Gimenez and G. Milligan
Figure 1 ER retention of FLAG-α1b-adrenoceptor TMI-TMIV-eYFP
Anti-FLAG immunocytochemistry (FLAG, A1, A2, red) or ER labelling (ER, B1, B2, red) of HEK-293 cells stably expressing either wild-type FLAG-α1b-adrenoceptor-eYFP (A1, B1) or
FLAG-α1b-adrenoceptor TMI-TMIV-eYFP (A2, B2). Anti-FLAG staining was performed on non-permeabilized cells and where detected represents receptor at the cell surface. This was only
observed in cells expressing wild-type FLAG-α1b-adrenoceptor-eYFP (A1), whilst parallel imaging of eYFP fluorescence (eYFP, A1, B1, A2,B2, green) confirmed cellular expression of both forms
of the receptor. Merging (Merge) of red and green images confirmed cell surface delivery only of FLAG-α1b-adrenoceptor-eYFP (Merge, A1, yellow) or merging of red and green images indicated
co-localization of FLAG-α1b-adrenoceptor TMI-TMIV-eYFP with the ER marker (Merge, B2, yellow). Blue represents nuclear staining with the DNA-binding dye Hoechst 33342. The scale bar in B2
corresponds to 100μm.
lysates of cells expressing FLAG-α1b-adrenoceptor-eYFP indi-
cated this construct could be detected as three predominant
polypeptides of apparent masses corresponding to 75, 105 and
160 kDa (Figure 3A, upper panel). Two further polypeptides
identified by the anti-FLAG antibodies were unrelated to the
FLAG-α1b-adrenoceptor-eYFP polypeptide, as these were also
detected in lysates of parental, non-transfected HEK-293 cells
(Figure 3A). By contrast with FLAG-α1b-adrenoceptor-eYFP, the
FLAG-α1b-adrenoceptor TMI-TMIV-eYFP mutant was present
only as the 75 and 160 kDa forms (Figure 3A). We have prev-
iously demonstrated that, in transiently transfected cells, the
105 kDa form of FLAG-α1b-adrenoceptor-eYFP corresponds to
the terminally N-glycosylated form of the receptor [18]. We
confirmed that this was also the case in HEK-293 cells stably
expressing FLAG-α1b-adrenoceptor-eYFP, because treatment of
lysates from these cells with NGaseF resulted in loss of the
105 kDa band (Figure 3B) and its replacement by the form with
an apparent mass of 75 kDa (Figure 3B), whereas treatment
with EndoH did not alter the mobility of the 105 kDa band
(resultsnotshown).Furthermore,cellsurfacebiotinylationassays
demonstrated that the terminally N-glycosylated form of FLAG-
α1b-adrenoceptor-eYFP was the only form present at the cell
surface (Figure 3A, lower panel). Treatment of cells expressing
FLAG-α1b-adrenoceptor-eYFP with prazosin (10−7M, 16 h) had
little effect on the glycoslyation pattern (Figure 3A, upper
panel)orlevelsofcellsurfaceFLAG-α1b-adrenoceptor-eYFPthat
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TMIV of α1bAR in dimerization and function
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Figure 2 The α1-adrenoceptor antagonist prazosin promotes cell surface delivery of FLAG-α1b-adrenoceptor TMI-TMIV-eYFP
(A)Intactcellanti-FLAGELISAscreeningoftheeffectofseveraldrugsknowntohaveaffinityfortheα1b-adrenoceptor.ELISAassayswereperformedoncellsstablyexpressingFLAG-α1b-adrenoceptor
TMI-TMIV-eYFP after an overnight treatment with 10−5M of each drug. Basal (−) staining reflects non-specific labelling of the cells. Statistics were performed using a one-way ANOVA with
the application of the Tukey post-test analysis:∗significantly greater than basal, +, significantly lower than the effect of prazosin, P <0.01 in each case. (B) Prazosin increases cell surface
FLAG-α1b-adrenoceptorTMI-TMIV-eYFPinaconcentration-dependentmanner.Intactcell,anti-FLAGELISAassayswereperformedasin(A)followingovernighttreatmentwithvaryingconcentrations
of prazosin. Results are expressed as mean of the fold increase of the absorbance detected in untreated cells (+
Figure 1 in fixed, non-permeabilized cells expressing either FLAG-α1b-adrenoceptor-eYFP (upper images) or FLAG-α1b-adrenoceptor TMI-TMIV-eYFP (lower images) that were treated overnight
with 10−7M prazosin. Imaging of eYFP (green) confirmed expression of both constructs and merging of the images (Merge) confirmed that prazosin treatment promoted cell surface delivery of
FLAG-α1b-adrenoceptor TMI-TMIV-eYFP (lower panels, right-hand image). Blue staining represents nuclear staining with the DNA-binding dye Hoechst 33342.
−S.E.M.). (C) Anti-FLAG immunocytochemistry (α-FLAG, red) was performed as in
could be detected via biotinylation (Figure 3A, lower panel).
By contrast, equivalent treatment of cells expressing FLAG-
α1b-adrenoceptor TMI-TMIV-eYFP with prazosin resulted in the
appearance of the 105 kDa form (Figure 3A, upper panel), its
detection as a cell surface biotinylated polypeptide (Figure 3A,
lower panel) and the capacity of NGaseF to modify the 105kDa
polypeptide form of FLAG-α1b-adrenoceptor TMI-TMIV-eYFP
such that it now migrated as the 75 kDa species (Figure 3B).
Intactcell[3H]prazosinbindingstudiesofcellsexpressingFLAG-
α1b-adrenoceptorTMI-TMIV-eYFP,pre-treatedwithprazosinand
then extensively washed, also confirmed marked increases of cell
surface FLAG-α1b-adrenoceptor TMI-TMIV-eYFP from almost
undetectable levels (Figure 3C).
Inordertoassessmorefullythecellularlocationofthepharma-
cological chaperone action of prazosin, cells were pre-treated
with combinations of prazosin and BFA, a toxin that collapses the
Golgi apparatus and therefore blocks the transport of proteins
beyond the ER and on to the cell surface. Co-treatment of
cells expressing FLAG-α1b-adrenoceptor TMI-TMIV-eYFP with
prazosinandBFAresultedinthelossofappearanceofthe105 kDa
band corresponding to the terminally N-glycosylated cell surface
formofthereceptor(Figure4).However,incellsexpressingeither
wild-type or the mutant α1b-adrenoceptor constructs that had
previously been treated with prazosin, BFA treatment resulted in
theappearanceofadistinctbandwithanapparentmassof90 kDa
(Figure 4) that is likely to correspond to a form of the receptor
containingincompletelyprocessedN-linkedoligosaccharides.As
such, prazosin acts at a point prior to the final production of the
terminally N-glycosylated forms of the receptor in the Golgi and
allows this step to occur.
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166
M. Canals, J. F. Lopez-Gimenez and G. Milligan
Figure 3
reaches the cell surface
Only the terminally N-glycosylated form of the α1b-adrenoceptor
(A) Upper panel: cell lysates of parental HEK-293 cells (HEK) and cells stably expressing
eitherFLAG-α1b-adrenoceptor-eYFP(α1bwt)orFLAG-α1b-adrenoceptorTMI-TMIV-eYFP(α1b
TMI-TMIV) that were treated with (+) or without (−) prazosin (10−7M, 12 h) were resolved
by SDS/PAGE and immunoblotted with anti-FLAG. Lower panel: cell surface biotinylation
of the above cells. After treatment with or without prazosin, cell surface α1b-adrenoceptors
were biotinylated and pulled down with streptavidin–agarose beads, resolved by SDS/PAGE
and immunoblotted with anti-FLAG antibody. (B) Mock (−) or NGaseF de-glycosylation (+)
followedbyanti-FLAGimmunoblottingofcelllysatesofFLAG-α1b-adrenoceptor-eYFP(α1bwt)
and FLAG-α1b-adrenoceptor TMI-TMIV-eYFP (α1bTMI-TMIV) cells that were treated with (+)
orwithout(−)prazosin(10−7M,12 h)wasperformedtoidentifymatureandimmatureformsof
the receptor. (C) Intact cell-specific [3H]prazosin binding studies (0.4nM final) were performed
in cells stably expressing FLAG-α1b-adrenoceptor TMI-TMIV-eYFP that had previously been
treated with or without 10−7M prazosin for an overnight period and then extensively washed.
∗∗P <0.001 (Student’s t test).
Functional rescue of the FLAG-α1b-adrenoceptor TMI-TMIV mutant
Our previous studies in transiently transfected HEK-293 cells
demonstrated that the α1b-adrenoceptor TMI-TMIV mutant was
unabletogenerateanintracellularCa2+mobilizationsignal[18].It
was unclear,however,if this reflectedits absencefrom theplasma
membrane or was inherently due to the introduced mutations.
To assess this, we delivered the stably expressed FLAG-
α1b-adrenoceptor TMI-TMIV-eYFP to the cell surface by pre-
treatmentwithprazosin.However,theuseofprazosinasapharm-
acological chaperone for the rescue of FLAG-α1b-adrenoceptor
TMI-TMIV-eYFP to the cell surface implies the occupancy of the
Figure 4
ceptor
BFA treatment prevents full N-glycosylation of the α1b-adreno-
Lysates of cells stably expressing either FLAG-α1b-adrenoceptor-eYFP (α1bwt) or FLAG-α1b-
adrenoceptor TMI-TMIV-eYFP (α1bTMI-TMIV) that were treated with prazosin (PRZ) and/or
Brefeldin A (BFA) as indicated (10−7M and 5 μg/ml for 12h respectively) were resolved by
SDS/PAGE and immunoblotted with anti-FLAG.
receptorbindingsitebythisantagonist.Itwasthereforenecessary
to remove the antagonist completely following its effect as a
pharmacological chaperone to subsequently assess receptor
functionality. We extensively washed the treated cells with fresh
media in the absence of prazosin for at least 12 h. To assess the
effectivenessofprazosinwashout,saturationbindingexperiments
with [3H]prazosin were then performed on membranes from
prazosin-treated and untreated cells expressing either wild-type
or the TMI-TMIV mutant FLAG-α1b-adrenoceptor-eYFP. Such
saturation experiments indicated no differences in terms of Kd
or Bmax for FLAG-α1b-adrenoceptor-eYFP (results not shown),
suggesting that the prazosin used for the pre-treatment had
been completely removed. Following optimization of the
washout conditions, intracellular Ca2+mobilization assays were
performed in cells expressing FLAG-α1b-adrenoceptor-eYFP
or FLAG-α1b-adrenoceptor TMI-TMIV-eYFP with and without
prazosin pre-treatment. In cells that had not been pre-treated with
prazosin, the α1-adrenoceptor agonist phenylephrine caused a
transient elevation of intracellular [Ca2+] only in cells expressing
FLAG-α1b-adrenoceptor-eYFP (Figure 5A). By contrast, in cells
that had been pre-treated with prazosin, phenylephrine now
resulted in elevation of intracellular [Ca2+] in cells expressing
either FLAG-α1b-adrenoceptor-eYFP or FLAG-α1b-adrenoceptor
TMI-TMIV-eYFP (Figure 5B).
Pharmacological characterization of the α1b-adrenoceptor
TMI-TMIV mutant
We recently reported, in transiently transfected cells, that, when
compared with the wild-type α1b-adrenoceptor, FLAG-α1b-ad-
renoceptor TMI-TMIV-eYFP displays an atypical pharmacolo-
gical profile.The mutant displayed
binding affinity for the agonist phenylephrine [18]. In
agreement with the data in transiently transfected cells, in
membrane preparations of HEK-293 cells stably expressing
FLAG-α1b-adrenoceptor TMI-TMIV-eYFP, phenylephrine was
significantly more potent in competing for the binding
of [3H]prazosin than in membranes from cells expressing
FLAG-α1b-adrenoceptor-eYFP (Figure 6A and Table 1). This
higher affinity of phenylephrine for FLAG-α1b-adrenoceptor
TMI-TMIV-eYFPwasreflected
mentation of potency of this agonist in both Gαq/11[35S]GTP[S]
binding and intracellular Ca2+mobilization assays. In both cases
phenylephrine was approx. 10-fold more potent at the FLAG-α1b-
adrenoceptor TMI-TMIV-eYFP mutant than at the FLAG-
α1b-adrenoceptor-eYFP wild-type (Figures 6B and 6C, and
significantlyhigher
in anequivalent aug-
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TMIV of α1bAR in dimerization and function
167
Figure 5
FLAG-α1b-adrenoceptor TMI-TMIV-eYFP is only observed following prazosin
treatment
Agonist-mediated elevation of calcium in cells expressing
Cells stably expressing FLAG-α1b-adrenoceptor-eYFP (?) or FLAG-α1b-adrenoceptor
TMI-TMIV-eYFP (?) were loaded with Fura-2 and stimulated with 10−5M phenylephrine
at the indicated time. (A) Untreated cells and (B) cells pre-treated with prazosin (10−7M,
12 h). Alterations in Fura-2 fluorescence ratios report changes in intracellular Ca2+levels. A
representative of 3 independent experiments is shown.
Table 2). This was despite the intracellular Ca2+mobilization
experiments being performed in intact cells pre-treated with
prazosin to facilitate receptor delivery to the cell surface, whereas
the [35S]GTP[S] binding experiments were performed with total
membranes from untreated cells. Of interest, the [35S]GTP[S]
binding results demonstrate that FLAG-α1b-adrenoceptor TMI-
TMIV-eYFP is able to couple to Gαq/11 G-proteins within
the intracellular compartment(s) where it is retained and
therefore is an immature protein. This is consistent with recent
reports indicating that the β2-adrenoceptor becomes G-protein-
associated prior to membrane delivery [24]. Although not
explored exhaustively, this variation in agonist affinity/potency
was not restricted to phenylephrine. For example, adrenaline
also displayed higher affinity for FLAG-α1b-adrenoceptor TMI-
TMIV-eYFP (pKi=7.2) than for FLAG-α1b-adrenoceptor-eYFP
(pKi=6.3) in [3H]prazosin competition binding experiments.
Toexamineifsuchdifferencesinaffinityandpotencyofphenyl-
ephrine for FLAG-α1b-adrenoceptor TMI-TMIV-eYFP and
FLAG-α1b-adrenoceptor-eYFP corresponded to differences in
Figure 6
and FLAG-α1b-adrenoceptor TMI-TMIV-eYFP
Pharmacological comparisons of FLAG-α1b-adrenoceptor-eYFP
(A) Ligand-binding characteristics of the wild-type and mutant α1b-adrenoceptor. The capacity
ofphenylephrinetocompeteforbindingwith[3H]prazosintoFLAG-α1b-adrenoceptor-eYFP(?)
andFLAG-α1b-adrenoceptorTMI-TMIV-eYFP(?)wasassessed.(B)Stimulationof[35S]GTP[S]
binding by phenylephrine in Gαq/Gα11 immunoprecipitates from membranes of HEK-293
cells stably expressing FLAG-α1b-adrenoceptor-eYFP (?) and FLAG-α1b-adrenoceptor
TMI-TMIV-eYFP (?). (C) Intracellular calcium mobilization induced by phenylephrine
in cells stably expressing FLAG-α1b-adrenoceptor-eYFP (?) and FLAG-α1b-adrenoceptor
TMI-TMIV-eYFP previously treated with 10−7M prazosin (?). Complete removal of prazosin
after the overnight period was assessed by testing the response of the wild-type receptor after
treatment and washout (open circles). All graphs are representative results from at least three
independent experiments.
receptor glycosylation/maturation states, we initially performed
[3H]prazosin/phenylephrine competition binding experiments
usingtotalcellmembranespreparedfromcellsexpressingFLAG-
α1b-adrenoceptor-eYFP that were treated or not with the
glycosylation inhibitor tunicamycin, membranes expressing
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Page 8

168
M. Canals, J. F. Lopez-Gimenez and G. Milligan
Table 1
adrenoceptor TMI-TMIV-eYFP than for the wild-type receptor
Binding affinity of phenylephrine is higher for FLAG-α1b-
Membrane preparations of HEK-293 cells stably expressing either FLAG-α1b-eYFP or
FLAG-α1b-TMI-TMIV-eYFPreceptorsandpre-treatedornotwithprazosinwereusedtoperform
[3H]prazosin/phenylephrine competition binding assays as described in the Experimental
section. Values are means+
Significant differences from FLAG-α1b-eYFP:∗P <0.05;∗∗P <0.001 (Student’s t test)
−S.E.M of the indicated number of independent experiments.
pKi(high)pKi(low)n
FLAG-α1b-eYFP
FLAG-α1b-TMI-TMIV-eYFP
FLAG-α1b-TMI-TMIV-eYFP (prazosin treatment)
7.09+
8.64+
9.09+
−0.52
−0.17∗
−0.42∗
4.88+
6.33+
5.90+
−0.18
−0.17∗∗
−0.20∗∗
6
12
5
Table 2
Ca2+mobilization via FLAG-α1b-adrenoceptor TMI-TMIV-eYFP
Phenylephrine has higher potency for activation of G-proteins and
[35S]GTPγS:membranepreparationsofHEK-293cellsstablyexpressingeitherFLAG-α1b-eYFP
or FLAG-α1b-TMI-TMIV-eYFP receptors were used to perform [35S]GTPγS binding assays as
described in the Experimental section. Ca2+mobilization: HEK-293 cells stably expressing
either FLAG-α1b-eYFP or FLAG-α1b-TMI-TMIV-eYFP receptors untreated or treated with
prazosin (10−7M, 16 h) were used to perform intracellular calcium mobilization assays using a
FlexStation as described in the Experimental section. Values are means+
independent experiments.∗P <0.05 (Student’s t test).
−S.E.M of three
pEC50
n
[35S]GTPγS
FLAG-α1b-eYFP
FLAG-α1b-TMI-TMIV-eYFP
Ca2+mobilization
FLAG-α1b-eYFP
FLAG-α1b-eYFP (prazosin treatment)
FLAG-α1b-TMI-TMIV-eYFP (prazosin treatment)
5.33+
6.45+
−0.13
−0.8∗
−0.13
−0.12
−0.14∗
3
3
7.23+
7.02+
8.45+
6
6
4
FLAG-α1b-adrenoceptor-eYFP that were subjected to NGaseF
treatment, or membranes from cells expressing FLAG-α1b-
adrenoceptor TMI-TMIV-eYFP treated with prazosin. No differ-
encesinphenylephrineaffinitywereobservedwhenthewild-type
FLAG-α1b-adrenoceptor-eYFP was not terminally N-glycosyl-
ated by either inhibiting de novo glycosylation with tunicamycin
or deglycosylating the receptor with NGaseF (results not shown).
Similarly, when FLAG-α1b-adrenoceptor TMI-TMIV-eYFP was
encouraged to mature and become terminally N-glycosylated
by prazosin treatment, no differences in phenylephrine binding
parameters were observed either (Table 1). Together, all these
results lead us to conclude that the atypical pharmacological
profileofthemutantreceptorforphenylephrineandotheragonists
is inherent to the mutations in TMI and TMIV rather than a
consequence of defective maturation of the mutant receptor.
The conformational change induced by prazosin affects the
oligomeric organization of the α1b-adrenoceptor
The TMI and TMIV mutations in the α1b-adrenoceptor result not
only in defective receptor maturation and delivery to the plasma
membrane, but also in alteration of quaternary structure, with
the TMIV mutations being critical for this effect [18]. To assess
if the rescue of cell surface expression produced by treatment
with prazosin was associated with a change in conformation
and/or the oligomeric organization of the receptor, we employed
single cell 3-colour FRET imaging [18]. Forms of the FLAG-
α1b-adrenoceptor TMI-TMIV mutant C-terminally tagged with
each of eCFP, eYFP and dsRed2 were transiently co-expressed
in HEK-293 cells. Sequential eCFP to dsRed2 FRET was then
Figure 7
binding alters the oligomeric structure of the FLAG-α1b-adrenoceptor TMI-
TMIV mutant
Sequential 3-colour FRET imaging demonstrates that prazosin
C-terminally eCFP, eYFP, and dsRed2 forms of FLAG-α1b-adrenoceptor (wt) or FLAG-α1b-
adrenoceptor TMI-TMIV (TM) were co-expressed in HEK-293 cells and eCFP to dsRed2 FRET
was measured as detailed in Lopez-Gimenez et al. [18]. In experiments involving FLAG-α1b-
adrenoceptor TMI-TMIV, controls were provided by replacing the eYFP-tagged construct with
theequivalentconstructtaggedwithY67CeYFP(Y67C)whichisnotfluorescentandhenceacts
asneitheraFRETenergyacceptor(fromeCFP)nordonor(todsRed2).FRETdataarereportedas
normalizedFRET(FRETN)asdescribedbyLopez-Gimenezetal.[18].InthiscontextFRETN=1.0
represents an absence of energy transfer (and is the anticipated value when employing Y67C
eYFP). FRETNvalues greater than 1.0 reflect the occurrence of sequential FRET. In a number
of experiments, cells expressing the eCFP, eYFP and dsRed2 forms of FLAG-α1b-adrenoceptor
TMI-TMIVweretreatedwithprazosin(10−7M,12h)(TM+PRZ)priortomeasurementofFRET
signals.
measured following pre-treatment with prazosin or vehicle.
Controls expressed FLAG-α1b-adrenoceptor TMI-TMIV linked
to Y67C-eYFP, a non-fluorescent variant of eYFP that therefore
can act neither as a FRET acceptor from eCFP nor as a FRET
donor to dsRed2, along with the eCFP and dsRed2 tagged forms
of FLAG-α1b-adrenoceptor TMI-TMIV. As described previously
[18], differences in eCFP to dsRed2 FRET signals between cells
co-expressing the receptor tagged with each of eCFP, eYFP and
dsRed2 and cells co-expressing the receptor tagged with each of
eCFP, Y67C-eYFP and dsRed2 represent the FRET that has been
transferred sequentially from eCFP to eYFP and then to dsRed2,
rather than directly from eCFP to dsRed2, and reports directly on
theoligomericorganizationofthereceptor.ThemutationsinTMI
and TMIV affected the quaternary structural organization of the
α1b-adrenoceptor as shown by a marked decrease in sequential
eCFP to eYFP to dsRed2 FRET signal compared with those
produced following expression of the equivalent forms of the
wild-type α1b-adrenoceptor (Figure 7). However, after treatment
with prazosin of cells expressing the three FRET-competent
forms of the FLAG-α1b-adrenoceptor TMI-TMIV mutant, the
sequentialFRETsignal(i.e.eCFPtodsRed2FRET)wasincreased
(Figure7)andnowwasnotsignificantlylowerthanthesequential
FRET signal for the wild-type receptor. This probably reflects
changes of conformation induced by prazosin which may induce
modifications in orientation of or distances between, the FRET
fluorophores and hence increase the energy transfer. These
results provide a correlation between the effectiveness of cell
surfacedeliveryoftheα1b-adrenoceptorandtheprazosin-induced
recovery of the quaternary structural organization of FLAG-α1b-
adrenoceptor TMI-TMIV.
The α1b-adrenoceptor is delivered to the cell surface as a
dimer/oligomer
The α1b-adrenoceptor TMI-TMIV mutant receptor acts as a
dominant-negative for the wild-type α1b-adrenoceptor by limiting
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TMIV of α1bAR in dimerization and function
169
Figure 8
does not bind [3H]prazosin with significant affinity
Myc-D125Aα1b-adrenoceptor-Y67C-eYFP is well expressed but
Membranes from mock transfected (HEK) HEK-293 cells and cells transfected to express
Myc-α1b-adrenoceptor-Y67C-eYFP (α1bwt) or Myc-D125Aα1b-adrenoceptor-Y67C-eYFP
(α1bD125A)wereemployedinspecific[3H]prazosin(10nM)bindingstudies.Inset:membranes
of these cells were resolved by SDS/PAGE and immunoblotted to detect the Myc epitope.
itscellsurfaceexpressionwhenbothformsareco-expressed[18].
We therefore assessed if the pharmacological chaperone action
of prazosin on α1b-adrenoceptor TMI-TMIV would concurrently
traffic co-expressed, and hence ER-retained, wild-type α1b-
adrenoceptor to the plasma membrane as part of a dimeric/oligo-
meric complex with α1b-adrenoceptor TMI-TMIV. To perform
these studies we generated a form of Myc-α1b-adrenoceptor-
Y67C-eYFP that is unable to bind prazosin due to introduction of
a D125A (D3.32A) mutation into the receptor [25] (Figure 8).
Although unable to bind prazosin with significant affinity,
Myc-D125Aα1b-adrenoceptor-Y67C-eYFP was expressed to
similar levels as Myc-α1b-adrenoceptor-Y67C-eYFP (Figure 8).
Myc-D125Aα1b-adrenoceptor-Y67C-eYFP was delivered to the
surface of HEK-293 cells when expressed alone (Figure 9), but
was trapped intracellularly when co-expressed with FLAG-α1b-
adrenoceptor TMI-TMIV-eYFP (Figure 9). Treatment of cells
co-expressing Myc-D125Aα1b-adrenoceptor-Y67C-eYFP and
FLAG-α1b-adrenoceptorTMI-TMIV-eYFPwithprazosinresulted
in delivery of both FLAG-α1b-adrenoceptor TMI-TMIV-eYFP
andMyc-D125Aα1b-adrenoceptor-Y67C-eYFPtothecellsurface
(Figure 9), despite Myc-D125Aα1b-adrenoceptor-Y67C-eYFP
being unable to bind prazosin. This is consistent with the ligand
allowing cell surface trafficking of a dimer/oligomer containing
copies of both FLAG-α1b-adrenoceptor TMI-TMIV-eYFP and
Myc-D125Aα1b-adrenoceptor-Y67C-eYFP.
DISCUSSION
The results reported herein provide novel evidence on the capa-
city of the α1b-adrenoceptor to dimerize/oligomerize in the ER
and to traffic to the cell surface as a quaternary complex. Support-
ing previous studies in which mutations in TMI and TMIV
of the α1b-adrenoceptor resulted in both alteration of receptor
quaternary structure and intracellular retention [18], we now
demonstrate that the ability of α1-adrenoceptor antagonists to act
as pharmacological chaperones and recover the α1b-adrenoceptor
TMI-TMIV mutant to the cell surface is correlated with the
ability of the ligands to produce conformational changes that
alter the quaternary structure of the mutant receptor to a form
that resembles the wild-type. The binding of such ligands also
promoted the correct series of steps in terminal N-glycosylation
and maturation to allow cell surface delivery of the mutant α1b-
adrenoceptor. However, it remains uncertain if these two effects
(i.e. generation of the correct quaternary structure and receptor
maturation) are linked causally or, if so, which is the initial
event. Synthesis of GPCRs in the ER, as for other proteins, is a
tightly regulated process. This concludes with a correctly folded
polypeptide which, after further maturation steps in the Golgi,
is trafficked to the cell surface. The ER is the main organelle of
quality control over mis-folded unstable proteins and therefore
functions to prevent the transport and delivery of potentially
non-functional units. Protein folding is a highly complex process
that requires interactions of newly synthesized proteins with ER-
residing chaperones. Proteins that fail to undergo correct folding
areeventuallyroutedforproteosomaldegradation.Mutationsthat
affect folding and surface delivery have been described for a
number of GPCRs and pathological states (see [13,17] for re-
views). Small molecule GPCR ligands able to enter cells and
bind to and assist with folding and subsequent plasma membrane
delivery of mutant GPCRs are described as pharmacological
chaperones.Herewealsoreportthatanoligomerization-defective
α1b-adrenoceptor mutant can be trafficked from the ER to the cell
surface by the antagonist prazosin.
MutantGPCRsthatareretainedintheERareoftenabletoactas
dominant-negativesforcellsurfacedeliveryofthewild-typeform
of the same receptor. This could reflect major folding defects that
result in the exposure of inappropriate domains and, hence, cause
non-specific protein aggregation. However, rather than being
severelymis-foldedandhenceunabletobindα1-adrenoceptorlig-
ands, the mutant that we have generated binds antagonist ligands
with ‘wild-type’ affinity and actually binds agonist ligands with
higher affinity than the wild-type α1b-adrenoceptor. Furthermore,
we have previously shown that this mutant does not interfere
with the cell surface trafficking of all co-expressed GPCRs [18],
indicating that the relevant effect is a selective one. This provides
clear evidence that the dominant-negative effect of the mutant
α1b-adrenoceptor on the co-expressed wild-type α1b-adrenoceptor
truly reflects the formation of ER-retained dimers/oligomers. Be-
cause prazosin treatment of cells expressing the mutant receptor
concurrently allowed a co-expressed, otherwise wild-type, α1b-
adrenoceptorthatisunabletobindligandtoreachthecellsurface,
this indicates that the mature α1b-adrenoceptor must traffic to
the cell surface as a dimeric/oligomeric complex. Increased cell
surface expression of the mutant receptor could also potentially
have been a consequence of antagonist-mediated stabilization
of immature forms of the mutant receptor that might saturate
the ER quality control system and hence result in trafficking
to the cell surface. However, the differential N-glycosylation
pattern following prazosin treatment allowed us to eliminate this
possibility. As the inability of a receptor to reach the cell surface
oftenlimitsitsresponsetoagonists,itislogicaltothinkthatonceit
reachestheplasmamembraneitmayrecoveritsfunctionality[26].
In the case of the α1b-adrenoceptor mutant, we demonstrated that
the lack of receptor at the plasma membrane was the cause of the
absence of response to agonist, because when the mutant, which
actually,althoughunexpectedly,bindsagonistligandswithhigher
affinity than the wild-type receptor, was recovered to the cell
surfacebyprazosintreatment,receptorresponsewasalsorestored
and with potency for agonist ligands higher than for the wild-type
receptor.
Although the current data on the α1b-adrenoceptor provide
strong evidence of the dimeric/oligomeric nature of the cell-
surface-deliveredformsofthisGPCR,itremainstobeestablished
if this is universally true. For example, although a number of
studies have previously indicated GPCR dimerization to occur
prior to plasma membrane delivery [4,19,27], a number of other
studies have suggested that this is not inherently required and
that dimerization-deficient GPCRs and GPCR monomers can
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M. Canals, J. F. Lopez-Gimenez and G. Milligan
Figure 9
adrenoceptor-Y67C-eYFP to the cell surface
Prazosin-induced cell surface delivery of FLAG-α1b-adrenoceptor TMI-TMIV-eYFP also recovers co-expressed and ER-trapped Myc-D125Aα1b-
HEK-293 cells were transfected to express Myc-D125Aα1b-adrenoceptor-Y67C-eYFP (α1bD125A, A), FLAG-α1b-adrenoceptor TMI-TMIV-eYFP (α1bTMITMIV, B and C), or both
Myc-D125Aα1b-adrenoceptor-Y67C-eYFP and FLAG-α1b-adrenoceptor TMI-TMIV-eYFP in a 1:3 ratio (D–G). Immunocytochemistry (Epitope) was performed on non-permeabilized cells using
either anti-Myc (A, E and G) or anti-FLAG (B–D and F) antibodies and a secondary antibody conjugated to Alexa594 (red) whilst monitoring eYFP (YFP, green) provided a measure of
FLAG-α1b-adrenoceptorTMI-TMIV-eYFPexpressionanddistribution.NucleiwereidentifiedwithHoechst33342nucleardye(blue).Co-expressionofFLAG-α1b-adrenoceptorTMI-TMIV-eYFPwith
Myc-D125Aα1b-adrenoceptor-Y67C-eYFP resulted in intracellular retention of the D125A version of the receptor (E). After prazosin (PRZ) treatment (10−7M, overnight), both N-terminal epitopes
were detected at the cell surface (F and G).
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TMIV of α1bAR in dimerization and function
171
reach the cell surface [28,29]. It remains possible that there are
variations in the mechanisms of trafficking of different members
of the GPCR superfamily and, if so, it will be important to
understand the general rules that govern this process.
Studies exploring the contribution of TMs to the interactions
that generate GPCR quaternary structure have produced conflict-
ing data (see [2] for review). Indeed, detailed analysis of the
literature provides evidence for contributions of all TMs, apart
from TMIII, in various GPCRs. Furthermore, in a number of
studies contributions of multiple TMs were implicated [30,31].
SuchdatapotentiallyprovidesupportforGPCRsexistingasmore
complex oligomeric structures rather than simple dimers. How-
ever,inrecenttimes,aseriesofstudieshaveconcentratedattention
on the importance of TMIV as a dimeric/oligomeric interface for
rhodopsin family GPCRs [2]. Most importantly for the present
study, Guo et al. [32] replaced residues all along TMIV of the
dopamine D2 receptor with cysteines and then performed cross-
linking experiments in the presence of agonist or inverse agonist
ligands. Differences in the rate of cross-linking of individual sites
were consistent with the binding of antagonist/inverse agonists
(as well as agonists) altering the detailed structure of TMIV and
with the concept of ligand binding being communicated between
protomers across the dimer/oligomer interface [32]. A number of
studies have also attempted to interfere with GPCR dimerization
by targeting TMIV. One strategy has been to employ peptides
corresponding to specific TMs. Wang et al. [33] demonstrated
that a synthetic peptide corresponding to TMIV of the chemokine
CXCR4 receptor reduced FRET signals between co-expressed
energy-transfer competent forms of this receptor and blocked
a number of CXCR4-mediated effects. The contribution of
individual amino acids within transmembrane helices to GPCR
dimerization has, however, been difficult to explore or to gener-
alize. Our selection of amino acids in TMIV and in TMI was
based on the self-association of receptor fragments containing
theseelements[30]andontheinformaticandbiochemicalexperi-
ments of Hernanz-Falcon et al. [28] on the potential interface(s)
of the chemokine CCR5 receptor dimer/oligomer.
In conclusion, by employing a α1b-adrenoceptor mutant that
has altered quaternary structure and is retained in the ER, we
provideclearevidencethateffectivedimerization/oligomerization
is required to allow a GPCR to pass protein quality control in the
ER and demonstrate that pharmacological chaperones can alter
thedefectivequaternaryorganizationofaGPCRmutanttoensure
correctprocessingandcellsurfacedeliveryofaERpre-assembled
dimeric/oligomeric complex of the α1b-adrenoceptor.
FUNDING
ThesestudiesweresupportedbytheMedicalResearchCouncil[grantnumberG9811527]
and the Wellcome Trust [grant number AL 070185 MA].
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Received 13 June 2008/27 August 2008; accepted 3 September 2008
Published as BJ Immediate Publication 3 September 2008, doi:10.1042/BJ20081227
c ?The Authors Journal compilation c ?2009 Biochemical Society
The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/)
which permits unrestricted non-commerical use, distribution and reproduction in any medium, provided the original work is properly cited.
© 2008 The Author(s)