In the present study, we aimed to determine the contributions of these lipid-facing residues to recep-tor function and oligomerization by systemically generating chimeric com-plement fac
Trang 1importance of lipid-facing residues in transport
competence
Jeffery M Klco1,*, Saurabh Sen1,*,, Jakob L Hansen2,3,*, Christina Lyngsø2,3,
Gregory V Nikiforovich4,, Soren P Sheikh5and Thomas J Baranski1
1 Departments of Medicine and Molecular Biology & Pharmacology, Washington University School of Medicine, St Louis, MO, USA
2 Laboratory for Molecular Cardiology, Danish National Research Foundation Centre for Cardiac Arrhythmia, The Heart Centre, Copenhagen University Hospital, Denmark
3 Laboratory for Molecular Cardiology, Danish National Research Foundation Centre for Cardiac Arrhythmia, Department of Neuroscience and Pharmacology, University of Copenhagen, Denmark
4 Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO, USA
5 The Laboratory of Molecular and Cellular Cardiology, Department of Biochemistry, Pharmacology and Genetics, University Hospital of Odense, Denmark
Keywords
BRET; C5aR; G protein-coupled receptor;
lipid-facing; transmembrane helix
Correspondence
T J Baranski, Departments of Medicine
and Molecular Biology & Pharmacology,
Washington University School of Medicine,
Campus Box 8127, 660 South Euclid
Avenue, St Louis, MO 63110, USA
Fax: +1 314 362 7641
Tel: +1 314 747 3997
E-mail: baranski@wustl.edu
*These authors contributed equally to this
work
Present addresses
Neurology, CNET, University of Alabama at
Birmingham, AL, USA
MolLife Design LLC, St Louis, MO, USA
(Received 14 January 2009, revised 3 March
2009, accepted 12 March 2009)
doi:10.1111/j.1742-4658.2009.07002.x
Residues that mediate helix–helix interactions within the seven transmem-branes (TM) of G protein-coupled receptors are important for receptor biogenesis and the receptor switch mechanism By contrast, the residues directly contacting the lipid bilayer have only recently garnered attention
as potential receptor dimerization interfaces In the present study, we aimed to determine the contributions of these lipid-facing residues to recep-tor function and oligomerization by systemically generating chimeric com-plement factor 5a receptors in which the entire lipid-exposed surface of a single TM helix was exchanged with the cognate residues from the angio-tensin type 1 receptor Disulfide-trapping and bioluminescence resonance energy transfer (BRET) studies demonstrated robust homodimerization of both complement factor 5a receptor and angiotensin type 1 receptor, but
no evidence for heterodimerization Despite relatively conservative substitu-tions, the lipid-facing chimeras (TM1, TM2, TM4, TM5, TM6 or TM7) were retained in the endoplasmic reticulum⁄ cis-Golgi network With the exception of the TM7 chimera that did not bind ligand, the lipid-facing chimeras bound ligand with low affinity, but similar to wild-type comple-ment factor 5a receptors trapped in the endoplasmic reticulum with brefel-din A These results suggest that the chimeric receptors were properly folded; moreover, native complement factor 5a receptors are not fully com-petent to bind ligand when present in the endoplasmic reticulum BRET oligomerization studies demonstrated energy transfer between the wild-type complement factor 5a receptor and the lipid-facing chimeras, suggesting that the lipid-facing residues within a single TM segment are not essential for oligomerization These studies highlight the importance of the lipid-facing residues in the complement factor 5a receptor for transport competence
Abbreviations
AT1, angiotensin type 1; BFA, brefeldin A; BRET, bioluminescence resonance energy transfer; C5aR, complement factor 5a receptor; CaR, calcium-sensing receptor; CCR5, CC chemokine receptor 5; CHO, Chinese hamster ovary; CI, confidence interval; CuP, cupric
orthophenanthroline; EndoH, endo-b-N-acetylglucosaminidase H; GFP2, green fluorescent protein 2; GPCR, G protein-coupled receptor; IP 3, inositol 1,4,5-triphosphate; Rluc, Renilla luciferase; TM, transmembrane helix; YFP, yellow fluorescence protein.
Trang 2G protein-coupled receptors (GPCRs) are seven
transmembrane (TM) spanning receptors that catalyze
the exchange of GTP for GDP on the a subunit of
heterotrimeric G proteins, ultimately leading to the
activation of multiple intracellular signaling cascades
[1] The seven a-helical domains of GPCRs are
orga-nized into a tightly packed barrel-like structure, as
demonstrated by the high-resolution structure of
bovine rhodopsin in the inactive state [2,3] and the
structure of b-adrenergic receptor [4,5], and opsin
bound to a transducin peptide [6,7] Each a-helix has
a surface with a strong packing moment and the vast
majority of the packing moments are oriented toward
the helix bundle [8] These intramolecular interactions
between the TMs are considered to stabilize the
inac-tive state of the receptor Disruption of the involved
amino acids often has a functional consequence For
example, in the complement factor 5a receptor
(C5aR), interfering with a hydrophobic pocket
between TM3, TM6 and TM7 can induce
constitu-tive activity [9,10]
Less is known about the contributions of the
lipid-facing residues in the TM helices with respect to the
structure and function of GPCR A correlated
muta-tion analysis of rhodopsin-like GPCRs revealed that a
number of correlated mutations map to the lipid-facing
surfaces of the TMs, suggesting a functional
signifi-cance [11] Our saturation mutagenesis analyses of the
seven TMs of the C5aR functionally mapped essential
residues on lipid facing surfaces in TM2, TM4, TM6
and TM7 [9,12] These residues may be important in
protein–lipid interactions with respect to aiding
mem-brane insertion and stability Alternatively, these
resi-dues may mediate the many protein–protein
interactions observed for GPCRs, including those with
endoplasmic reticulum (ER) chaperones such as
caln-exin [13] to regulate membrane expression or the
inter-action of the calcitonin-receptor-like receptor and the
receptor-activity modifying proteins to dictate the
ligand specificity of the receptor [14]
Another possibility is that the lipid-facing residues
may mediate GPCR oligomerization Although
classi-cally viewed as monomers, biochemical and
biophysi-cal evidence of GPCR oligomerization has become
available at a surprising pace in recent years [15,16]
Most reports, especially for receptors in the rhodopsin
family of GPCRs, suggest that the oligomerization
interface resides in the TMs, although a conserved
oligomerization interface has yet to be identified
Indeed, different TMs have been frequently implicated:
TM1 in the yeast GPCR Ste2 [17], TM4 in the
dopa-mine D2 receptor [18,19], TM5 in the adenosine A2A
receptor [20] and TM6 in both the b2-adrenergic
recep-tor [21,22] and the leukotriene B4 receprecep-tor [23] Other studies [16,24,25], including our own previous work [26], have implied that more than one helix is responsi-ble and that GPCRs likely form larger oligomeric complexes through multiple oligomeric interfaces Taken together, these studies, in which the majority of the TMs were implicated in oligomerization, suggested the need for a comprehensive and unbiased analysis of the TM helices
To systematically evaluate the contributions of the individual TMs of the C5aR to receptor function and oligomerization, we generated chimeras of the C5aR and the rat angiotensin type 1 (AT1) receptor in which only residues on the proposed lipid-exposed face of the
TM helices were exchanged The side chains making intramolecular contacts with the rest of the TM bundle were not disrupted, aiming to minimize alterations to the overall receptor 3D structure Surprisingly, five to six substitutions on the outer face of TM1, TM2, TM4, TM5, TM6 or TM7 led to retention of the chimeric C5aRs in the ER The chimeric receptors displayed weaker binding affinity than the wild-type receptor at the plasma membrane; however, the bind-ing affinities are similar to the wild-type receptor that
is located in the ER This suggests that the decrease in binding affinity is more likely to be a product of recep-tor localization in the ER and not the result of an overall structural alteration Despite all of the chime-ras being retained in the ER, all of the individual lipid-facing chimeras demonstrated energy transfer with the wild-type C5aR These data are consistent with the studies mentioned above, suggesting that GPCRs use more than just a single oligomerization interface, most likely to generate multimeric GPCR complexes, at the same time as emphasizing the overall importance of the lipid-facing residues in the receptor life cycle
Results
To monitor the contributions of the TM helices to receptor function, a chimeric C5aR strategy was devised to exchange residues only on the lipid-exposed regions of the TMs An important aim of these stud-ies is to determine which lipid-facing residues partici-pate in C5aR oligomerization The strategy employs substituting the lipid-facing residues from one GPCR into the corresponding TM helix of the C5aR and monitoring whether oligomerization is affected TM3 was avoided because, in rhodopsin, it has the highest helix packing value via its contacts with TM2, TM4, TM5, TM6 and TM7; TM3 also has the lowest lipid accessibility surface area [8] A chimeric strategy was
Trang 3favored over an alanine or tryptophan scan, which
has successfully been used to study the TM helices of
other membrane proteins, such as the lactose
perme-ase [27], because we suspected that individual amino
acid mutations into the hydrophobic faces of the
heli-ces might not be sufficient to alter the packing or
oligomerization properties of the receptor The
inter-pretation of these studies relies on selecting a chimeric
partner that does not oligomerize with the C5aR We
chose the angiotensin AT1 receptor, which has been
reported previously not to interact with the
chemoki-ne receptor, CC chemokichemoki-ne receptor 5 (CCR5) [28]
The AT1R homo-oligomerizes [29] and forms
hetero-oligomers with the AT2 receptor [30], b2-adrenergic
receptor [31], D1 dopamine receptor [32] and the
bradykinin receptor [33], although this interaction has
not consistently been observed [34] The C5aR has
been shown to form homo-oligomers [35–37] and
het-ero-oligomers with CCR5 [28] In the present study,
oligomerization between AT1R and C5aR was
evalu-ated first by disulfide trapping, which uses cysteine
residues as collisional probes to assess for proximity
We have previously used this technique to
demon-strate homo-oligomerization of C5aR [26] As with
C5aR, exposure of membranes from Chinese hamster
ovary (CHO)-K1 cells stably expressing AT1R with a
carboxy terminal yellow fluorescence protein (YFP) to
the oxidation catalyst cupric orthophenanthroline (CuP) produced disulfide-linked oligomers and decreased the amount of monomeric AT1R-YFP (Fig 1A) Indeed, a significantly greater fraction of
AT1R underwent cross-linking compared to the C5aR Furthermore, AT1R appears to undergo spon-taneous cross-linking in the absence of CuP, which is
in good agreement with our previous observations [29] Our previous studies on C5aR demonstrated that fusion of YFP to the carboxy terminus did not alter the cross-linking kinetics or receptor activity [26], sug-gesting that the observed cross-linking in AT1R is dependent on the receptor and not YFP In cells
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0
100 200 300
400 C5aR-Luc/C5aR-GFP 2
C5aR-Luc/C5aR-GFP 2 /WT-C5aR
GFP 2 /Rluc ratio
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
GFP 2 /Rluc ratio
0 100 200 300
400 C5aR-Luc/C5aR-GFP 2
AT 1 R-Luc/C5aR-GFP 2
CaR-Luc/C5aR-GFP 2
A
B
C
Fig 1 Oligomerization of C5aR and AT1R (A) Total membranes
were prepared from CHO-K1 cells stably expressing C5aR, AT1
R-YFP, or both C5aR and AT 1 R-YFP Membranes were treated with
1.5 m M CuP and the reaction was terminated with NEM and EDTA
after 10 min The samples were resolved by nonreducing
SDS ⁄ PAGE and immunoblotted (IB) with anti-C5aR serum (left) or
GFP serum (right) In this experimental set-up, the GFP
anti-body cross reacts with the YFP for the detection phenomenon (B)
BRET 2 saturation curves COS-7 cells were transiently transfected
with a fixed amount of C5aR-Rluc, AT 1 R-Luc or CaR-Luc and
increasing amounts of C5aR-GFP 2 BRET 2 -ratios, total
lumines-cence and total fluoreslumines-cence were measured, as described in the
Experimental procedures For titration experiments, all of the
trans-fections contained 0.3 lg of Rluc-tagged receptor and 0.1–4 lg of
the GFP 2 -tagged receptor (C) The specificity of the C5aR
homo-oli-gomer BRET signal was analysed by transiently transfecting a fixed
amount of C5aR-Rluc and increasing amounts of C5aR-GFP2alone
or in combination with untagged C5aR In this experiment, the
specificity of the C5aR homodimer BRET signal was tested by
cotransfecting the titrations with 3 lg of untagged C5a receptor In
(B) and (C), data represent at least 10 transfections performed on
three experimental days To illustrate the specificity of the BRET
signal more clearly, we have chosen to report the GFP2⁄ Rluc ratios
that are less than 1 The full spectra and quantification are included
in the Supporting information (Fig S1 and Table S1).
Trang 4stably expressing both C5aR ( 40 kDa) and
AT1R-YFP ( 90 kDa), no hetero-oligomeric
com-plexes ( 130 kDa) were present after CuP addition,
despite normal patterns of homo-oligomeric
cross-linking for C5aR and AT1R-YFP Because the rate of
cross-linking depends on the proximity of the side
groups, the flexibility of the structures containing the
cysteine probes, and the oxidizing environment, it is
possible that hetero-oligomers might be less suitable
partners for cross-linking Therefore, a negative result
in this assay does not preclude that the C5aR and
AT1R form hetero-oligomers
We next used bioluminescence resonance energy
transfer (BRET2) technology to evaluate further the
hetero-oligomerization potential of AT1R and C5aR
As shown in Fig 1B, robust energy transfer is
observed between C5aR molecules tagged with Renilla
luciferase (Rluc) and green fluorescent protein 2
(GFP2) The BRET50for this interaction is 0.17 [95%
confidence interval (CI) = 0.12–0.22], where BRET50
is defined as the GFP2⁄ Rluc ratio at which 50% of
the maximum BRET value is reached By contrast,
AT1R-Rluc coexpressed with C5aR-GFP2
demon-strated a right-shifted BRET2 signal and a reduction
in the maximum BRET response compared to the
C5aR–C5aR pair (BRET50= 3.3, 95% CI = 1.7–4.8;
Fig 1B)
To control for specificity of the BRET signal, we
performed two sets of experiments First we controlled
for ‘bystander’ energy transfer Accordingly, we
coex-pressed C5aR-GFP2 with the calcium-sensing receptor
(CaR-Luc) The CaR is a member of the metabotropic
glutamate receptor family, which has been shown to
form strong homodimers, and therefore would not be
expected to form oligomers with a rhodopsin family
member [38] When coexpressed with the C5aR-GFP2,
the CaR-Luc demonstrated a low maximum BRET
signal, which is similar to that observed for the
C5aR-GFP2⁄ AT1R-Rluc coupled with a BRET50value
of 0.59 (95% CI = 0.32–0.85) Second, we performed
the BRET titration curve in the presence of untagged
wild-type C5aR and observed a significant right shift
in the signal between C5aR-Rluc and C5aR-GFP2
(BRET50= 0.87, 95% CI = 0.40–1.35 versus BRET50
= 0.17, 95% CI = 0.12–0.22; Fig 1C) The maximal
BRET signal was not decreased by coexpressing
untagged C5aR (see Fig S1), which might reflect the
fact that the C5aRs do not form simple dimers but
rather assemble in larger oligomeric structures
The BRET results combined with the absence of
cross-linking suggest that little if any oligomerization
occurs between C5aR and AT1R and that the AT1R is
a suitable chimeric partner for C5aR
Selection of lipid-exposed residues in C5aR Similar to all members of the rhodopsin family of GPCRs, C5aR, rhodopsin and AT1R share many con-served amino acids within the TM bundle [39], allow-ing for an alignment of the TM sequences with high confidence (Fig 2) Furthermore, it is expected that the orientation of the TM bundle witnessed in the high-resolution structure of rhodopsin is similar in other rhodopsin family GPCRs This prediction was validated by the recently determined structures of the
b2-adrenergic receptors [4,5] For our studies, we used the X-ray structure of the dark-adapted conformation
of bovine rhodopsin as a template for modeling the orientation of the lipid-facing residues in the C5aR and AT1R Five to seven lipid-exposed residues in each helix were selected (Fig 2)
A 3D model of the TM bundle of C5aR was then generated to validate the selections of the lipid-facing residues The model was constructed as described in the Experimental procedures Briefly, the low-energy conformations of each individual TM were assembled into a TM bundle using the X-ray structure of dark-adapted rhodopsin as a template The resulting 3D model of the TM region of the C5aR differed from rhodopsin by a rms value of 2.40 A˚, mostly as a result
of less dense packing of TM1 with the other helices, causing a slight shift of TM1 relative to the rest of the bundle (Fig 3A) At the same time, the main kinks in the TM helices of rhodopsin (e.g the functionally important kink in TM6) were preserved in the 3D model of C5aR Also, the 3D model of the TM bundle
of C5aR differed from the recently published X-ray structure of the b2-adrenergic receptor [4,5] by a rms value of only 2.69 A˚, although the 3D model was not built by aligning to this particular X-ray structure The identified residues in C5aR were then changed
to the residue found at the cognate position in the
AT1receptor, one TM at a time Of the 39 total posi-tions targeted in the helices, C5aR and AT1R have an identical side chain at eight positions Ultimately, six changes were made in the TM1 lipid-facing chimeric receptor, whereas five substitutions were introduced in the TM2, TM4, TM5, TM6 and TM7 chimeras (Table 1) The 3D models generated for the resulting chimeras built by the same methodology to generate the wild-type C5aR TM model verified that the selected residues point into the lipid bilayer (Fig 3B) Furthermore, no significant changes in residue–residue interactions within the TM bundle were observed for the six lipid-facing chimeras Therefore, we assume that critical intramolecular interactions important for receptor activity and helix packing are preserved We
Trang 5also suggest that the interaction of the chimeras with
the membrane should be unperturbed, in large part
because AT1R has a similar complement of lipid-facing
residues Furthermore, 18 of the 31 changes occurred
between the hydrophobic residues isoleucine, leucine,
phenylalanine and valine A statistical analysis of the
lipid-exposed surface of membrane proteins with
high-resolution structures found that these four residues
demonstrate the strongest preference for interacting
with lipids in the hydrocarbon core of the lipid bilayer
[40] In TM7, for example, all five of the substitutions
involved isoleucine, leucine or phenylalanine
Analysis of chimeric receptors
Stable transfection of the six chimeric receptors with
a carboxy-terminal YFP fusion in CHO-K1 cells
revealed that all of the chimeric receptors were expressed, although their migration on SDS⁄ PAGE was different from that of the wild-type receptor (Fig 4A) C5aR is glycosylated on an asparagine resi-due in the amino terminus [41] and the carbohydrate character is dependent on the receptor location in the secretory pathway Endo-b-N-acetylglucosaminidase H (EndoH) removes high-mannose oligosaccharides, and glycoproteins sensitive to EndoH treatment are found
in the ER or the cis-Golgi [42,43] Further processing
in the Golgi generates EndoH-resistant complex oligo-saccharides; therefore, the susceptibility to EndoH can
be used as a marker for transport through the secre-tory pathway The majority of the chimeric receptors were sensitive to EndoH, suggesting that these recep-tors were found predominantly in the ER By contrast, the wild-type receptor had a significant proportion of
Fig 2 Alignment of rat AT 1 R, human C5aR and bovine rhodopsin The outward facing locations selected for substitution are shown in bold Positions with greater than 60% conservation in the rhodopsin family [39] are shown at the top in italics TM heli-ces in rhodopsin are underlined; residue numbering corresponds to rhodopsin sequence The alignment was performed
by CLUSTALW analysis.
Trang 6EndoH-resistant species (Fig 4A) Moreover, the
lipid-facing chimeras were frequently expressed at higher
levels and migrated as high-molecular weight
aggre-gates These slower-migrating species were sensitive to
EndoH, as demonstrated by separation on low
percentage SDS⁄ PAGE (S Sen & T J Baranski,
unpublished results) It is unclear whether these larger
species represent specific higher-ordered structures,
such as dimers, or whether they are nonspecific
aggre-gates of the chimeric C5aRs These findings were
supported by strong perinuclear and intracellular
staining by fluorescence microscopy for all of the chi-meras in a pattern consistent with the endoplasmic reticulum, which is not seen for the wild-type C5aR (Fig 4B) Together with the high levels of EndoH-sen-sitive receptors in cell lysates, the substitutions in the chimeric receptors appear to disrupt the ability of C5aR to reach the cell surface Surprisingly, this effect was observed for all of the TMs that were evaluated Although the TM4 chimera was expressed in CHO-K1 cells at lower levels, as assessed by western blotting, fluorescence microscopy revealed similar levels of expression compared to the other TM chi-meras This difference most likely reflects the hetero-geneous expression of receptors in cells stably expressing the TM4 chimera Furthermore, transient expression in COS-7 cells revealed comparable levels
of expression for all the TM chimeras, as well as simi-lar EndoH sensitivities (Fig 5) Transient expression in COS-7 cells also resulted in a significant fraction of the chimeric receptors, as well as the wild-type receptor, migrating as higher-molecular weight, EndoH-sensitive complexes As in CHO-K1 cells, the significance of these ER complexes remains unclear
A
B
Fig 3 Molecular model of C5aR (A)
Sche-matic representation of spatial arrangement
of the TM helices in C5aR (shaded ribbons)
and rhodopsin (tubes) View from the side
of the membrane; the extracellular surface
is on the top TM helices are color-coded
as: TM1, white; TM2, blue; TM3, cyan;
TM4, green; TM5, red; TM6, yellow; TM7,
magenta (B) Sketch of the TM bundle of
C5aR (helices shown as shaded ribbons)
extracted from the 3D models of the
lipid-facing chimeras The lipid-lipid-facing residues
after substitution are shown and color-coded
according to their helix: TM1, white; TM2,
blue; TM4, green; TM5, red; TM6, yellow;
TM7, magenta The view is from both the
extracellular surface (left) and intracellular
surface (right) perpendicular to membrane
plane For clarity, the residues are not
labeled For mutations, see Table 1.
Table 1 Substitutions introduced at lipid-exposed locations in
C5aR.
W60I
Trang 7Competitive binding studies on the wild-type and chimeric receptors showed that the receptors were capable of binding ligand, although with a weaker affinity than the wild-type C5aR (Fig 6 and Table 2) The only exception was the TM7 chimera, which dem-onstrated no detectable binding To determine whether the decrease in binding affinity in the other chimeras was the result of protein instability and misfolding or was merely a byproduct of the localization of chimeras
in the ER, cells expressing the wild-type C5aR were treated with brefeldin A (BFA) to disrupt receptor maturation and ER to Golgi transport The apparent
Kd values for the BFA-treated wild-type receptor are
in the same range as that of the chimeric receptors (Table 2) Furthermore, the wild-type receptor demon-strates two distinct binding populations and a two-site competition binding analysis verified two receptor populations with distinct binding characteristics The high-affinity population (Kd1 and Bmax1) is consistent with the known binding characteristics of the C5aR and most likely reflects the receptor population at the plasma membrane The lower affinity population (Kd2 and Bmax2) is consistent with the binding kinetics of the BFA-treated wild-type receptor, as well as the ER retained chimeras, which suggests that this lower affin-ity population may comprise the proportion of the wild-type C5aR in the ER Thus, all the mutant chime-ric receptors display a lower binding affinity similar to the BFA-treated wild-type receptor and, based on our localization data, fail to reach the plasma membrane This internal receptor population is properly folded, but probably not assembled correctly for transport to the plasma membrane These findings suggest that the lipid-facing residues examined in the present study are not essential for monomeric receptor folding (with the exception of the TM7 residues), but rather are vital for receptor maturation and subcellular transport
Of note, the TM1-mutated C5aRs display higher affinity versus the low affinity sites of the wild-type C5aR and the other TM-mutated receptors (compare 13.5, 47.9 and 15.7 nm versus 150–600 nm; Fig 6
EndoH
100 75
100
*
+
75
50
37
50
37
– +
Fig 5 Expression and activity in COS-7 cells (A) Cell lysates were treated with (+) and without ( )) EndoH C5aR-YFP with EndoH-resistant complex oligosaccharides (*) and EndoH-sensitive high-mannose oligo-saccharides (+) are shown Western blots were performed with anti-C5aR serum.
A
B
Fig 4 Stable expression of lipid-facing chimeras in CHO-K1 cells.
(A) Cell lysates were treated with (+) and without ( )) EndoH
C5aR-YFP with resistant complex oligosaccharides (*) and
EndoH-sensitive high-mannose oligosaccharides (+) are shown Western
blots were performed with anti-C5aR serum (B) Fluorescence
microscopy of wild-type C5aR or lipid-facing chimeras.
Trang 8and Table 2) and the total number of binding sites is
lower than for the other TM-mutated receptors or the
low affinity site of the wild-type C5aR The molecular
basis for this interesting difference is not known It is
also possible that some of the mutated residues in the
TM contribute directly to ligand binding We do not
favor this interpretation because our modeling of the
C5aR places the TM1 residues (I38, V42, V46, L49,
L53 and W60) on the lipid-facing portion of the TM
Furthermore, mapping of the essential residues in
TM1 of the C5aR did not identify lipid-facing
resi-dues; however, the scan identified D37 and A40,
which, in our model of the C5aR, are positioned on
one face of the a-helical surface of TM1, pointing
favorably for a potential interaction with ligand
toward the center of the helical crevice [12]
Nonethe-less, if TM1 is either disordered or highly flexible, it is
possible that some of the lipid-facing residues might
contribute directly to the binding affinity of the
recep-tor for C5a or that these residues might affect the con-formation of D37 and A40 or other residues involved
in ligand binding
To investigate whether the mutant C5aRs that did reach the cell surface were able to activate G proteins,
we performed inositol 1,4,5-triphosphate (IP3) accumu-lation assays in COS-7 cells using a small molecule C5aR agonist (W5Cha) that is incapable of traversing the plasma membrane These studies demonstrated ligand stimulated activation for all of the chimeras, with the exception of the TM7-mutated C5aR (Fig 7) However, the levels of IP3 accumulation were consis-tently lower than the wild-type receptor, which likely reflects the smaller amount of mutated C5aRs at the plasma membrane Based on the EndoH treatment (Fig 5), it is difficult to determine the percentage of the mature receptors that were targeted appropriately
to the plasma membrane Nonetheless, the ability of the chimeras to induce IP3 accumulation after ligand
Fig 6 Binding analysis of the wild-type and lipid-facing chimeras Competition binding analysis of the wild-type and lipid-facing chimeras was performed with isolated membranes as described in the Experimental procedures The graphs are representative of a typical experi-ment performed at least in duplicate and repeated three times independently Calculations were performed using GRAPHPAD PRISM software.
In the BFA experiment, the compound was added 8 h post-transfection at a concentration of 10 lgÆmL)1 (A) Wild-type C5aR; (B) wild-type C5aR treated with BFA (10 lgÆmL)1); (C) TM1 chimera; (D) TM2 chimera; (E) TM4 chimera; (F) TM5 chimera; (G) TM6 chimera.
Trang 9treatment argues that, for at least the fraction of the
receptors reaching the plasma membrane, the
intro-duced substitutions do not drastically alter the overall
molecular architecture of the receptors Interestingly, the TM7 chimera was neither able to activate G pro-teins, nor bind ligand, despite the fact that the amino acid substitutions in TM7 were the most conservative versus the other TMs (all L, I or F; Table 1) The inability of TM7 to tolerate even conserved hydro-phobic substitutions demonstrates a unique folding requirement for TM7 relative to the other TMs (excluding TM3, which was not included in the present study) Similarly, saturation mutagenesis studies dem-onstrated that TM7 tolerated the fewest substitutions
in the functional mapping of the seven TMs in the C5aR [9,12]
Oligomerization of the lipid-facing chimeras Receptor biogenesis has frequently been linked to GPCR oligomerization, suggesting that defects in olig-omerization as a result of the introduced amino acid substitutions may be responsible for the observed transport incompetence Unfortunately, an evaluation
of the oligomerization potential of receptors in the ER
by disulfide trapping is complicated by the presence of high-molecular aggregates subsequent to resolution on
Table 2 Binding parameters for the wild-type and mutant constructs Apparent K d values and the approximate relative B max were directly derived from the raw data of the homologous competition-binding assay by fitting into competition binding models (one site ⁄ two site) and taking the best-fit R 2 values Values are represented from each experiment, where the experiments were repeated two or three times at least in duplicate and the standard errors are representative of data from within the experiment NA, only a single population of low-affinity sites were demonstrated for these receptors; ND, not detectable.
Construct
Fig 7 IP3 signaling assay IP3signaling activity of the wild-type
C5aR and lipid-facing chimeras COS-7 cells were transiently
transfected with Ga16 plus the wild-type C5aR or and lipid-facing
chimera and treated with 1 l M W5Cha Each bar represents the
mean ± SE of at least two independent trials.
Trang 10SDS⁄ PAGE (Fig 4A) BRET, however, has been used
to characterize GPCR oligomerization in the ER in
previous studies [22,44] BRET2 saturation curves were
generated in which the wild-type C5aR containing a
carboxy terminal Rluc was coexpressed with increasing
amounts of the lipid-facing chimeras fused to GFP2 in
COS-7 cells Two different energy transfer patterns
were observed: (a) a slope and maximal BRET signal
similar to the wild-type receptor, such as TM2, TM4
and TM7 (Fig 8A) or (b) a right-shifted energy
trans-fer relative to the wild-type receptor, such as the TM1,
TM5 and TM6 chimeras (Fig 8B) However,
quantifi-cation of the BRET50 values for this group did not
demonstrate significant differences compared to the
wild-type–wild-type interaction (see Table S1) As
pre-viously shown (Fig 5), a significant fraction of both
the wild-type C5aR and the chimeras are in the ER when expressed in COS-7 cells To address the possibil-ity that lipid facing-mutated receptors are aggregating and therefore interacting nonspecifically with wild-type C5aR, we performed additional BRET experiments with the TM7-mutated C5aR-GFP2 and AT1R-Luc, or CaR-Luc The rationale for these experiments is that if the TM7-mutated C5aR, which appeared to be the least well folded considering its failure to bind ligand, forms large aggregates in the ER, then we might expect that these aggregates would also trap AT1R and CaR that are folding and being processed in the ER We found no evidence for any interactions between AT1R or CaR and the TM7-mutated C5aR (see Fig S2) Although we cannot rule out that TM7-mutated C5aR is misfolded, the BRET signal is specific for this mutant and the wild-type C5aR
Discussion Much is known about how the seven TM helix bundle
of GPCRs packs and reorganizes during receptor acti-vation This helix packing is mediated by intramolecu-lar interactions among amino acids whose side chains are oriented away from the lipid bilayer into the helix core Less is known, however, about the roles of the amino acids that point away from the bundle These residues would be expected to be important in mem-brane insertion, thus regulating protein stability and folding, and in protein–protein interactions, such as receptor oligomerization To systematically evaluate each TM with a notable lipid exposed surface in C5aR, we employed a ‘lipid-facing chimera’ approach
in which only five to six residues predicted to orient away from the TM helix bundle were altered A poten-tial advantage to this novel approach was to minimize the likelihood of altering the overall 3D structure of the receptors, which is a common side effect of swap-ping entire TM helices For example, chimeras of the
a1b-adrenergic receptor in which each TM was replaced in entirety by the corresponding helix from the b2-adrenergic receptor were primarily retained in the ER [45], likely secondary to global receptor misfolding To our knowledge, this is the first study
in which chimeric GPCRs were generated that exchanged only the lipid-exposed residues
The binding data reported in the present study on both the wild-type receptor and the chimeric receptors illustrate that GPCRs can assume a ligand-binding con-formation in the endoplasmic reticulum; however, the overall binding is not as avid as that observed for recep-tors at the plasma membrane The low affinity of C5aR
in the ER might illustrate a more general concept;
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
0
100
200
300
400
Wild-type
TM1
TM5
TM6
Wild-type
TM2
TM4
TM7
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
0
100
200
300
400
A
B
Fig 8 Oligomerization of lipid-facing chimeras BRET 2 saturation
curves of COS-7 cells transiently transfected with a fixed amount
of C5aR-Rluc and increasing amounts the GFP 2 tagged lipid-facing
chimeras Data represent at least ten transfections performed on
three experimental days (A) BRET 2 saturation curves of lipid-facing
chimeras (TM2, TM4 and TM7) demonstrating BRET 2 values similar
to the wild-type C5aR (solid black) (B) BRET2saturation curves of
lipid-facing chimeras (TM1, TM5 and TM6) with decreased BRET 2
values compared to the wild-type C5aR (solid black).