Complement factor 5a receptor chimeras reveal the importance of lipid-facing residues in transport competence Jeffery M. Klco 1, *, Saurabh Sen 1, * , , Jakob L. Hansen 2,3, *, Christina Lyngsø 2,3 , Gregory V. Nikiforovich 4, à, Soren P. Sheikh 5 and Thomas J. Baranski 1 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 àMolLife 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 AT 1 , 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; GFP 2 , 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. 2786 FEBS Journal 276 (2009) 2786–2800 ª 2009 The Authors Journal compilation ª 2009 FEBS G 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 b 2 -adrenergic recep- tor [21,22] and the leukotriene B4 receptor [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 (AT 1 ) 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 J. M. Klco et al. Functional importance of lipid-facing residues in TMs of C5aR FEBS Journal 276 (2009) 2786–2800 ª 2009 The Authors Journal compilation ª 2009 FEBS 2787 favored 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 AT 1 receptor, which has been reported previously not to interact with the chemoki- ne receptor, CC chemokine receptor 5 (CCR5) [28]. The AT 1 R homo-oligomerizes [29] and forms hetero- oligomers with the AT 2 receptor [30], b 2 -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 AT 1 R 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 AT 1 R 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 AT 1 R-YFP (Fig. 1A). Indeed, a significantly greater fraction of AT 1 R underwent cross-linking compared to the C5aR. Furthermore, AT 1 R 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 AT 1 Ris 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 BRET (x1000) 0 100 200 300 400 C5aR-Luc/C5aR-GFP 2 AT 1 R-Luc/C5aR-GFP 2 CaR-Luc/C5aR-GFP 2 BRET (x1000) A B C Fig. 1. Oligomerization of C5aR and AT 1 R. (A) Total membranes were prepared from CHO-K1 cells stably expressing C5aR, AT 1 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 anti-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 fluorescence 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 lgof 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-GFP 2 alone 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 GFP 2 ⁄ Rluc ratios that are less than 1. The full spectra and quantification are included in the Supporting information (Fig. S1 and Table S1). Functional importance of lipid-facing residues in TMs of C5aR J. M. Klco et al. 2788 FEBS Journal 276 (2009) 2786–2800 ª 2009 The Authors Journal compilation ª 2009 FEBS stably expressing both C5aR ( 40 kDa) and AT 1 R-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 AT 1 R-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 AT 1 R form hetero-oligomers. We next used bioluminescence resonance energy transfer (BRET 2 ) technology to evaluate further the hetero-oligomerization potential of AT 1 R 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 (GFP 2 ). The BRET 50 for this interaction is 0.17 [95% confidence interval (CI) = 0.12–0.22], where BRET 50 is defined as the GFP 2 ⁄ Rluc ratio at which 50% of the maximum BRET value is reached. By contrast, AT 1 R-Rluc coexpressed with C5aR-GFP 2 demon- strated a right-shifted BRET 2 signal and a reduction in the maximum BRET response compared to the C5aR–C5aR pair (BRET 50 = 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-GFP 2 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-GFP 2 , the CaR-Luc demonstrated a low maximum BRET signal, which is similar to that observed for the C5aR-GFP 2 ⁄ AT 1 R-Rluc coupled with a BRET 50 value 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-GFP 2 (BRET 50 = 0.87, 95% CI = 0.40–1.35 versus BRET 50 = 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 AT 1 R and that the AT 1 Ris 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 AT 1 R 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 b 2 -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 AT 1 R. 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 b 2 -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 AT 1 receptor, one TM at a time. Of the 39 total posi- tions targeted in the helices, C5aR and AT 1 R 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 J. M. Klco et al. Functional importance of lipid-facing residues in TMs of C5aR FEBS Journal 276 (2009) 2786–2800 ª 2009 The Authors Journal compilation ª 2009 FEBS 2789 also suggest that the interaction of the chimeras with the membrane should be unperturbed, in large part because AT 1 R 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. Functional importance of lipid-facing residues in TMs of C5aR J. M. Klco et al. 2790 FEBS Journal 276 (2009) 2786–2800 ª 2009 The Authors Journal compilation ª 2009 FEBS EndoH-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-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. TM1 TM2 TM4 TM5 TM6 TM7 I38V I73V G151L A201G K242R L284I V42T V80L I155V V205T A249L F288I V46I L84C A158I V208I I253F I291F L49V I91L G162L I220T V260I I295L L53F S95Y I169L F224L M264L I298L W60I J. M. Klco et al. Functional importance of lipid-facing residues in TMs of C5aR FEBS Journal 276 (2009) 2786–2800 ª 2009 The Authors Journal compilation ª 2009 FEBS 2791 Competitive 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 K d 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 (K d1 and B max1 ) 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 (K d2 and B max2 ) 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 Mock WT TM1 TM2 TM4 TM5 TM6 TM7 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 EndoH-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. Functional importance of lipid-facing residues in TMs of C5aR J. M. Klco et al. 2792 FEBS Journal 276 (2009) 2786–2800 ª 2009 The Authors Journal compilation ª 2009 FEBS and 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 (IP 3 ) 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 IP 3 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 IP 3 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 lgÆ mL )1 ); (C) TM1 chimera; (D) TM2 chimera; (E) TM4 chimera; (F) TM5 chimera; (G) TM6 chimera. J. M. Klco et al. Functional importance of lipid-facing residues in TMs of C5aR FEBS Journal 276 (2009) 2786–2800 ª 2009 The Authors Journal compilation ª 2009 FEBS 2793 treatment 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 High affinity sites Low affinity sites K d1 (nM) B max1 (pmolÆmg )1 ) K d2 (nM) B max2 (nmolÆmg )1 ) C5aR wild-type 1.05 ± 0.07 13.15 440 ± 45 5.48 0.67 ± 0.01 8.73 305 ± 26 4.17 1.05 ± 0.08 12.92 163 ± 54 2.01 Wild-type (BFA) NA NA 395 ± 17 3.25 < 0.0008 0.005 598 ± 18 4.10 0.31 ± 0.21 0.93 749 ± 29 8.79 TM1 NA NA 13.5 ± 2.0 0.62 47.9 ± 1.2 0.78 15.7 ± 4.8 0.37 TM2 NA NA 222 ± 10 1.99 233 ± 14 2.40 504 ± 27 3.34 TM4 NA NA 282 ± 22 5.26 294 ± 25 2.07 150 ± 33 1.13 TM5 NA NA 310 ± 34 3.29 289 ± 16 2.92 596 ± 26 7.56 TM6 NA NA 956 ± 37 4.70 332 ± 21 2.49 TM7 ND ND Fig. 7. IP 3 signaling assay. IP 3 signaling activity of the wild-type C5aR and lipid-facing chimeras. COS-7 cells were transiently transfected with Ga 16 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. Functional importance of lipid-facing residues in TMs of C5aR J. M. Klco et al. 2794 FEBS Journal 276 (2009) 2786–2800 ª 2009 The Authors Journal compilation ª 2009 FEBS SDS ⁄ PAGE (Fig. 4A). BRET, however, has been used to characterize GPCR oligomerization in the ER in previous studies [22,44]. BRET 2 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 GFP 2 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 BRET 50 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-GFP 2 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 a 1b -adrenergic receptor in which each TM was replaced in entirety by the corresponding helix from the b 2 -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 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 BRET (x1000) 0 100 200 300 400 A B BRET (x1000) 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) BRET 2 saturation curves of lipid-facing chimeras (TM1, TM5 and TM6) with decreased BRET 2 values compared to the wild-type C5aR (solid black). J. M. Klco et al. Functional importance of lipid-facing residues in TMs of C5aR FEBS Journal 276 (2009) 2786–2800 ª 2009 The Authors Journal compilation ª 2009 FEBS 2795 [...]...Functional importance of lipid-facing residues in TMs of C5aR namely, that GPCRs in the ER are not fully competent to bind ligand and this might be the basis for a qualitycontrol or fail-safe mechanism to prevent unwanted receptor activation The lower binding affinity of the receptors in the ER may be the result of several mechanisms: (a) the cholesterol content of the ER membrane is different from that of the. .. and the high-affinity binding state of the receptor might only be achieved in presence of membrane bound cholesterol, as observed for the oxytocin receptors [46]; (b) receptors in the ER may not be associated with G proteins and thus may be incapable of high-affinity ligand binding; and (c) the oligomerization state of receptors in the ER may be different from those of the matured population at the surface... bundle for C5aR was the X-ray structure of darkadapted rhodopsin (Protein Databank entry: 1F88, chain A) At the stage of helical packing, the dihedral angles F and W were ‘frozen’ at the values previously obtained by energy minimization of the individual helices The resulting 3D model of the TM region of the C5aR differed from the corresponding region of rhodopsin by a rms value of ˚ 2.40 A Acknowledgements... thawed on ice, the protein concentration was determined using BSA as standard The binding reaction was set up using the Binding Buffer (Hanks buffer, 25 mm Hepes, pH 7.5, BSA 0.1%) containing the membrane fraction and cold C5a (concentration varying between 10)11 and 10)5 m) The reaction was initiated upon addition of 100 pm of [125I]C5a After incubation for 45 min, the reaction was terminated through... Packing of the 7TM bundle for the 3D model(s) of C5aR was performed according to a previously described procedure [56] Packing consisted of minimization of the sum of all intra- and interhelical interatomic energies in the multidimensional space of parameters that included the ‘global’ parameters (i.e those related to movements of individual helices as rigid bodies; namely, translations along the coordinate... split into two portions of approximately 0.5 · 106 cells The first portion was used to examine the GFP2 levels, by fluorescent measurements, and the Rluc expression by measuring the Coelenterazine h induced lumi- Functional importance of lipid-facing residues in TMs of C5aR nescence The second portion of the cells was submitted to DeepBlueC excitation, and the luminescence at the dual bands (515 ⁄ 30... directed from the first to the last Ca-atom; the Y-axis was perpendicular to X and went through the Ca-atom of the ‘middle’ residue of each helix; and the Z-axis was built perpendicular to X and Y to maintain FEBS Journal 276 (2009) 2786–2800 ª 2009 The Authors Journal compilation ª 2009 FEBS 2797 Functional importance of lipid-facing residues in TMs of C5aR the right-handed coordinate system The above... [49], the vasopressin and oxytocin receptors [44], C5aR [35] and 5-HT2C [50], demonstrating that GPCR oligomerization is a constitutive process occurring early in receptor biogenesis Furthermore, for the GABAB receptor, oligomerization in the ER is a prerequisite for transport to the plasma membrane [51] The results obtained in the present study also suggest that oligomerization occurs early in the biosynthetic... biosynthetic process However, our results demonstrate that oligomerization is not sufficient for membrane localization In addition, we anticipated that some of the lipid-facing chimeras would disrupt oligomerization, thereby identifying potential oligomerization surfaces Surprisingly, substituting the lipid-facing residues of any of the TMs does not significantly alter oligomerization of the C5aR, as... at the surface of the membrane Importantly, the subcellular localization and binding studies also demonstrated that the lipid facing residues are important, although not absolutely essential, for trafficking the C5aR to the plasma membrane Retention of GPCRs in the ER has been linked to defects in receptor oligomerization [47] A number of studies have now observed GPCR oligomerization in the ER, such . oligomerization. These studies highlight the importance of the lipid-facing residues in the complement factor 5a receptor for transport competence. Abbreviations AT 1 ,. 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