C-Terminal Interaction Is Essential for Surface Trafficking But Not for Heteromeric Assembly of GABA_B Receptors

MATERIALS AND METHODS

Generation of mutant expression plasmids. All constructs were subcloned into the cytomegalovirus-based eukaryotic expression vector pCI (Promega, Madison, WI). To allow detection of transiently expressed receptors, the N-terminal 16 residues of rat GABA_B(1a) (Kaupmann et al., 1997) were replaced by 36 residues encoding the mGlu5 signal peptide, M¹VLLLILSVLLLKEDVRGSAQS²², followed by the c-myc epitope, TRE²⁵QKLISEEDL³⁴TR. The GABA_B(1) signal peptide was replaced by the mGlu5 signal peptide because the latter is known to accurately release the N-terminal hemagglutinin (HA) epitope tag (Ango et al., 1999). Likewise the N-terminal 41 residues of rat GABA_B(2) (Kaupmann et al., 1998a) were replaced by the mGlu5 signal peptide followed by the HA epitope, TRY²⁵PYDVPDYA³³TR. The presence of the c-myc and HA epitope was found to affect neither the pharmacology nor the expression level of recombinant receptors. A cassette strategy was used to engineer all mutations and truncations. Artificial XhoI and EcoRV sites were introduced by silent site-directed mutagenesis of G²⁶⁹¹C and A²⁶⁹⁴G in GABA_B(1a)and C²⁵⁰⁸T in GABA_B(2), respectively. Chimera R1CR2 was made by overlap extension PCR. GABA_B(1a) was amplified using a forward primer (bp 1941–1958) and a chimeric reverse primer containing GABA_B(1a) (bp 2539–2559) and GABA_B(2) (bp 2224–2244) sequences. The GABA_B(2) C-terminal tail was amplified by using chimeric primer complementary to the one described above and a reverse primer (bp 2803–2823) with an in-frame 4xHis tag and a stop codon followed by an XhoI site. The two PCR products were joined by PCR and subcloned into the ClaI (bp 2245)/XhoI sites of GABA_B(1a). Chimera R2CR1 was made as described above, with primers containing both GABA_B(2) (bp 2194–2223) and GABA_B(1a) sequences (bp 2560–2588). The GABA_B(1a) C-terminal tail was amplified using reverse primer (bp 2863–2880) with an in-frame 4xHis tag and a stop codon followed by an NotI site. The 4xHis tags were added to monitor expression of the R2T749_his, R1CR2_his, and R2CR1_his constructs. We alternatively used the HA tag at the N terminus of R2T749. R2T749_his and R2T749_ha show no overt differences in expression levels, as well as in pharmacological and functional properties. The final PCR product was subcloned into the RsrII (bp1661)/NotI sites of GABA_B(2). The GABA_B(1a) C-terminal truncations R1I860, R1T872, and R1K886 were constructed by ligating complementary oligonucleotides with appropriate overhangs intoBclI²⁵⁷⁶/EcoRI (3′ cloning site). Using XhoI/EcoRI sites we made R1L915, R1L921, and R1P928. By ligating complementary oligonucleotides into EheI²⁷⁷¹/EcoRI sites we made R1G940 and R1G952. GABA_B(1a) alanine scanning from residue R⁹²² to P⁹²⁸ and mutants with S⁹²³→D, RR^922/924→AA, and RRR^922/924/925→AAA changes were constructed by site-directed mutagenesis and subcloned intoXhoI/EcoRI. The GABA_B(1a)LZ1 (bp 2650–2751) (Kammerer et al., 1999) mutant R1[PPP] (LIV^897/901/908→PPP) was made using the primer 5′-GAA AAC CGA GAA CCC GAG AAG ATC CCC GCT GAG AAA GAG GAG CGC CCC TCT GAA CTG CG-3′. The LZ1 deletion mutant R1ΔLZ was generated from wild-type (WT) GABA_B(1a) using complementary 44mer oligonucleotides flanking LZ1 (bp 2626–2649 and 2752–2772). We used overlap extension PCR to construct R1LZ2 by amplifying LZ2 (bp 2354–2454) (Kammerer et al., 1999) with chimeric primers complementary to WT GABA_B(1a) and GABA_B(2) (bp 2353–2373, 2431–2454). The amplified LZ2 domain was then inserted into R1ΔLZ. The GABA_B(2) C-terminal truncation R2T749 was made by ligating complementary oligonucleotides with appropriate overhangs in MluI (bp 2136)/XbaI (3′ cloning site); truncations R2L791, R2E811, and R2P820 were constructed by taking advantage of PshAI (bp 2368)/XbaI sites. CR2 (R⁷¹⁴-L⁹⁴¹) and CR2P820 (R⁷¹⁴-P⁸²⁰) were made by removing the N-terminal and transmembrane domain (TMD)1–6 domains from the WT and R2P820 GABA_B(2) constructs by MluI digestion and religation, thereby preserving the previously inserted HA tag. R2[PPP] with mutation of LIL^798/802/805→PPP was made using primer 5′-GAA AAC CAC CGC CCG CGG ATG AAG CCC ACA GAG CCG GAC AAA GAC TTG-3′. The LZ2 deletion in R2ΔLZ was made using complementary 48mer oligonucleotides flanking LZ2 (bp 2311–2334 and 2437–2460). We made chimera R2LZ1 by inserting LZ1 into WT GABA_B(2). PCR using a forward primer duplicating sequences complementary to GABA_B(2) (bp 2311–2334) and GABA_B(1a) (bp 2650–2672) amplified LZ1; reverse primer corresponded to chimeric GABA_B(1a) (bp 2729–2751) and GABA_B(2) sequences (bp 2437–2460). All constructs were verified by sequencing.

Cell culture and radioligand binding assays. Mammalian cells were purchased from the American Type Culture Collection (Rockville, MD) and cultured in Earle's MEM (Life Technologies, Basel, Switzerland) supplemented with 10% fetal calf serum and 2 mml-glutamine. Cells were harvested for membrane preparation 2 d after transfection. Preparation of cell lysates and [¹²⁵I]CGP64213 binding assays was performed as described (Kaupmann et al., 1997). [¹²⁵I]CGP64213 and [¹²⁵I]CGP71872 were radiolabeled (ANAWA, Wangen, Switzerland) to a specific activity of 2000 Ci/mmol. All ligands were synthesized in house.

Immunoblot. Cellular membranes were solubilized in Laemmli sample buffer (125 mm Tris, pH 6.8, 1% SDS, 25 mm DTT, 5% glycerol/bromphenol blue). Proteins (10–20 μg per lane) were separated by SDS-PAGE using 4–15% gradient gels and transferred onto Immobilon-P polyvinylidene difluoride membranes (Millipore AG, Volketswil, Switzerland) by standard wet electrophoretic transfer. Blocking was overnight in NET-G buffer (150 mm NaCl, 50 mmEDTA, 500 mm Tris HCl, pH 7.4, 0.5% v/v Triton X-100, 2.5% w/v gelatin) at 4°C. Incubation with primary and secondary antibodies was for 45 min at room temperature. Polyclonal rabbit antisera Ab174.1, Ab176a, and C22 were as described (Malitschek et al., 1998; Kaupmann et al., 1998a,b). Polyclonal antisera N22 was derived against the GABA_B(2) N-terminal peptide A¹⁶⁴-L²¹⁵ and produced as described (Malitschek et al., 1998). Antibodies were diluted in NET-G buffer as follows: Ab174.1 (1:2000), Ab176a (1:2000), N22 (1:2000), C22 (1:3000), and goat anti-rabbit IgG H+L POD (1:3000; Bio-Rad, Glattbrugg, Switzerland). Detection was with enhanced chemiluminescent Western Blot reagents (Amersham, Duebendorf, Switzerland) and exposure to Biomax maximum resolution x-ray films (Kodak, Cambridge, UK).

Surface labeling experiments. Given that [¹²⁵I]CGP71872 cannot permeate the plasma membrane, photoaffinity labeling of intact cells reveals receptor/ligand interactions that occur at the extracellular binding site (Malitschek et al., 1999). HEK293 cells were transferred to six-well plates 6 hr after transfection. After an additional 36 hr incubation, cells were washed twice with cold HEPES buffer, pH 7.6 (Life Technologies). Half of the cells were used for photoaffinity labeling of surface receptors (S), the other half for labeling of receptors in the cell homogenate (H). For labeling of surface receptors, living cells were incubated in the dark for 1 hr at room temperature with 0.8 nm[¹²⁵I]CGP71872. Cells were washed with cold HEPES buffer to remove unbound [¹²⁵I]CGP71872 before UV cross-linking (Kaupmann et al., 1997). Photoaffinity-labeled cells were collected, and the radioactivity was determined in a gamma counter (Packard, Zurich, Switzerland). Photoaffinity labeling of receptors in the membrane fraction was as described (Kaupmann et al., 1997). For SDS-PAGE, the pellets of cells and cell lysates were resuspended in HEPES buffer containing 0.1% SDS. An aliquot was taken for protein determination (Micro BCA Protein Assay Reagent Kit, Pierce, Rockford, IL). Twenty micrograms of total protein of cell and membrane fraction were loaded onto a 4–15% gradient gel. Photoaffinity-labeled proteins were detected using autoradiography. The radioactivity incorporated into the surface (S) and homogenate (H) fraction was normalized to the respective amount of protein in each fraction before the percentage S/H ratio was calculated. Presumably because of the differences in the radiolabeling procedure for surface and homogenate receptors, the percentage S/H sometimes exceeds the theoretical value of 100%.

Immunofluorescence studies. HEK293 cells were grown on eight-well glass slides coated with poly-d-lysine (Becton Dickinson, Basle, Switzerland) and transfected (0.05 μg of GABA_B(1a)/0.2 μg of GABA_B(2) DNA in 5 μl of H₂O) with FuGene6 (Roche Molecular Biochemicals, Rotkreuz, Switzerland). In control experiments the empty pCI vector substituted for GABA_B plasmids. Dissociated cerebellar granule cells were isolated from 7-d-old mice and plated on poly-l-ornithine-coated 35 mm glass coverslips (Van-Vliet et al., 1989); after 7 d in culture, cells were transfected (Ango et al., 1999) with c-myc and HA-tagged GABA_B(1a) and GABA_B(2)constructs, respectively. Cells were fixed 48 hr after transfection with 4% paraformaldehyde in PBS for 20 min and blocked with 1% BSA and 2% heat-inactivated goat serum in PBS for 1 hr. Cell surface receptor expression was assayed by labeling cells with either anti-c-myc (clone 9E10, Roche Molecular Biochemicals; 1:200) or anti-HA (clone 12CA5, Roche Molecular Biochemicals; 1:80) monoclonal antibodies for 2 hr. After extensive washing, cells were incubated for 1 hr with secondary Cy3-conjugated affinity-purified goat anti-mouse IgG (1:400, Jackson ImmunoResearch Laboratories, West Grove, PA). Intracellular receptor expression was assayed with permeabilized cells (0.4% saponin added to the blocking/washing buffers and to the primary and secondary antibody solutions). Immunofluorescence microscopy was with a ZeissAxioplan microscope using a 20× Plan-Neofluar objective. Images were collected with a Hamamatsu 3 CCD digital camera.

Measurement ofΔ[Ca²⁺]_I by fluorometry. For measurement of Δ[Ca²⁺]_i all transfections included Gα_qo5 to couple GABA_B receptors to PLC (Franek et al., 1999). HEK293 cells (1.5 × 10⁶) were electroporated (250 V, 300 μF; BioRad Gene Pulser) with 5 μg of cDNA constructs in a total volume of 150 μl buffer (50 mmK₂HPO₄, 20 mm CH₃COOK, 20 mm KOH, pH 7.4). Transfected cells were resuspended in culture medium and split into three wells of 12-well plates that were coated with 100 mg/ml poly-d-lysine. Twenty-four hours after transfection, the cells were incubated at room temperature for 1 hr in a HEPES buffer, pH 7.6 (Life Technologies) containing 10 μg/ml of the calcium indicator fura-2 AM, 0.5% Pluronic F-127 (Molecular Probes, Eugene, OR), and 1% (v/v) dimethyl sulfoxide. Glass coverslips carrying dye-loaded cells were mounted into a perfused cuvette (2 ml/min) in a fluorescence spectrophotometer (F-4500, Hitachi). Changes in [Ca²⁺]_i were monitored by measuring the ratio of fura-2 fluorescence (510 nm) excited at 340 and 380 nm, switched at 1.6 Hz. The viability of transfected cells was tested by application of 10 μm ATP. Control experiments showed no changes in [Ca²⁺]_i levels after application of 1 mm GABA in HEK293 cells transfected with pcDNA1 vector and in HEK293 cells expressing GABA_B(1)+Gα_qo5, GABA_B(2)+Gα_qo5, or Gα_qo5. No quantitative comparison between experiments was made, because the signal amplitude depends on the transfection efficiency.

Fluorometric image plate reader. Transfected HEK293 cells were plated into poly-d-lysine coated 96-well plates (Becton Dickinson). After transfection (48–72 hr), cells were loaded for 45 min with 2 μm fluo-4 AM (Molecular Probes) in HBS (Life Technologies) containing 50 μm probenecid (Sigma, Buchs, Switzerland). Plates were washed and transferred to a FLIPR (Molecular Devices, Crawley, UK). Concentration–response curves were recorded with three to eight wells per concentration as a change of fluorescence over baseline (ΔF/F). Data were fitted using Igor Pro (Wavemetrics) with a logistic equation using weighted nonlinear regression. Curves and data points were normalized to their asymptotic maximal values or for negative subunit combinations to the respective values of the positive controls measured on the same plate.

Electrophysiology. Concatemers of Kir3.1/3.2 subunits (Kaupmann et al., 1998b) were cotransfected with GABA_BR cDNA combinations into HEK293 cells. Whole-cell patch-clamp recordings were performed at room temperature 48–72 hr after transfection in a bath solution consisting of (in mm): 135 NaCl, 5.4 KCl, 1.8 CaCl₂, 1 MgCl₂, 10 glucose, 5 HEPES, pH 7.4. Patch pipettes were pulled from borosilicate glass capillaries, heat-polished to give a tip resistance of 3–5 MΩ, and filled with 140 mm KCl, 2 mm MgCl₂, 1 mm EGTA, 1 mmNa₂ATP, 100 μm cAMP, 100 μm GTP, and 5 mm HEPES, pH 7.3. Currents were elicited at a holding potential of −90 mV by application of GABA in a high potassium extracellular buffer (25 mm KCl, 115.6 mm NaCl). Data were recorded with an Axopatch 200B using pClamp6.

RESULTS

Previous studies indicated that GABA_Breceptor assembly and trafficking are intricately linked processes and that functional activity of the GABA_B(1,2)heteromer correlated with GABA_B(1) cell surface expression (Couve et al., 1998; White et al., 1998). Functional expression in heterologous systems therefore provides a fast and sensitive means of identifying structural elements that are necessary for receptor maturation. In this study we constructed chimeric, truncated, and point mutated GABA_B(1) and GABA_B(2) mutants and coexpressed them in HEK293 cells and cultured neurons. Immunoblot analysis was used to verify that all receptor mutants were expressed. For functional analysis we measured Gα_qo5 signaling to phospholipase C (PLC), as manifested by GABA-elicited increases in cytosolic calcium ([Ca²⁺]_i) (Franek et al., 1999) and Gβγ stimulation of Kir3.1 + 3.2 type K⁺ channels (I_{Kir3.1 + 3.2}) (Kaupmann et al., 1998b). Surface expression was studied using immunocytochemistry and quantified using photoaffinity labeling with the membrane-impermeable GABA_B(1)-specific ligand [¹²⁵I]CGP71872 (Kaupmann et al., 1997). The surface expression and functional data are summarized in the Table.

DISCUSSION

To date, the physiological significance of GPCR dimerization remains largely unknown. GABA_B receptors are currently the only example of where function depends on dimerization. GPCRs other than GABA_B, such as the serotonin (Xie et al., 1999), opioid (Jordan and Devi, 1999), dopamine D₁/adenosine A₁ receptors (Ginés et al., 2000) and angiotensin II AT₁/bradykinin B2 (AbdAlla et al., 2000) have recently been shown to assemble from distinct subunits. Heteromerization can change pharmacological properties and potentiate signal transduction. This raises the possibility that heteromerization is a relevant and more common principle among GPCRs. Conceptually, this largely increases the already vast repertoire of GPCRs and may significantly change our view of how these receptors operate. Understanding the mechanisms that govern assembly of functional heteromeric GPCRs is therefore a fundamental problem and may reveal dimerization partners that otherwise are difficult to discover.

For any protein that oligomerizes in the ER, the proper tertiary and quartenary structures must be achieved before the release. Sorting mechanisms are in place to discriminate between the unassembled subunits and completed oligomers. These mechanisms include, for example, the exposure and masking of short discrete ERR/R motifs. The best-characterized signals are the luminal KDEL sequences of soluble and type II transmembrane proteins (Munro and Pelham, 1987) and the cytoplasmic KK sequences of type I membrane proteins (Teasdale and Jackson, 1996). Other signals include RR (Schutze et al., 1994) or RXR (Zerangue et al., 1999) in the cytoplasmic domain of the retained protein.

It was shown that GABA_B(2) can target to the cell surface by itself (Marshall et al., 1999). In contrast, GABA_B(1) is retained in the ER in the absence of coexpressed GABA_B(2), and heterodimerization results in a profound enhancement of surface expression (Couve et al., 1998; Marshall et al., 1999). The mechanism of plasma membrane targeting was not understood. We now show that a four amino acid motif in GABA_B(1), RSRR, tightly inhibits the surface expression of monomers. The RSRR motif combines two known ERR/R signals: RXR (Zerangue et al., 1999) and RR (Schutze et al., 1994). One would assume that proteins possessing this enhanced motif would be retained more efficiently in the ER than those featuring the minimal motif.

GABA_B(2) was found in yeast two-hybrid screens using the GABA_B(1) C terminus as a bait, suggesting that the leucine zippers were responsible for heteromerization of the GABA_B(1,2) complex (White et al., 1998; Kammerer et al., 1999; Kuner et al., 1999). We show that a GABA_B(2) C-terminal fragment, CR2P820, is sufficient to reverse the ER retention imposed by the RSRR motif (Fig.4D). The close proximity of the RSRR motif to the C-terminal LZ1 suggests that the coiled-coil interaction is responsible for masking the ERR/R signal. In support of such an involvement, shielding of the ERR/R signal is lost once serial deletions of the C terminus of GABA_B(2) progress into the leucine zipper domain (Fig. 4C) or when the LZ2 is deleted in R2ΔLZ2 (Fig. 5B). Surprisingly, however, mutation of the leucine zipper in R1[PPP] still allows the formation of functional surface receptors together with WT GABA_B(2) and R2[PPP] (Fig. 5D,E). These latter results suggest that some shielding of the ERR/R signal still occurs when leucine zipper interactions are partly or completely destabilized. It does not imply, however, that in WT receptors the coiled-coil interaction is not involved in masking the ERR/R signal. Our conclusion from these seemingly conflicting results is that the leucine zipper interaction is involved in, but not absolutely necessary for, masking the ERR/R signal. Accordingly, functional interactions between GABA_B(1) and GABA_B(2) can still be obtained when leucine zipper interactions are weakened or prevented by mutation or deletion. From a therapeutic point of view, it appears that drugs that disrupt the coiled-coil interaction will not necessarily reduce the number of GABA_B receptors expressed at the cell surface. Possibly the coiled-coil domains also serve functions other than GABA_B surface trafficking and support interaction with additional proteins, such as recently shown for ATF-4 (Nehring et al., 2000).

Human GABA_B(1) polymorphisms were described, but none of them involve the LZ1 domain or the RSRR motif (Sander et al., 1999). Known GABA_B(1) splice variants conserve the ERR/R signal and the LZ1 domain (Isomoto et al., 1998) (GeneBank accession number AJ012187). Moreover, two C-terminal splice variants of GABA_B(2) (our unpublished data) do not alter the LZ2 domain. This emphasizes the importance of the coiled-coil domains for receptor maturation. We did not observe any trafficking of GABA_B(1) to the surface when coexpressed with several family 3 GPCRs. However, it has been shown recently that ionotropic and metabotropic receptors are able to undergo a physical association (Liu et al., 2000). Proteins with unrelated structure therefore may traffic GABA_B(1) to the cell surface. Although GABA_B(1) monomers do not efficiently couple through Gα and Gβγ, it is possible that once at the cell surface GABA_B(1) signals through G-protein independent-pathways, as shown for the mGlu receptors (Heuss et al., 1999). The interdependence between heteromerization, exit of GABA_B(1) from the ER, and functional activity makes it difficult to separate the role of individual receptor domains. However, no obvious functional properties of heterologous GABA_B receptors are lost when both C termini are deleted. It follows that the primary determinants for heteromeric receptor assembly and function do reside in the N-terminal domain and TMD.

The ER trafficking signal identified here was first characterized in K_ATP channels (Zerangue et al., 1999). This signal was proposed to serve as a quality control mechanism that ensures only the surface expression of properly assembled octameric channels. Because for WT subunits only GABA_{B(1, 2)} complexes are shown to be capable of robust signal transduction, a similar mechanisms may exist that prevents monomeric GABA_B(1) from expressing on the cell surface. Mutations leading to truncation of the C terminus of SUR1 are one cause of a severe, recessive form of persistent hyperinsulinemic hypoglycemia of infancy, most likely because aberrant K_ATPchannels are formed (Zerangue et al., 1999). Because GABA_B(1) monomers are functionally inert when expressed on the plasma membrane, they would not have obvious harmful effects. An important aspect of GPCR regulation is the control of the number of receptors expressed on the cell surface. The ERR/R signal identified here is poised to play a dynamic role in controlling the level of surface expression of heterodimeric receptors. Slow trafficking rates may create a large intracellular pool of GABA_B(1) protein that is ready to go to the cell surface in the event that GABA_B(2) is provided. For the GABA_B receptors, ERR/R may therefore represent a means to rapidly regulate surface expression levels, as proposed similarly for Homer1b and mGlu5 (Roche et al., 1999).

After submission of this manuscript a similar study reporting the identification of the RSRR motif in GABA_B(1) was published (Margeta-Mitrovic et al., 2000). The authors of that study reached conclusions that are mostly similar to ours. However, our data do not support the conclusion that “interaction of GABA_B(1) and GABA_B(2)through the C-terminal coiled-coil α helices seems to be required not only for the shielding of the retention/retrieval signal RSRR in GABA_B(1) but also for the functional activity of the fully assembled complex.” Margeta-Mitrovic et al. (2000) base their statement on an experiment with point mutations in the coiled-coil and RSRR regions of GABA_B(1). Although they observe surface expression of that GABA_B(1) mutant, they do not see function on coexpression with GABA_B(2). In contrast, we observe in all of our experiments with either deleted (Figs.1C,D, 2C,E,3B,D,E,4C,D) or mutated (Fig.5B,C,D,E) coiled-coil domains, functional heteromeric receptors whenever the two subunits are coexpressed at the cell surface. This shows convincingly that the N-terminal domain and TMD of the two subunits are sufficient to assemble a functional heteromeric complex provided GABA_B(1) exits the ER.