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The EMBO Journal vol.13 no.2 pp.342-348, 1994
Domains involved in the specificity of G protein activation in phospholipase C-coupled metabotropic glutamate receptors J.-P.Pin', C.Joly, S.F.Heinemann2 and J.Bockaert Centre CNRS-INSERM de Pharmacologie-Endocrinologie, rue de la Cardonille, 34094 Montpellier Cedex 05, France and 2Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, PO Box 85800, San Diego, CA 92138, USA 'Corresponding author Communicated by D.Strosberg
G protein-coupled glutamate receptors (mGluR) have recently been characterized. These receptors have seven putative transmembrane domains, but display no sequence homology with the large family of G proteincoupled receptors. They constitute therefore a new family of receptors. Whereas mGluRl and mGluR5 activate phospholipase C (PLC), mGluR2, mGluR3, mGluR4 and mGluR6 inhibit adenylyl cyclase (AC) activity. The third putative intracellular loop, which determines the G protein specificity in many G protein-coupled receptors, is highly conserved among mGluRs, and may therefore not be involved in the specific recognition of G proteins in this receptor family. By constructing chimeric receptors between the AC-coupled mGluR3 and the PLCcoupled mGluRlc, we report here that both the Cterminal end of the second intracellular loop and the segment located downstream of the seventh transmembrane domain are necessary for the specific activation of PLC by mGluRlc. These two segments are rich in basic residues and are likely to be amphipathic a-helices, two characteristics of the G protein interacting domains of all G protein-coupled receptors. This indicates that whereas no amino acid sequence homology between mGluRs and the other G protein-coupled receptors can be found, their G protein interacting domains have similar structural features. Key words: calcium signal/G protein interaction/mGluR/ phospholipase C
Introduction The main excitatory neurotransmitter in the mammalian brain, glutamate (Glu), acts on two classes of receptors (Monaghan et al., 1989). One class corresponds to ligandgated channels and comprises three main pharmacologically defined receptor types named according to their specific agonist: N-methyl-D-aspartate (NMDA), a-amino-3hydroxy-5-methyl-isoxazole-4-propionate (AMPA) and kainate. The second class of Glu receptors corresponds to metabotropic receptors (mGluR) which are coupled to G proteins and modulate the production of intracellular messengers (Sladeczek et al., 1985; Nicoletti et al., 1986; Sugiyama et al., 1987; Schoepp et al., 1990) or the opening 342
of ion channels (Charpak et al., 1990; Lester and Jahr, 1990; Fagni et al., 1991; Trombley and Westbrook, 1992) Six different cDNAs coding for such receptors (mGluRl -6) have now been isolated (Houamed et al., 1991; Masu et al., 1991; Abe et al., 1992; Nakanishi, 1992; Tanabe et al., 1992). These cDNAs code for proteins that have no amino acid sequence homology with any other known G proteincoupled receptors, so that these proteins constitute a totally new family of receptors. All mGluRs contain seven hydrophobic segments which could correspond to transmembrane a-helices. In contrast to other seven transmembrane domain (TMD) receptors, the mGluRs possess a very long N-terminal extracellular domain, and the seven TMD are separated by short intraand extracellular loops. The C-terminal end, presumably intracellular, is variable in length, ranging from 51 (mGluR3) to 359 (mGluRla) amino acid residues. Expression of these receptors revealed that mGluRl and mGluR5 activate phospholipase C (PLC) (Houamed et al., 1991; Masu et al., 1991; Abe et al., 1992), while mGluR2, mGluR3, mGluR4 and mGluR6 are negatively coupled to adenylyl cyclase (AC) (Tanabe et al., 1992, 1993; Thomsen et al., 1992). The regions involved in the specificity of recognition and activation of G proteins by other 7-TMD receptors have been extensively studied (Ostrowski et al., 1992; Savarese and Fraser, 1992). The N- and C-terminal amphipathic domains of the third intracellular loop (i3) have been shown to play a critical role in determining the specific activation of G proteins (Kobilka et al., 1988; Lechleiter et al., 1990; Wess et al., 1990; Wong et al., 1990; Cotecchia et al., 1992). Although the first and second intracellular loops as well as the region downstream of the seventh TMD are also involved in the receptor G protein interaction (Konig et al., 1989; Franke et al., 1990; Wong et al., 1990; Liggett et al., 1991), the importance of i3 has been strengthened by showing that synthetic peptides derived from this region can directly activate G proteins (Cheung et al., 1991; Okamoto et al., 1991; Okamoto and Nishimoto, 1992). The third intracellular loop of all mGluRs is short and is the most conserved intracellular domain making it unlikely to be involved in the specificity of activation of G proteins. A main difference between mGluRs negatively coupled to AC and those coupled to PLC is the length of their Cterminal intracellular domain. The very long C-terminal domain found only in PLC-coupled mGluRs (mGluRl and 5) is, however, probably not involved in the specific interaction with PLC-activating G proteins, since two alternatively spliced forms of mGluRl (mGluRlb and c) which lack this domain can also activate PLC (Pin et al., 1992; Tanabe et al., 1992). By constructing chimeric receptors between mGluRlc and mGluR3 we have examined the role played by the second intracellular loop (i2) as well as the region located downstream of the seventh TMD (i4) of mGluRlc in determining its specific coupling to PLC. © Oxford University Press
Results
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G protein interaction domains of mGluRs
Alignment of the intracellular loop sequence of six cloned mGluRs reveals that both the first (il) and second (i2) intracellular loops contain amino acid residues that are conserved in the PLC-coupled mGluRs (mGluRl and mGluR5) and different from those conserved in the ACcoupled mGluRs (mGluR2-4, mGluR6) (Figure la). The sequence of the intracellular domain located downstream of TMD VII (i4) is more variable in AC-coupled mGluRs, but highly conserved in PLC-coupled mGluRs. It is therefore possible that these domains play a role in the specific activation of G proteins. Because the regions involved in the specific recognition of G proteins in most receptors are likely to be amphiphilic a-helices (Strader et al., 1989; Cheung et al., 1991; Okamoto et al., 1991; Sukumar and Higashijima, 1992), we also looked for such segments among
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the intracellular domains of mGluRs. In the PLC-coupled mGluRs, both the N- and C-terminal domains of i2 have a high c-helix amphipathicity value, as shown for mGluRlc (Figure lb). The same is true for the segment located downstream of TMD VII (i4, Figure 1). These amphipathic segments of mGluRl and mGluR5 contain many basic residues (Figure la) and therefore fulfill some of the criteria established for G protein activation domains of 7-TMD receptors (Okamoto and Nishimoto, 1992). Interestingly, in AC-coupled mGluRs, the N-terminal portion of i2 is the only intracellular segment within the 7-TMD region likely to be an amphiphilic a-helix, as shown for mGluR3 (Figure lb). To test the possibility that putative amphiphilic a-helices of mGluRs are involved in the specific activation of G proteins, we constructed chimeric receptors between mGluR3 and mGluRlc, a truncated isoform of mGluRla which has a C-terminal intracellular domain similar in length to that of AC-coupled mGluRs. Different segments of mGluR3 were exchanged with their homologues from mGluRlc. The ability of the resulting chimeric receptors to activate PLC upon Glu stimulation was examined by injecting their corresponding in vitro synthesized transcripts into Xenopus oocytes. In these cells, it is well established that
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Fig. 1. Sequence comparison of mGluRs. (a) Sequence alignment of the intracellular loops of cloned mGluRs. The loop located between TMD I and II is called il. Loop i2 is located between TMD HI and IV, and loop i3 between TMD V and VI. The sequence located downstream of TMD VII is referred to as i4. Conserved residues among five of the receptors are boxed. Residues which are conserved only in mGluRs negatively coupled to AC (mGluR2-4, mGluR6) are boxed in black. Gaps are indicated by '-'. (b) Hydrophobicity and a-helix amphipathicity plots of mGluRlc (top) and mGluR3 (bottom). Only the sequence downstream of amino acid residue 500 is represented. The top curve corresponds to the hydrophobicity plot according to Kyte and Doolittle (1982) using a window of 11 residues. The positions of the putative TMDs are indicated by the black boxes. The bottom curve corresponds to the (x-helix amphipathicity plot according to Eisenberg et al. (1984) using a window of 11 residues. Segments with an ax-helix amphipathicity value >0.4 are underlined with hatched boxes.
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Fig. 2. Schematic representation and coupling to PLC of the different chimeric receptors. (a) Schematic representation of the chimeric receptors constructed and analyzed in this study. Hatched boxes, mGluRlc segments; open boxes, mGluR3 sequences; black boxes, positions of the TMDs; il, i2 and i3, intracellular loops. The table on the right panel summarizes the ability of the different receptors to activate PLC. In the last column values are means A SEM of the peak current induced by 200 AM Glu. Only responsive oocytes were included in the calculation. Asterisk indicates that identical responses were measured whether the 3'UTR of the chimera was that of mGluR3 or that of mGluRlc, so that the data obtained with both constructs have been pooled. Plus sign indicates that only constructs with the 3'UTR of mGluRlc have been analyzed. (b) The horizontal bars correspond to the segments of mGluRlc and mGluR3 interchanged in the chimeras. The names of these segments correspond to those used in the names of the chimeric receptors. For example, R3/1-(i2,C4) is a chimeric receptor corresponding to mGluR3 carrying the i2 and C4 segments of mGluRlc. The C2 segment corresponds to the entire C-terminal end of mGluRlc.
343
J.-P.Pin et al.
G protein-coupled receptors, only those coupled to PLC activate Ca2+-dependent Cl- channels by releasing Ca2+ from internal stores (Hirono et al., 1987). The subsequent current can be easily recorded using the two electrode voltage-clamp technique. As previously reported, mGluRlc clearly activates a Clcurrent when expressed into oocytes (Pin et al., 1992), and as expected the AC-coupled mGluR3 does not generate such a response (Figures 2 and 3), even when PLC-activating G proteins of the Gq family are co-expressed with this receptor (data not shown). Replacing the second intracellular loop of mGluR3 with that of mGluR1 is not sufficient to enable chimeric receptor R3/1-(i2) (for the nomenclature of the chimeric receptors, see Materials and methods) to activate PLC. Similarly, chimeric receptor R3/lc-(C2), which possesses the Cterminal intracellular domain of mGluRlc, does not activate PLC (Figure 2). However, when both i2 and the C-terminal domain of mGluR3 are replaced by their equivalent domains of mGluRlc, the resulting chimeric receptor R3/lc-(i2,C2) induces an inward current when activated by Glu (Figures 2 and 3). We have also replaced the second intracellular loop of mGluRl with that of mGluR3. No responses could be detected upon Glu application in oocytes previously injected with the cRNA coding for this chimeric receptor (data not shown). This further strengthens the importance of i2 in the specific coupling to PLC. In order to delineate more precisely the region in i2 involved in the specific activation of PLC-coupled G protein, the N- or C-terminal parts of i2 were exchanged between mGluR3 and mGluRl. These chimeras also carried the Cterminal intracellular domain of mGluRlc (Figure 2). Among the four chimeras constructed, only R3/lc-(i2d,C2) was able to activate PLC, indicating that the 16 C-terminal residues of mGluRl i2 are necessary for PLC coupling. Similarly, we also tried to diminish the length of the Cterminal intracellular domain exchanged between mGluR3 and mGluRlc. Two chimeric receptors were constructed in among
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which the 19 (C4 segment, see Figure 2b) or the 28 (C5 segment, Figure 2b) N-terminal amino acid residues of the C-terminal tail of mGluR3 are replaced by their mGluRl homologues. These receptors also contain the entire i2 of mGluRl. Both chimeras activate PLC, indicating that the extreme C-terminal end of mGluRlc is not necessary for PLC coupling (Figures 2a and 3). Although the 19 residues located downstream of TMD VII appear sufficient for the activation of PLC, the responses induced by this chimeric receptor are delayed and much smaller than those induced by the chimera containing the entire C-terminal end of mGluRlc (Figure 3). Only when the 28 N-terminal residues of this intracellular tail are present is the chimeric receptor able to induce responses similar in amplitude and shape to those induced by R3/lc-(i2,C2) (Figures 2a and 3). Similar results were obtained with R3/1-(i2d,C4) and R3/1-(i2d,C5) carrying only the 16 C-terminal amino acid residues of the mGluRl i2 (Figures 2a and 3). In order to verify that responses induced by these chimeric receptors are due to the activation of PLC and the consequent release of Ca2+ from internal stores, we examined their Ca2+ dependence. Intracellular injection of EGTA totally abolishes the response induced by R3/lc-(i2,C2), indicating that it results from an increase in intracellular Ca2+ (Figure 4a). This is not due to Ca2+ entry, because the response is not modified in the absence of extracellular Ca2+ (Figure 4a). We also verified that the channel responsible for this response is a Cl- channel, as always observed for responses induced in oocytes by PLC-coupled receptors (Hirono et al., 1987). The reversal potential of the current induced by Glu is shifted towards more positive values when external Cl- concentrations are decreased 2500
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R3/1-(i2d,C4) -(i2,C5)
R3/1 -(i2d,C5)
Fig. 3. Typical traces obtained with the different wild type and chimeric receptors. Xenopus oocytes were injected with 10 ng of in vitro synthesized transcript and maintained in Barth medium at 18°C. After 3-10 days, oocytes were voltage-clamped at -70 mV using the two electrode voltage-clamp technique. The chamber containing the oocyte was perfused with Glu (200 iM) for 30 s starting 3 s after the beginning of the trace: horizontal bar, 16 s; vertical bar, 400 nA. 344
current depends on an increase in intracellular Ca2+. The maximal inward currents induced by 200 leM Glu were recorded from 5-10 oocytes injected with 10 ng of R3/1-(i2,C2) cRNA. Experiments were performed on control oocytes or on oocytes injected with either 20 nl H20 (H20) or 20 nl of 10 mM EGTA (EGTA) 15-0 min before recordings. Control oocytes were also recorded in the absence of extracellular Ca2+ (Ca-free), oocytes being perfused with a Barth medium devoid of added Ca2+ and containing 2 mM EGTA for 1.5 min before being stimulated with 200 1eM Glu in the same solution. Results are means + SEM of 5-10 determinations performed on different oocytes. (b) Variation of the reversal potential (Vrev) as a function of the extracellular Cl- concentrations. The calculated slope (-54) is close to the value expected according to the Nerst equation (-58 at 20°C) for a Cl- current. The reversal potential of the response elicited by Glu in R3/lc-(i2,C2) cRNA-injected oocytes was measured using 10 consecutive 1 second ramps from -60 to +40 mV in Barth's solutions containing various Cl- concentrations (90, 55 and 35 mM, Cl- being replaced by gluconate). Each point represents the means SEM of 5-10 determinations performed on different
oocytes.
G protein interaction domains of mGluRs
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needed to reach the maximal response (time to peak value) being 12.8 1.0 s (n 123). This is observed regardless of the maximal amplitude of the response (Figure 5a). In contrast, responses induced by R3/lc-(i2,C2) are rapid and often consist of a single peak which is rarely followed by long-lasting oscillations (Figure 3). The responses always reach their maximal value within the first 10 s (Figure 5b), regardless of the maximal amplitude of the response. The time to peak value [5.8 i 0.4 s (n 51)] is significantly smaller than that measured for mGluRlc-induced responses (P
