(Received for publication, May 8, 1995; and in revised form, November 13, 1995 )
From the
Metabotropic glutamate receptors (mGluR) share no sequence
homology with any other G-protein-coupled receptors (GPCRs). The
characterization of their G-protein coupling domains will therefore
help define the general rules for receptor-G-protein interaction. To
this end, the intracellular domains of mGluR3 and mGluR1, receptors
coupled negatively to adenylyl cyclase and positively to phospholipase
C, respectively, were systematically exchanged. The ability of these
chimeric receptors to induce Ca signals were examined
in Xenopus oocytes and HEK 293 cells. The chimeric receptors
that still possessed the second intracellular loop (i2) of mGluR3
induced little or no Ca
signals, even though these
proteins were targeted correctly to the plasma membrane. Consistent
Ca
signals could be recorded only with chimeric
mGluR3 receptors that contains i2 and at least one other intracellular
domain of mGluR1. However, most intracellular domains of mGluR3 have to
be replaced by their mGluR1 equivalent to produce optimal coupling to G
protein. These observations indicate that i2 of mGluR1 is a critical
element in determining the transduction mechanism of this receptor.
These results suggest that i2 of mGluRs may play a role similar to i3
of most other GPCRs in the specificity of coupling to the G-proteins.
Moreover, as in many other GPCRs, our data revealed cooperation between
the different mGluR intracellular domains to control efficient coupling
to G-proteins.
Receptors coupled to G-proteins (GPCRs) ()constitute
a large family of receptor proteins with seven transmembrane domains.
Their agonist recognition site has been localized in a cavity formed by
the seven transmembrane domains in receptors activated by small ligands
like catecholamines (1) and peptides(2, 3) .
Their effector domains, the domains interacting with and activating the
G-proteins, have been studied extensively(4, 5) . It
has been proposed that their third intracellular loop (i3) plays a
critical role for the activation of
G-proteins(6, 7, 8, 9, 10, 11, 12) .
However, the binding and selective recognition of the G-protein depends
not only on the sequence of the i3 loop but is also influenced by other
intracellular
domains(13, 14, 15, 16, 17, 18, 19) .
The cloning of 8 metabotropic glutamate receptors (mGluR1-8)
and Ca-sensing receptors revealed the existence of a
new GPCR family(20, 21) . Whereas mGluR1,
mGluR5(22, 23, 24) , and the
Ca
-sensing receptors (25, 26) activate phospholipase C, the other mGluRs
inhibit adenylyl
cyclase(27, 28, 29, 30, 31, 32) .
Although mGluRs also possess 7 putative TMD, they do not show sequence
homology with the other GPCRs. Several lines of evidence suggest that
mGluRs do not operate like the other GPCRs. Their large (about 600
residues) extracellular N-terminal domains are homologous to bacterial
periplasmic binding proteins and constitute the agonist recognition
domain(33, 34) . Their intracellular domains also show
striking differences with the corresponding domains of the other GPCRs.
For example, i3 is very short and highly conserved among mGluRs and
Ca
-sensing receptors(21) , whereas it is the
longest and more variable loop in all other GPCRs. The DRY (or ERW)
tripeptide at the N-terminal end of i2 that is conserved in most GPCRs
and plays a critical role in G-protein coupling (13, 35) is absent in mGluRs. Nevertheless, and in
spite of these differences, mGluRs and Ca
-sensing
receptors probably activate the same set of heterotrimeric G-proteins
as all other GPCRs. The understanding of how mGluRs interact with and
activate G-proteins will therefore help define the general rules for
receptor-G-protein interaction.
In our preliminary analysis of rat mGluR domains involved in G-protein coupling selectivity, we only examined the role of i2 and the C-terminal tail of mGluR1 in the coupling to PLC(36) . In contrast to what has been reported for many GPCRs in which only one short segment plays a critical role in G-protein coupling, we found that the simultaneous exchange of those two domains of the adenylyl cyclase-coupled mGluR3 with those of mGluR1 generated a chimeric receptor which coupled to PLC. However, the kinetics of the responses induced in Xenopus oocytes by this chimeric receptor differed from those of the wild type mGluR1, suggesting the involvement of other intracellular domains in G-protein coupling. The aim of the present work was therefore: 1) to examine whether only one, like in other GPCRs, of the two previously identified domains was critical for the PLC coupling and 2) to examine whether other intracellular domains (i1 and i3) could also influence the coupling of mGluR1 to PLC.
Figure 2:
Summary
table of the chimeric receptors constructed and analyzed in this study. a, wild type receptors mGluR1a and mGluR1c. b, R3/1
chimeric receptors in which i2 has not been replaced by that of mGluR1. c, R3/1 chimeric receptors with at least i2 of mGluR1a; d, chimeric mGluR3 receptors with the C-terminal domain of
mGluR1c. e, mGluR1a chimeric receptors with i2 and i3 of
mGluR3, respectively. In the first column are the names of the chimeric
receptors with their respective scheme. The mGluR3 sequences are in white, mGluR1 segments are in black, and the specific
mGluR1c sequence is represented by the hatched box. The small black rectangles on top of each scheme indicate the
position of the seven transmembrane domains. In the second column are
the means ± S.E. of the maximal current amplitude (I in nanoamperes) obtained upon stimulation
with 300 µM Glu of oocytes injected with 10 ng of cRNA.
Values are from determinations (n) made on different oocytes (n) giving a response out of a total (t) of recorded
oocytes. In the third column are the means ± S.E. of the
time-to-peak values for responses with I
smaller
than 1000 nA. To obtain responses in this range of maximal amplitudes,
some experiments were conducted with oocytes injected with smaller
amounts of cRNA (3-0.5 ng). * indicates that the corresponding
value is statistically different (t test, p <
0.01) from that measured with mGluR1a.
The sequence of the domains
exchanged between mGluR1 and mGluR3 is the following: i1 of mGluR1 TLIF . . . SSRE
i1 of mGluR3
ITVF . . . SGRE
; i2 of mGluR1
RIAR . . . AWAQ
, i2 of mGluR3
CIAR . . . PSSQ
; i3 of mGluR1
NVPA
, i3 of mGluR3
KCPE
; C1 of mGluR1a
NSNGK . .
. stop inserted at the C-terminal end of mGluR3; C2 of mGluR1a or
mGluR1c
KPERN . . . stop, C2 of mGluR3
QPQKNV . . . stop.
24 h after transfection, HEK 293 cells grown on coverslips were washed three times in PBS, fixed for 15 min at room temperature in 3% paraformaldehyde in PBS, and washed three times in PBS. The fixed cells were then permeabilized with ice-cold methanol for 2 min. After washing four times with PBS, the cells were incubated for 1 h at room temperature in PBS containing 3% bovine serum albumin and rabbit anti-mGluR1a (1:500), anti-mGluR1c (1:1000), or anti-mGluR3 (1:2000) antibody. Cells were washed in PBS containing 3% bovine serum albumin, and bound primary antibodies were detected with a secondary antibody (1/200 dilution of fluorescein-labeled goat anti-rabbit IgG) for 30 min at room temperature. Cells were washed thoroughly, and the coverslips were mounted in 1,4-Diazabicyclo[2.2.2]octane (100 mg/ml) in PBS:glycerol (1:1). Fluorescence microphotographs were prepared on a Zeiss Axioskop microscope.
In our preliminary analysis of mGluR domains involved in G-protein coupling, we examined only the role of i2 and the C terminus of mGluR1c in determining the coupling to PLC(36) . However, sequence alignment of the mGluR intracellular domains also revealed that several residues in i1 and i3 are conserved in adenylyl cyclase-coupled mGluRs, but different in PLC-coupled mGluRs. For example, Arg-618, Ser-625, and Ser-627 (numbers correspond to the mGluR1 sequence) in the first intracellular loop of mGluR1 and mGluR5, are replaced by Asn, Ala, and Gly, respectively, in the adenylyl cyclase-coupled mGluRs (Fig. 1). Moreover, the neutral residue Ala-779 in the middle of i3 in PLC-coupled mGluRs is replaced by a glutamic acid residue in all adenylyl cyclase-coupled mGluRs (Fig. 1). Finally, the C-terminal intracellular domains show little homology between mGluRs(21) . A role for the C terminus in G-protein coupling is strengthened by the observation that mGluR1 splice variants, mGluR1a, mGluR1b, and mGluR1c, which have different C-terminal intracellular domains, are all coupled to PLC but have slightly different functional properties (37, 40) (see Fig. 2a). In mGluR1b and mGluR1c, the C-terminal 318 residues of mGluR1a are replaced by 20 and 11 residues, respectively(32, 37) . These observations suggested that additional intracellular domains may somehow be involved in the control of the interaction between mGluRs and the G-protein.
Figure 1:
Sequence alignment of the
intracellular domains of mGluRs and Ca-sensing
receptors. i1, i2, and i3 correspond to the
first, second, and third intracellular loops, respectively, and C
corresponds to the N-terminal segment of the C-terminal intracellular
domain. Residues conserved in all mGluRs are boxed. Residues
conserved in most mGluRs negatively coupled to adenylyl cyclase
(mGluR2-4 and -6-8) are boxed in black, and the
corresponding residues in PLC-coupled mGluRs are in bold.
Under the sequence of the Ca
-sensing receptors,
residues found to be identical at the equivalent position in at least
one adenylyl cyclase-coupled mGluR are indicated with -, those
found in at least one PLC-coupled mGluR are indicated with +,
those conserved in most mGluRs are boxed and indicated with
*.
In
order to analyze the exact role of the different intracellular domains
of mGluR1 in its coupling to PLC, we exchanged the equivalent domains
of the adenylyl cyclase-coupled mGluR3 with those of mGluR1a or
mGluR1c. The coupling to PLC of the resulting chimeric receptors was
first analyzed after transient expression in Xenopus oocytes.
In these cells, it is well established that activation of PLC results
in the activation of a Ca-activated chloride current
due to the IP3-induced release of intracellular
Ca
(41) . Not only the amplitude of the
current was measured, but also the time needed to reach the maximal
current after the response began (time-to-peak value). This later
parameter has previously been shown to be a functional characteristic
of a given receptor, not directly related to the level of expression of
this receptor(7, 11, 37, 42) . This
parameter can therefore be considered as a more accurate measure of the
efficiency of coupling of the receptor to this transduction pathway in
oocytes(7, 11, 37, 42, 43, 44) .
As shown in Fig. 2b, none of the chimeric receptors that still possess the second intracellular loop of mGluR3 elicited consistent responses when expressed in Xenopus oocytes. Although responses were recorded from some oocytes expressing the chimeric receptor R3/1a-(i1,i3,C2) (see ``Materials and Methods'' and Fig. 2for the nomenclature of chimeric receptors), these were very small and observed in only 8 out of 55 recorded oocytes. In contrast, many chimeric receptors that possess i2 of mGluR1 activated chloride current upon stimulation with 1 mM Glu (Fig. 2, c and d). Taken together, these results suggested that, in contrast to what has been reported for most GPCRs, i2 rather than i3 of mGluR1a plays a critical role in determining the PLC coupling of this receptor. To further support this conclusion, we constructed chimeric mGluR1a receptors with the second or third intracellular loops of mGluR3. The exchange of i3 in mGluR1a did not prevent the receptor from activating the chloride current when expressed in oocytes (Fig. 2e and Fig. 5), whereas no responses could be measured with the mGluR1a chimeric receptor with i2 of mGluR3.
Figure 5: Immunofluorescence detection of different wild type and chimeric receptors expressed in HEK 293 cells using an antibody directed against the C terminus of mGluR1a. a, mGluR3; b, mGluR1a; c, R3/1a-(C2); d, R3/1a-(i1,C2); e, R3/1a-(i1,i3,C2); f, R3/1a-(i2,C2); g, R3/1a-(i1,i2,C2); h, R3/1a(i2,i3,C2).
However, exchanging i2 of mGluR3 with that of mGluR1 was not sufficient to generate a receptor able to activate a chloride current in Xenopus oocytes. Glu-induced responses could be recorded if, in addition to i2, at least another intracellular loop (i1 and/or i3) was simultaneously exchanged (Fig. 2c and 3c). Chimeric receptors that contain i2 and the end of the C-terminal domain (C1 domain) of mGluR1a did not generate responses upon stimulation with Glu (Fig. 2c). However, the exchange of both i2 and the entire C-terminal intracellular domain of mGluR1a generated a chimeric receptor activating PLC in oocytes (Fig. 2c). In contrast to the responses induced by the wild type mGluR1a that were fast and transient (Fig. 2a and Fig. 3a), responses generated by this chimeric receptor R3/1a-(i2,C2) had time-to-peak values often greater than 10 s (Fig. 2c and Fig. 3d). As shown in Fig. 2c and Fig. 3, e and f, the additional exchange of either i1 or i3 in R3/1a-(i2,C2) generated receptors that induced fast responses in oocytes, similar to those induced by the wild type mGluR1a, regardless of the maximal amplitude of the response (Fig. 3, e and f). This suggests that most intracellular domains of mGluR1a have to be present to recover the functional coupling to the G-protein of the wild type mGluR1a. Similarly, among the chimeric receptors with the C terminus of mGluR1c, only the R3/1c-(i1,i2,i3,C2) receptor that contains all intracellular domains of mGluR1c, induced responses similar to those generated by mGluR1c (Fig. 2d).
Figure 3:
Analysis of the kinetics of the responses
induced by mGluR1a (a), mGluR1c (b), and the chimeric
receptors R3/1-(i1,i2,i3) (c), R3/1a-(i2,C2) (d),
R3/1a-(i1,i2,C2) (e), and R3/1a-(i2,i3,C2) (f). In
each case are presented: a serpentine scheme of the wild type and
chimeric receptors, with the mGluR1 sequences in black and the
mGluR3 sequence in white; a typical trace obtained upon
stimulation with 300 µM Glu. In each graph, the
time-to-peak values of individual responses are plotted against I. Scale bars: vertical, 200
nA; horizontal, 16 s.
The pharmacological profile of R3/1c-(i2,C2) previously has been studied extensively, and full agonist dose-response curves were constructed(36) . This revealed that the chimeric receptors had a pharmacology very similar to that of mGluR3. In the present study, we verified that the new R3/1 chimeric receptors constructed had a pharmacology similar to that of mGluR3, but distinct from that of mGluR1. As shown in Fig. 4, the potent agonist of mGluR1, quisqualate, did activate R1a/3-(i3) chimeric receptors, but not R3/1 receptors. In contrast, L-CCG-I which is a potent agonist at mGluR3, activated R3/1 chimeric receptors, but not R1a/3-(i3). Finally, 1S,3R-ACPD that activates both mGluR1 and mGluR3 activated all chimeric receptors tested.
Figure 4: Pharmacological analysis of chimeric receptors. Responses induced by 300 µM Glu, 1 µM quisqualate (Quis), 15 µML-CCG-I, and 10 µM 1S,3R-ACP dehydrogenase on R1a/3-(i3) (a), R3/1-(i1,i2,i3) (b), R3/1a-(i2,C2) (c), and R3/1a-(i1,i2,C2) (d) are presented.
The expression and coupling
to Ca signaling of chimeric receptors was also
examined in mammalian cells. After transient expression in HEK 293
cells, all chimeric and wild type receptors analyzed were found to be
expressed at the plasma membrane level as illustrated by
immunohistochemistry using antibodies directed against the C terminus
of either mGluR1a (Fig. 5), mGluR3, or mGluR1c (data not shown).
None of these antibodies gave immunostaining in mock-transfected cells
or cells expressing a receptor with a different C terminus ( Fig. 5and data not shown). The chimeric receptors that did not
activate chloride current in Xenopus oocytes did not induce
Ca
signal in HEK 293 cells when activated with 1
mM Glu (Fig. 6). In contrast, Glu induced clear
Ca
signals in HEK 293 cells expressing chimeric
receptors that were functional in oocytes (Fig. 6).
Figure 6:
Glu-induced Ca signals
in HEK 293 cells expressing wild type or chimeric receptors. a, mGluR1a; b, mGluR3; c, R3/1a-(C2); d, R3/1a-(i1,C2); e, R3/1a-(i1,i3,C2); f,
R3/1a-(i2,C2); g, R3/1a-(i1,i2,C2); h,
R3/1a-(i2,i3,C2). Traces show time course of
[Ca
]
monitored by the
ratio of fura-2 fluorescence exited at 340 and 380 nm, while cells were
superfused for 1 min with 1 mM Glu.
The present study was aimed at examining the role of all
intracellular domains of mGluR1a and mGluR1c in determining their
coupling to PLC-activating G-proteins. For that purpose, the different
intracellular domains of the adenylyl cyclase-coupled mGluR3 were
systematically exchanged by their mGluR1a or mGluR1c equivalent. Our
results revealed that all chimeric receptors able to strongly activate
the chloride current in Xenopus oocytes, or to induce
Ca signals in transfected HEK 293 cells, possess the
second intracellular loop of mGluR1. The importance of i2 in
determining the transduction mechanism of mGluR1 was strengthened by
showing that the exchange of i2 of mGluR1a with that of mGluR3
abolishes its coupling to PLC. Although the absence of a radioactive
ligand with high enough affinity prevented us from estimating the
receptor density in the transfected cells, immunofluorescence studies
indicated that all chimeric receptors including those that did not show
a Glu-induced Ca
signal were properly targeted to the
plasma membrane of HEK 293 cells. Whether some of these receptors
retained their ability to inhibit adenylyl cyclase has not been
examined yet.
These data indicate that, within the intracellular
domains of mGluR1, i2 plays a critical role in the activation of
PLC-coupled G-protein. Considering this and our previous
studies(36) , our data indicate that, within i2, the 16
C-terminal residues are necessary for optimum coupling to PLC. That
such a short segment plays a critical role in G-protein coupling in
mGluRs is supported by our recent observation that only few residues in
the subunit of G-proteins play a critical role in their selective
interaction with mGluRs. As observed with other G
-coupled
GPCRs(45) , G
-coupled mGluRs can activate PLC when
co-expressed with a chimeric G
subunit that has its 5
C-terminal residues replaced by those of G
. (
)Finally, it is interesting to note that the C-terminal
portion of i2 is likely to fold into an amphiphilic
helix(36) . Such a secondary structure has been proposed to be
important for the receptor domains involved in G-protein
activation(46, 47, 48, 49, 50, 51) ,
although this may not be a general rule(52) .
As previously
reported, the exchange of the second intracellular loop of mGluR3 with
that of mGluR1 is not sufficient to obtain a chimeric receptor able to
activate the chloride current in Xenopus oocytes. The
additional exchange of another intracellular domain is necessary. We
previously reported that the additional exchange of the C terminus of
mGluR1c was sufficient to allow the chimeric receptor to activate PLC
in oocytes(36) . Similar results were obtained with the
chimeric receptor with i2 and the entire C-terminal intracellular
domain of mGluR1a. Our data also revealed that the simultaneous
exchange of i2 and either i3 or i1 plus i3 also generated chimeric
receptors able to activate chloride currents in oocytes, even though
the C-terminal domain was not exchanged. Therefore, all intracellular
domains of mGluRs play a role in G-protein activation by facilitating
the action of i2. However, none of the specific residues found in i1,
i3, or the C-terminal intracellular domain of PLC-coupled mGluRs are
absolutely required for the activation of PLC. In agreement with this
conclusion, the sequences of i1 and i3 of the
Ca-sensing receptors (that activate PLC) are almost
identical with those of adenylyl cyclase-coupled mGluRs (Fig. 1).
As reported for other receptors expressed in Xenopus oocytes (7, 11, 37, 42, 43, 44) , the responses elicited by the R3/1 chimeric receptors can be either fast (small time to peak values) or more slowly generated (larger time to peak values). This difference is observed whatever the maximal amplitude of the responses and is therefore unrelated to the level of expression of the receptors. The kinetic of the response appears therefore as a good measure of the efficacy of coupling of a given receptor to this transduction cascade(7, 11, 44) . Interestingly, most of the intracellular domains have to be present in the R3/1 chimeras to recover the time to peak value of the corresponding wild type receptor (mGluR1a or mGluR1c). Taken together, these results suggest that the coupling efficacy of mGluRs is determined by the combination of all their intracellular domains. Accordingly, a cooperation between several intracellular domains in controlling either the specificity or the efficacy of coupling to G-proteins has already been reported for other GPCRs(8, 14, 16, 19) .
In conclusion, our results indicate that i2 of mGluR1 plays a critical role in the G-protein coupling selectivity of this receptor and may therefore be regarded as the equivalent of i3 of most other GPCRs. We show, moreover, that the coupling and activation of the G-protein involve a cooperation between all intracellular domains of mGluR1. Therefore, although the mGluR share no sequence homology with the other GPCRs, similar mechanisms have been selected during evolution for their coupling to G-proteins.