From the Division of Molecular Medicine, Department of Medicine,
Weill Medical College and Graduate School of Medical Sciences of
Cornell University, New York, New York 10021 and the
Department of Physiology and Biophysics, Mount Sinai
School of Medicine, New York, New York 10029
Received for publication, August 29, 2000, and in revised form, September 29, 2000
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ABSTRACT |
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Because charged residues at the
intracellular ends of transmembrane helix (TMH) 2 and TMH3 of G
protein-coupled receptors (GPCRs) affect signaling, we performed
mutational analysis of these residues in the constitutively signaling
Kaposi's sarcoma-associated herpesvirus GPCR (KSHV-GPCR). KSHV-GPCR
contains the amino acid sequence Val-Arg-Tyr rather than the
Asp/Glu-Arg-Tyr ((D/E)RY) motif at the intracellular end of TMH3.
Mutation of Arg-143 to Ala (R143A) or Gln (R143Q) abolished
constitutive signaling whereas R143K exhibited 50% of the basal
activity of KSHV-GPCR. R143A was not stimulated by agonist, whereas
R143Q was stimulated by growth-related oncogene- Kaposi's sarcoma-associated herpesvirus
(KSHV)1 (human herpesvirus 8)
is a In a previous report (10), we tested the hypothesis that the
amino-terminal extracellular domain of KSHV-GPCR may serve as a
"tethered ligand" that constitutively activates the receptor. Although we found that the amino terminus was important for high affinity binding of chemokines, we showed that the amino terminus was
not important for constitutive signaling. This observation was recently
confirmed (11). Therefore, other domains within KSHV-GPCR are involved
in causing the receptor to signal constitutively. Rosenkilde et
al. (11) identified residues at the extracellular loop/fifth
transmembrane helix (TMH5) and TMH6 boundaries, which when mutated
decreased agonist stimulation and residues in TMH2, which when mutated
decreased basal signaling. We sought to identify additional domains
within KSHV-GPCR that cause the receptor to signal constitutively. In
particular, we were interested in discovering mutations that could lead
to increased basal signaling so as to use computer models of these
receptors to delineate the structural changes that constitute KSHV-GPCR activation.
The highly conserved amino acid sequence, Asp/Glu-Arg-Tyr ((D/E)RY), at
the intracellular end of TMH3 of other GPCRs of the rhodopsin family
has been shown to be important in receptor activation. It was initially
shown that the charged pair of Glu-Arg was needed for rhodopsin
activation because double mutants of these residues failed to activate
transducin (12). With In this study, we have performed mutational analysis of the residues at
the intracellular end of TMH2 and of nonconserved Asp residues in TMH2
and TMH3 in KSHV-GPCR. We have focused primarily on the effects of
these residues on constitutive signaling, because it is easier to
understand the intramolecular interactions that lead to conformational
changes involved in receptor activation in the absence of agonist
binding. Arg-143 in KSHV-GPCR was determined to be critical for
signaling. Moreover, mutation of Asp-83 caused increased basal
signaling, and mutations at these two positions acted synergistically
to increase constitutive signaling. A model is presented that predicts
these site-specific substitutions act independently to activate
KSHV-GPCR.
Construction of KSHV-GPCR Mutants--
All KSHV-GPCR mutants
were constructed by PCR using pcDNA 3.1-KSHV-GPCR as template
unless otherwise stated. V142N, R143A, R143K, R143Q, Y144A, and Y144F
were constructed using the following oligonucleotides:
(a)
5'-TACTTGGATATCTTCAGTGTTGTGTGCGTCAGTCTAAACAGGTACCTCCTGGTGGCATATTCTACG-3'; (b)
5'-TACTTGGATATCTTCAGTGTTGTGTGCGTCAGTCTAGTGGCGTACCTCCTGGTGGCATATTCTACG-3'; (c)
5'-TACTTGGATATCTTCAGTGTTGTGTGCGTCAGTCTAGTGAAGTACCTCCTGGTGGCATATTCTACG-3'; (d)
5'-TACTTGGATATCTTCAGTGTTGTGTGCGTCAGTCTAGTGCAGTACCTCCTGGTGGCATATTCTACG-3'; (e)
5'-TACTTGGATATCTTCAGTGTTGTGTGCGTCAGTCTAGTGAGGGCCCTCCTGGTGGCATATTCTACG-3'; and (f)
5'-TACTTGGATATCTTCAGTGTTGTGTGCGTCAGTCTAGTGAGGTTCCTCCTGGTGGCATATTCTACG-3', respectively, as sense primers, and (g)
5'-CTAGTCTAGAGATCCTCCGGAGAGGCTAGATTAAATTAAGGGGGAAGGGCA-3' as the antisense primer. EcoRV and XbaI
restriction sites were used to clone the PCR fragments into pcDNA
3.1-KSHV-GPCR. All the mutants were confirmed by sequencing.
For mutants V142D, D132A, and D132N, the following antisense
oligonucleotides were used: (h)
5'-CAGGAGGTACCTGTCTAGACTGACGCACACAACACTGAAGAT-3'; (i)
5'-CAGGAGGTACCTCACTAGACTGACGCACACAACACTGAAGATAGCCAAGTAGACATATAAATAGTA-3'; and (j)
5'-CAGGAGGTACCTCACTAGACTGACGCACACAACACTGAAGATATTCAAGTAGACATATAAATAGTA-3', respectively, and (k)
5'-ATCCG-GCTAGC-AAGCTT-GAATTC-TCCGGA-CCACC-ATGGCGGCCGAGGATTTCCTAACCATCTTCTTA-3' as the sense primer. HindIII and KpnI
restriction sites were used to clone the PCR products into pcDNA
3.1-KSHV-GPCR, and the mutants were confirmed by sequencing.
Overlapping PCRs were performed to generate D83A and D83N KSHV-GPCRs.
In both constructions the same sense and antisense primers were used:
(l)
5'-CAAGTG-GAATTC-TCCGGA-AGATCT-AATATT-GTTAAC-CACC-ATGGCGGCCGAGGATTTCCTAACCATCTTC-3' and (m) 5'-ACTGCTTCTTGGGCCAGGAACGCGTAG-3'. The two
overlapping primers were as follows: (n)
5'-CGGGCAGGAGCGATAGCTATACTGCTCCTGGGTATCTGCCTAAAC-3' and (o)
5'-ACCCAGGAGCAGTATAGCTATCGCTCCTGCCCGCGATCGGTGCTT-3' for D83A; and
(p) 5'-CGGGCAGGAGCGATAAATATACTGCTCCTGGGTATCTGCCTAAAC-3' and
(q) 5'-ACCCAGGAGCAGTATATTTATCGCTCCTGCCCGCGATCGGTGCTT-3'
for D83N. EcoRI and EcoRV restriction sites
were used to clone the PCR products into pcDNA 3.1-KSHV-GPCR, and
the mutants were confirmed by sequencing.
The double mutant D83A/D132A was generated using primer k
and i as sense and antisense primer, respectively, using
D83A KSHV-GPCR as the template. HindIII and KpnI
restriction sites were used to clone the PCR products into pcDNA
3.1-KSHV-GPCR, and the mutants were confirmed by sequencing.
D83A/V142D, D83A/R143K, and D83A/R143Q double mutants were constructed
through digesting and ligating the respective single mutants using
EcoRI and EcoRV restriction sites.
Inositol Phosphate Accumulation--
COS-1 cells were
transfected using the DEAE-dextran method (5) with pcDNA 3.1 plasmids encoding the wild-type or mutant KSHV-GPCR. Mock transfectants
were cells transfected with plasmid not encoding a receptor. Various
amounts of plasmid DNA were used to attain different levels of receptor
expression. Transfected cells were re-seeded into 24-well plates
24 h after transfection in Dulbecco's modified Eagle's medium
with 5% Nu-Serum (Collaborative Research) and 1 µCi/ml
myo-[3H]inositol (PerkinElmer Life Sciences).
48 or 72 h after transfection, the medium was removed, and cells
were rinsed with Hank's balanced salt solution (HBSS) containing 10 mM HEPES, pH 7.4 (HHBSS). The cells were then incubated in
HHBSS containing 10 mM LiCl in the absence or presence of
growth-related oncogene Competition Binding--
Cells were transfected and seeded as
described above except that no
myo-[3H]inositol was added. 48 h after
reseeding, cells were rinsed once with cold HHBSS and then incubated in
cold HHBSS supplemented with 0.5% bovine serum albumin and 10 pM 125I-labeled Gro Computer Modeling--
Construction of the model of KSHV-GPCR
was as follows. The helical boundaries of KSHV-GPCR were determined
from a multiple alignment of the sequence of the receptor with those of
the nine known chemokine receptors. The conserved residues in TMH1
(Asn-65), TMH3 (Arg-143), TMH5 (Pro-233), and TMH7 (Pro-311) were used
to align the sequence with that of rhodopsin. Because TMH2, TMH4, and
TMH6 of KSHV-GPCR lacked the usual conserved residues of GPCRs of the
rhodopsin family, their boundaries were determined from the alignment
with the other chemokine receptors in which the conserved residues are
present. The assigned helical segments were superimposed on the C
The short loops, i.e. intracellular loops 1, 2, and 3 and
extracellular loop 1 were designed as turns such that the distance between the ends of the loops was approximately equal to the distances between the helices that the loops were connecting. Extracellular loop
2 was divided into two segments. The first extracellular loop 2 segment
spanned the sequence from the top of TMH4 to Cys-196, which putatively
forms a disulfide bridge with Cys-118 at the top of TMH3. The second
segment of extracellular loop 2 spanned the sequence from the disulfide
bridge to the point the loop connected with TMH5. Each of the segments
was designed as a turn. Because extracellular loop 3 is too long to
design as a turn, it was constructed by homology to a sequence
identified in a known structure of the protein 1EMS (Nit-Fragile
Histidine Triad Fusion Protein) with 77% similarity and 41% identity.
The loop has a distinct helical segment followed by a turn, and the
distance between the ends of the loop agrees well with the distance
between TMH6 and TMH7. The loops were connected manually and the entire
system was minimized with a distance-dependent dielectric
constant. The construction of the receptor was done with Insight and
the structure optimizations with CHARMM 26. The chemokine receptor
CXCR2 was constructed in a similar way using the aligned sequence from
the multiple alignment as above.
Statistical Analysis--
Statistical analyses were performed by
analysis of variance.
To show that Arg-143 at the intracellular end of TMH3 is involved
in signaling in KSHV-GPCR, Arg-143 was mutated to Ala, Gln, or Lys. The
three mutant receptors exhibited binding affinities for
125I-labeled Gro, and R143K, similar
to KSHV-GPCR, was stimulated further. These findings show that Arg-143
is critical for signal generation in KSHV-GPCR. In other GPCRs, Arg in
this position may act as a signaling switch by movement of its
sidechain from a hydrophilic pocket in the TMH bundle to a position
outside the bundle. In rhodopsin, the Arg of Glu-Arg-Tyr interacts with the adjacent Asp to constrain Arg outside the TMH bundle. V142D was
70% more active than KSHV-GPCR, suggesting that an Arg residue, which
is constrained outside the bundle by interacting with Asp-142, leads to
a receptor that signals more actively. Because the usually conserved
Asp in the middle of TMH2 is not present in KSHV-GPCR, we tested
whether Asp-83 at the intracellular end of TMH2 was involved in
signaling. D83N and D83A were 110 and 190% more active than KSHV-GPCR,
respectively. The double mutant D83A/V142D was 510% more active than
KSHV-GPCR. That is, cosubstitutions of Asp-83 by Ala and Val-142 by Asp
act synergistically to increase basal signaling. A model of KSHV-GPCR
predicts that Arg-143 interacts with residues in the TMH bundle and
that the sidechain of Asp-83 does not interact with Arg-143. These data
are consistent with the hypothesis that Arg-143 and Asp-83
independently affect the signaling activity of KSHV-GPCR.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-herpesvirus that has been implicated in the pathogenesis of
several human diseases including Kaposi's sarcoma (KS), primary effusion lymphoma, and a subset of multicentric Castleman's disease (1, 2). A G protein-coupled receptor (KSHV-GPCR) is encoded within the
genome of KSHV (3, 4). It has been shown that KSHV-GPCR exhibits
constitutive signaling via the phosphoinositide-specific phospholipase
C pathway (5). When expressed in mouse fibroblasts, KSHV-GPCR
stimulates cell proliferation, causes transformation, and promotes
angiogenesis mediated by vascular endothelial growth factor (6).
Recently, expression of this viral receptor was shown to induce the
formation of angioproliferative lesions in transgenic mice that were
similar to the lesions of Kaposi's sarcoma (7). Because persistently
activated GPCRs can function as human tumor genes (8), we suggested
that the constitutive signaling activity of KSHV-GPCR may play an
important role in KSHV-induced tumorigenesis (9). Understanding the
details of KSHV-GPCR structure that cause constitutive signaling,
therefore, may allow for the design of drugs to treat KS and primary
effusion lymphoma.
1B-adrenergic receptors, mutation
of the Arg to some amino acids caused inhibition of stimulated signaling whereas mutation to Lys increased basal signaling, and mutation of Asp to Ala caused a marked increase in basal activity (13,
14). In the receptor for gonadotropin-releasing hormone, which has an
Asp-Arg-Ser sequence at the intracellular end of TMH3, mutation of Arg
markedly decreased agonist-stimulated activation whereas the Asp to Asn
mutation was less active than the wild type (Asp
Ala did not express,
Ref. 15). In the CB2 cannabinoid receptor, mutation of Arg to Ala
inhibited agonist-stimulated signaling, Asp to Ala abolished signaling,
and Tyr to Ala inhibited signaling; there was no apparent effect on
basal signaling (16). Mutation of the Arg of the DRY sequence in the
oxytocin receptor caused the receptor mutant to be constitutively
active (17). With the histamine H2 receptor, which is constitutively
active, mutation of Asp caused increased constitutive signaling whereas mutation of Arg caused a decrease in signaling activity (18). The
sequence at the putative intracellular end of TMH3 in KSHV-GPCR is
Val-Arg-Tyr. It is particularly relevant, therefore, that mutation of
the Asp of the DRY sequence in the chemokine receptor CXCR2 (the human
receptor most homologous to KSHV-GPCR) to Val caused constitutive
activation of this receptor (19).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(Gro
), which is a chemokine agonist for
KSHV-GPCR (20, 21), for 2 h at 37 °C. Accumulated inositol
phosphates (IPs) were measured using ion-exchange chromatography as
described (22). IP accumulation was analyzed as
[3H]IPs/([3H]lipids + [3H]IPs).
(PerkinElmer Life
Sciences) in the absence or presence of various concentrations of
Gro
for 4 h at 4 °C. At the end of the incubation, cells
were washed twice with 1 ml of cold HHBSS containing 0.5 M
NaCl and lysed with 0.5 ml of 0.4 N NaOH. Cell lysates were
transferred to glass test tubes and counted in a gamma counter.
template of the TMH bundle designed by Baldwin et al.
(23).
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
that were within 2-fold of the affinity
of KSHV-GPCR (Figs. 1A and
2B). We measured constitutive
signaling activity of these receptors as ligand-independent signaling
that is directly related to the level of receptors expressed
transiently in COS-1 cells. Fig. 1B illustrates the results
from experiments in which the basal production of inositol phosphate
second messenger molecules was measured in populations of cells
expressing different levels of KSHV-GPCR and mutants of Arg-143. Basal
signaling was measured as the slope of the regression line of inositol
phosphate production versus receptor level. Mutation of
Arg-143 to either Gln or Ala abolished basal signaling
(p < 0.001) whereas mutation to Lys created a receptor
with 50% of the basal activity of KSHV-GPCR (p < 0.01)(Fig. 2A). Cells expressing R143A exhibited no
significant increase in signaling upon addition of Gro
whereas R143Q
was stimulated by 2-fold with Gro
, and R143K exhibited a response to
Gro
that was similar to KSHV-GPCR (Fig. 1C). These data
show that Arg-143 is important for basal and stimulated signaling by KSHV-GPCR.
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Fig. 1.
Effect of mutation of Arg-143 on KSHV-GPCR
binding, basal signaling, and Gro -stimulated
signaling. Human embryonic kidney EM cells were transfected with
plasmids encoding KSHV-GPCR, R143K, R143Q, or R143A. After 2 days,
125I-Gro
binding (A), basal inositol
phosphate (IP) production (B) and
Gro
-stimulated IP production (C) were measured as
described under "Experimental Procedures." For binding, the points
represent the mean ± S.D. of duplicate determinations in two
experiments. For basal IP production, the points represent the
mean ± S.D. of triplicate determinations in four experiments in
which both parameters were measured in the same cell populations. For
Gro
-stimulated IP production, the bars represent the
mean ± S.D. of triplicate determinations in a representative
experiment.
View larger version (46K):
[in a new window]
Fig. 2.
Comparison of the basal signaling activities
and binding affinities of KSHV-GPCRs. The experiments were
performed as described in the legend to Fig. 1. A, basal
signaling activity is defined as the slope of the line that represents
the regression analysis of inositol phosphate production
versus 125I-Gro binding. The dotted line is
basal signaling of KSHV-GPCR. B, equilibrium inhibitory
constants (Ki).
We next mutated Tyr-144 to Phe or Ala. Y144F and Y144A exhibited
binding affinities for 125I-labeled Gro (Fig.
2B), basal signaling activities (Fig. 2A), and
Gro
-stimulated signaling activities (data not shown) that were not
different from KSHV-GPCR. Thus, Tyr-144 is not important for KSHV-GPCR
binding and signaling.
In contrast to most GPCRs of the rhodopsin family, there is a Val
before Arg-Tyr at the intracellular end of TMH3 in KSHV-GPCR. As noted
above, mutation of the Asp in the DRY sequence of chemokine receptor
CXCR2 to Val caused the receptor to become more constitutively active
(19). With mutation of Val-142 to Asn and Asp in KSHV-GPCR, V142N
exhibited an affinity that was indistinguishable from KSHV-GPCR, and
V142D exhibited binding affinity for 125I-labeled Gro
that was within 2-fold of KSHV-GPCR (Fig. 2B). V142N
exhibited a basal level of signaling that was indistinguishable from
KSHV-GPCR (p > 0.1, Fig. 2A). Figs.
2A and 3 illustrate that V142D
is more basally active than KSHV-GPCR (p < 0.01). The
basal signaling activity of V142D, however, was only minimally
stimulated by Gro
(data not shown). These findings are different
from those reported by Rosenkilde et al. (11) who concluded
that V142D exhibited the same basal signaling activity as KSHV-GPCR. In
their analysis, however, Rosenkilde et al. (11) did not
compare basal signaling to receptor expression but compared it instead
to the amount of plasmid used in the transfection and assumed that the levels of receptor expression were similar. We have found that several
of the more active mutant receptors were expressed at lower levels than
KSHV-GPCR. Moreover, these levels varied from experiment to experiment.
We, therefore, determined receptor expression in each experiment to
measure basal signaling accurately.
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Based on data from experiments with other GPCRs, for example, the
1B-adrenerergic receptor (13, 14) and the receptor for
oxytocin (17), it has been suggested that the positive side chain of
the Arg of the (D/E)RY sequence may interact with a highly conserved
Asp residue in the mid-portion of TMH2. There is no Asp in the usual
position in TMH2 in KSHV-GPCR, but there is an Asp near the
intracellular end of TMH2 at position 83. With mutation of Asp-83 to
Ala and Asn, D83A and D83N exhibited affinities for 125I-labeled Gro
that were indistinguishable from
KSHV-GPCR (Fig. 2B). D83N exhibited basal signaling activity
that was 110% above KSHV-GPCR (p < 0.001), and D83A
exhibited activity that was 190% above KSHV-GPCR (p < 0.001)(Figs. 2A and 4). We
next constructed the double mutants D83A/R143K and D83A/R143Q to
determine whether these two mutations would interact to affect the
phenotype of the doubly mutated receptor. The hypothesis was that if
these mutations caused independent effects on basal signaling activity, then the basal activities of the double mutants would be at least the
sum of the individual mutations. D83A/R143K and D83A/R143Q exhibited
high affinity 125I-labeled Gro
binding that was similar
to KSHV-GPCR (Fig. 2B). D83A/R143K exhibited basal activity
that was 70% greater than KSHV-GPCR (p < 0.005), and
D83A/R143Q showed activity that was 30% above KSHV-GPCR
(p < 0.025, Fig. 2A). These levels of
signaling were equal to the addition of the effects of the individual
mutations and are consistent with the idea that these substitutions
caused effects that were independent of one another.
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Another negatively charged residue that could be part of a putative
hydrophilic pocket in the TMH bundle is Asp-132 in TMH3 in KSHV-GPCR.
With mutation of Asp-132 to Ala and Asn, D132A exhibited an affinity
that was within 2-fold of KSHV-GPCR and D132N exhibited an affinity
that was indistinguishable from KSHV-GPCR (Fig. 2B). D132A
exhibited activity that was 60% above KSHV-GPCR (p < 0.01), and D132N exhibited basal signaling activity that was similar to
KSHV-GPCR (p > 0.1)(Figs. 2A and 4). D132A
was not stimulated further by Gro whereas D132N was stimulated by
Gro
to the same extent as KSHV-GPCR (data not shown). We constructed
the double mutant D83A/D132A that exhibited similar basal signaling
activity as D83A (Fig. 2A) and was not stimulated by Gro
(data not shown). Thus, a negatively charged residue at position 132 was not as important as at position 83 in maintaining the wild-type
level of basal activity. Moreover, the lack of additive effects of
substituting Ala for Asp-83 and Asp-132 is consistent with the idea
that these substitutions affected basal signaling via similar
mechanisms (see below).
We next constructed the double mutant D83A/V142D. Although D83A/V142D
exhibited the same high affinity for 125I-labeled Gro as
KSHV-GPCR, the level of D83A/V142D expression was always lower than
KSHV-GPCR when the same amounts of plasmid DNA were used for
transfection. D83A/V142D exhibited basal signaling activity that was
510% greater than KSHV-GPCR (p < 0.001, Figs. 2A and 4). That is, the effect of having both mutations in
the same receptor was synergistic with regard to basal signaling
compared with D83A and V142D with individual mutations. This finding is consistent with the idea that these two substitutions act independently to activate the receptor (see below). The signaling activity of D83A/V142D was not further stimulated by Gro
(data not shown). D83A/V142D was the most active KSHV-GPCR receptor mutant we have found.
Because other constitutively active GPCR mutants have been shown to be
less stable than native, basally inactive receptors (18, 24), it is
possible that the low level of expression of D83A/V142D is caused by
its decreased stability.
Lastly, to determine whether these most constitutively active receptors
could be inhibited by the three chemokines found to be inverse agonists
of KSHV-GPCR (21, 25-27), we measured the effects of interferon
-inducible protein-10, stromal-derived factor-1 and viral monocyte
inflammatory protein-II. All three chemokines inhibited V142D and D83A
to an extent similar to inhibition of KSHV-GPCR but none of them had a
consistent effect on basal signaling by D83A/V142D (Fig.
5). We cannot provide an explanation for
these differences because the mechanism by which inverse agonists inhibit KSHV-GPCR signaling is not known. It seems, however, that the
ability of the inverse agonists to inhibit signaling by these receptors
was inversely related to the level of constitutive activity of the
receptor. Other KSHV-GPCR mutants have been found to be unresponsive to
inverse agonist inhibition but these were receptors with mutations in
their amino-terminal domains that caused loss of binding (10, 11).
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We constructed a computer model of KSHV-GPCR to help explain our
findings (Fig. 6). The model predicts
that Val-142 and Arg-143 in KSHV-GPCR are at the intracellular end of
TMH3. In rhodopsin (28), in which the sequence at the intracellular end
of TMH3 is ERY, as well as in our model of CXCR2 (not shown) in which the sequence is DRY, the side chain of Arg-143 interacts with the
negatively charged residue proximal to it. In KSHV-GPCR such an
interaction is impossible because Val-142 replaces the negatively charged residue. Thus, the side chain of Arg-143 has no partner to
stabilize its positive charge.
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Asp-83, which is predicted to be positioned at the intracellular end of TMH2 in KSHV-GPCR, is highly conserved in the chemokine receptor family. In other receptors of the rhodopsin family, this residue is an Asn. An examination of the model of KSHV-GPCR (Fig. 6) shows that this residue is directed inbetween TMH1 and TMH7 and is stabilized by a set of interactions with backbone N-H groups. It is also positioned close to Arg-78 in intracellular loop 1. The position and orientation of Asp-83 place it on the opposite side of TMH2 pointing away from TMH3. Thus, this residue shows no interaction with Arg-143 in TMH3. This is in full agreement with the position of the conserved Asn in TMH2 of rhodopsin as seen in its recent crystal structure (28). The model predicts that Arg-143 and Asp-83 do not interact. As described above, our experimental findings provide functional evidence that these residues do not interact also.
Because basal signaling by KSHV-GPCR is very active (5), we hypothesize that its structure is a model for the active states of GPCRs of the rhodopsin family including agonist-occupied receptors. Study of the structure-function relationship of constitutively active GPCRs (8) has the advantage of elucidating the three-dimensional structure of the active state of the receptor in the absence of a complicating ligand. This approach has been taken in a small number of instances. For example, we showed that models of the three-dimensional structure of constitutively active receptors for thyrotropin-releasing hormone (TRH) predict conformations similar to that of the TRH-occupied receptor (29). Lin et al. (30) predicted from a model of the luteinizing hormone receptor the changes in receptor structure that cause activation and used functional analysis of naturally occurring, constitutively active receptors to support their predictions. We suggest that further structure/function studies of basally active GPCRs will provide important insights into the mechanism of receptor activation.
In the study reported here, we constructed and characterized a number
of mutant receptors in which charged residues in TMH2 and TMH3 were
substituted to gain insight into the role of these residues in
signaling by KSHV-GPCR. As discussed in the Introduction, charged
residues within the (D/E)RY sequence at the intracellular end of TMH3
have been shown to play important roles in signaling in a number of
GPCRs (12-19). Charged residues in other domains within GPCRs have
been shown to be important for signaling also. For example, Perez and
coworkers (31, 32) presented evidence that disruption of a salt-bridge
between an Asp in TMH3 and a Lys in TMH7 may be involved in
1b-adrenergic receptor activation. Donohue et
al. (33) developed support for the idea that a salt-bridge between
an Asp at the extracellular loop 1/TMH2 boundary and an Arg at the
extracellular loop 3/TMH7 boundary constrained the gastrin-releasing
peptide receptor in conformation that is needed for coupling to G
proteins. We showed that an Arg residue in TMH6 of the TRH receptor was
involved in receptor activation (34). Here we found several different
phenotypes with regard to signaling properties of KSHV-GPCR mutants
substituted at charged residues even though all the mutant receptors
exhibited high binding affinity for Gro
. D132N exhibited basal and
Gro
-stimulated signaling activity indistinguishable from KSHV-GPCR.
R143Q had little basal activity but exhibited-fold stimulation by
Gro
that was similar to KSHV-GPCR whereas R143K had a 50% reduced
basal activity but was stimulated by Gro
to the same level as
KSHV-GPCR. In contrast, R143A did not signal basally and could not be
stimulated by Gro
. A number of mutant receptors such as D83A, D83N,
and D132A exhibited higher basal activity than KSHV-GPCR but were only
minimally stimulated by Gro
. Thus, some single site-specific
substitutions of charged residues led to receptors that were more
basally active than KSHV-GPCR. The double mutants D83A/R143K and
D83A/R143Q exhibited basal signaling activity that appeared to be equal
to the additive effects of the single mutations. That is, these
receptors exhibited basal activities that were intermediate between
those of D83A and R143K or R143Q. Perhaps the most interesting receptor
phenotype was that exhibited by the double mutant D83A/V142D. This
mutant had basal signaling activity that was markedly greater than
either single mutant D83A or V142D (see below). Mutations in other
domains of KSHV-GPCR have previously been shown to generate receptors with different phenotypes including receptors that do not bind ligands
with high affinity (10, 11), receptors that exhibit higher basal
signaling than KSHV-GPCR but are not stimulated by Gro
(11) such as
D83A, D83N, and D132A, and receptors that exhibit lowered basal
activity but could be stimulated by agonist (11), such as R143Q and
R143K. Thus, selective mutation of KSHV-GPCR can lead to different
phenotypes, similar to what has been reported with other GPCRs.
The phenotype of the double mutant D83A/V142D was unexpected. Before the construction of our model, we believed that Arg-143 and Asp-83 may interact, as has been proposed for the Arg of (D/E)RY and a highly conserved Asp in TMH2 of other GPCRs (13, 15), even though Asp-83 was not in the same position in TMH2 as the highly conserved Asp. Our findings, however, are consistent with the idea that Asp-83 at the intracellular end of TMH2 and Arg-143 at the intracellular end of TMH3 are important for KSHV-GPCR signaling but act independently of one another. We draw this conclusion based in particular on the observation that the double mutant D83A/V142D exhibits a higher level of constitutive signaling than would be expected by the addition of the increased signaling exhibited by the two single mutants D83A and V142D. Indeed, if Arg-143 and Asp-83 interact in KSHV-GPCR, we would expect that the double mutant would exhibit basal signaling activity equal to that of one of the individual mutant receptors. Moreover, our model is consistent with independent effects of substituting these two residues because the model of KSHV-GPCR predicts that the sidechain of Arg-143 resides in a hydrophilic pocket formed by helices 1, 2, and 7 within the TMH bundle and the sidechain of Asp-83 faces outside the bundle in a position on the side of TMH2 away from TMH3 near intracellular loop 1. That is, these two residues are predicted to be in the wrong orientation relative to one another and too far apart to interact. We can speculate on a mechanism to explain this phenotype that is consistent with the predictions of our model and with our data. It is possible that Arg-143 resides in the TMH bundle in a partially activated receptor and that movement out of the bundle caused by agonist stimulation in KSHV-GPCR or by interaction with Asp substituted at position 142 in V142D leads to further activation. This may occur because Arg-143 out of the TMH bundle interacts with residues in intracellular loop 3 to promote higher affinity coupling to G protein. Asp-83 interacts with residues in intracellular loop 1 in a partially activated receptor and upon agonist stimulation in KSHV-GPCR or substitution by Ala in D83A this interaction is lost, and the loss of this constraint allows intracellular loop 1 to couple to G protein more effectively. The synergism occurs because when both of these changes occur in the same receptor, the coupling between receptor and G protein is markedly enhanced. These observations support the concept that a receptor can exist in multiple conformational states along a continuum from completely inactive to fully active (35).
In conclusion, we have found that charged residues, Arg-143 and Asp-83,
at the intracellular ends of TMH2 and TMH3, respectively, upon mutation
affect signaling by KSHV-GPCR. We suggest that these residues are
directly involved in KSHV-GPCR signaling perhaps by mediating changes
in receptor conformation or by interacting with the G protein. It is
possible, however, that the signaling changes observed upon their
mutation may be caused by indirect effects. Arg at this position, which
is highly conserved in all GPCRs of the rhodopsin family, has been
shown to be important in signaling in all receptors in which the
residue has been studied. Asp at this position, which is found in all
chemokine receptors but not in most members of the rhodopsin family,
has not been studied in mammalian chemokine receptors. Substitutions
that we predict affect both of these residues in D83A/V142D lead to a receptor with more than 6 times the basal signaling activity of KSHV-GPCR but which no longer responds to stimulation by agonist or to
inhibition by inverse agonist. KSHV-GPCR is thought to be a pirated
chemokine receptor that has mutated to serve an important, but unknown
function(s) in the viral life cycle. Our findings show that additional
mutation of KSHV-GPCR can create a receptor that is more basally active
and not regulatable by chemokines. We suggest, therefore, that the
phenotype of high basal but chemokine-regulated signaling activity are
attributes of KSHV-GPCR that are important to KSHV survival.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Weill Medical College of Cornell University, 1300 York Ave., Rm. A328, New York, NY 10021. Tel.: 212-746-6275; Fax: 212-746-6289; E-mail: mcgersh@mail.med.cornell.edu.
Published, JBC Papers in Press, October 12, 2000, DOI 10.1074/jbc.M007885200
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ABBREVIATIONS |
---|
The abbreviations used are:
KSHV, Kaposi's
sarcoma-associated herpesvirus;
GPCR, G protein-coupled receptor;
TMH, transmembrane helix;
PCR, polymerase chain reaction;
HBSS, Hank's
balanced saline solution;
HHBSS, HEPES HBSS;
Gro, growth-related
oncogene-
;
IP, inositol phosphate.
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