(Received for publication, August 30, 1995; and in revised form, November 3, 1995)
From the
Constitutively active thrombin receptors were generated while
constructing chimeric receptors to identify the structural basis for
thrombin receptor agonist specificity. Substitution of eight amino
acids from the Xenopus receptor's second extracellular
loop (XECL2B) for the cognate sequence in the human thrombin receptor
was sufficient to confer robust constitutive activity. Smaller
substitutions within the XECL2B site yielded less constitutive
activation, and substitution of several unrelated sequences at this
site caused no activation. Expression of the XECL2B receptor caused
high basal Ca efflux in Xenopus oocytes and high
basal phosphoinositide hydrolysis and reporter gene induction in COS
cells. Of note, a mutant receptor in which all four of the Xenopus thrombin receptor's extracellular segments replaced the
cognate human sequences showed much less constitutive activity than
XECL2B and preserved responsiveness to agonist. This partial
complementation of the XECL2B phenotype by addition of other Xenopus extracellular structures suggests that the XECL2B
mutation causes constitutive activation by altering interactions among
the human receptor's extracellular domains. Thus, a change in an
extracellular loop of a G protein-coupled receptor can transmit
information across the cell membrane to cause signaling, perhaps via a
conformational change similar to that caused by agonist binding.
Indeed, the site of the activating mutation in XECL2B coincides with a
putative agonist-docking site, supporting the hypothesis that agonist
interactions with the thrombin receptor's extracellular loops
contribute to receptor activation.
A variety of naturally occurring and engineered mutations in G protein-coupled receptors have been found to cause constitutive activation. Their importance is underscored by the observation that activating mutations in G protein-coupled receptors underlie a variety of human diseases. The locations of activating mutations both within a single receptor and across receptors are widespread, with activating mutations reported in transmembrane domains 2, 3, 6, and 7; in cytoplasmic loops 1 and 3; and at the junction of transmembrane domain 2 and extracellular loop 1 (reviewed in (1, 2, 3, 4) ; see also (5, 6, 7, 8, 9) ). This diversity suggests that specific interactions maintain G protein-coupled receptors in their off-state(s) and that these interactions can be disrupted in a variety of ways. We recently generated a constitutively active thrombin receptor in the course of studying chimeric thrombin receptors designed to identify the receptor domains that distinguish the human from Xenopus thrombin receptor agonist peptides. Building human thrombin receptor sequence into the Xenopus receptor (specifically small regions of the receptor's amino-terminal exodomain near transmembrane domain 1 and its second extracellular loop) conferred human receptor-like agonist specificity(10, 11) . These same receptor regions, particularly receptor residues 260-268 in the second extracellular loop, were identified by a second approach that sought receptor mutations that complemented loss-of-function mutations in the agonist(11) . In an attempt to confer Xenopus receptor-like specificity to the human receptor, we built Xenopus receptor sequences into the human receptor, the converse of the chimera experiments just described. Strikingly, several of the resulting chimeras were constitutively active. The substitution responsible for activation mapped to residues 259-268 in the receptor's second extracellular loop, the same region previously identified as responsible for agonist specificity. These studies clearly show that mutation of a G protein-coupled receptor's extracellular domain can cause transmembrane signaling. The observation that the site of the activating mutation is one previously shown to be involved in agonist recognition suggests that the activating mutation may cause a conformational change similar to that caused by agonist docking and supports the hypothesis that agonist interactions with extracellular loops can contribute to transmembrane signaling.
Constructs
were as follows. XenAT replaced human receptor amino-terminal exodomain
residues 1-92 with the cognate Xenopus sequence; XECL1,
extracellular loop 1 residues 160-174; XECL2, extracellular loop
2 residues 244-268; XECL3, extracellular loop 3 residues
333-349; and XECL All = XenAT + XECL1 + XECL2
+ XECL3. XECL2A replaced residues 244-254, and XECL2B
replaced residues 255-268. Chimeras with more limited
substitutions within the XECL2B region were also tested; the specific
substitutions are indicated in Fig. 1B. Control
substitutions within the XECL2B region are indicated in Fig. 1D. The phosphorylation site mutant C-tail Ser/Thr
Ala is the ``C-tail alanine substitution'' mutant
described in (17) . The S42P mutation substitutes a proline at
the P
` position of the thrombin receptor cleavage site to
inhibit cleavage(15) .
Figure 1:
A, constitutive activity of
chimeric thrombin receptors expressed in Xenopus oocytes. The
wild-type human thrombin receptor (HTR) or chimeric thrombin
receptors in which the Xenopus thrombin receptor's
amino-terminal exodomain (XenAT) or extracellular loops (XECL1, XECL2,
or XECL2) were substituted for the cognate human sequences were
expressed in Xenopus oocytes (see ``Experimental
Procedures''). Basal Ca release was measured as an
index of each receptor's constitutive activity, and maximally
stimulated
Ca release (10 nM thrombin) was
measured as an index of receptor function. The level of surface
expression for each chimera was determined by antibody binding. All
chimeras used in these studies did express on the oocyte surface, in
general at 50-150% of wild-type receptor levels. Surface
expression of constitutively active chimeras was less than that of the
wild-type receptor per unit of cRNA injected (see Fig. 3); thus,
the high basal
Ca release in oocytes expressing these
chimeras was not due to high expression levels. B, effect of
more limited substitutions within the second extracellular loop. The
wild-type human thrombin receptor or chimeras in which the first or
second half (XECL2A and XECL2B) of the second extracellular loop was
replaced by cognate Xenopus receptor residues as well as
chimeras with the indicated more limited substitutions in the XECL2B
region were expressed in oocytes. Receptor expression and basal
activity were determined as described for A. C,
``control'' substitutions. The wild-type or XECL2B chimeric
receptors or mutant receptors in which other amino acids replaced those
altered in XECL2B were expressed in oocytes (see D). Data in A-C are means ± S.D. (n = 3) and
are expressed as (
Ca release per 10-min interval for each
chimera)/(
Ca release per 10-min interval for the wild
type). All are representative of at least three replicate experiments. D, location and sequence of activating and control
substitutions. Substitution of Xenopus thrombin receptor
sequence for the cognate human sequence between residues 259 and 268 of
the human thrombin receptor yielded a chimeric receptor with
constitutive activity. Additional chimeras with control substitutions
at these sites (SGA, Conservative, and Random) did not show constitutive activity and mediated
maximal responses to thrombin comparable to that elicited by the
wild-type receptor.
Figure 3:
Partial complementation of XECL2B by other Xenopus receptor extracellular domains. Xenopus oocytes were injected with the indicated amounts of cRNA encoding
wild-type or chimeric receptors in which either the entire second
extracellular loop (XECL2) or all Xenopus extracellular
domains (XECL All) replaced the cognate human structures. After culture
for 24 h, surface expression levels and basal and thrombin-stimulated Ca release were measured (see Fig. 1). Data shown
are means ± S.D. (n = 3); this experiment was
replicated four times. Basal signaling by the wild-type human thrombin
receptor (HTR) and XECL2B was different (p <
0.05), and basal signaling by XECL2B and XECL All was different (p < 0.05) at both cRNA levels.
Constitutively active thrombin receptors were generated serendipitously while studying chimeric receptors to identify receptor domains mediating agonist recognition. The human and Xenopus thrombin receptors each respond preferentially to peptide agonists representing their respective tethered ligand domains. Substitution of small segments of the human thrombin receptor's amino-terminal exodomain and second extracellular loop for the corresponding Xenopus thrombin receptor segments yielded a chimeric receptor with human receptor-like agonist specificity. The single substitution that yielded the greatest gain of responsiveness to human agonist replaced Xenopus receptor residues 259-262 in the second extracellular loop with the cognate human sequence. A second approach that sought human receptor mutations that complemented loss-of-function mutations in the human agonist peptide also identified the human thrombin receptor's second extracellular loop, particularly residues 260-268, as participating in agonist recognition (10, 11) .
An attempt to confer Xenopus thrombin receptor-like specificity to the human thrombin receptor by building Xenopus receptor sequence into the human receptor (a ``mirror image'' of the chimera experiments described above) yielded a surprise. Because substitution of the human amino-terminal exodomain and second extracellular loop into the Xenopus thrombin receptor had conferred human receptor-like specificity(10) , we constructed a human thrombin receptor containing the Xenopus receptor's amino-terminal exodomain and second extracellular loop. Strikingly, this chimera showed constitutive activity (Fig. 1).
To identify the
substitution(s) responsible for constitutive activation, we constructed
chimeras in which individual Xenopus thrombin receptor
extracellular domains were substituted for the cognate human sequences.
A chimera bearing the Xenopus thrombin receptor's second
extracellular loop alone (XECL2) caused constitutively high Ca release when expressed in Xenopus oocytes;
chimeras bearing other Xenopus thrombin receptor extracellular
domains did not (Fig. 1). The receptor's second
extracellular loop can be logically divided into two segments by
Cys
, which is thought to form a disulfide bridge with
Cys
at the extracellular end of transmembrane domain 3.
Substitution of the carboxyl-terminal segment of the Xenopus receptor's second extracellular loop for the corresponding
human sequence resulted in a chimera designated XECL2B with robust
constitutive activity when expressed in Xenopus oocytes (Fig. 1). XECL2B exchanged residues 259-262,
264-266, and 268 in the human thrombin receptor for the cognate Xenopus residues (Fig. 1). Smaller Xenopus-for-human amino acid substitutions within this region
yielded less robust constitutive activity, and irrelevant or
conservative substitutions instead of the Xenopus residues
introduced by XECL2B failed to cause constitutive activation (Fig. 1). Thus, specific sequence must be substituted at the
XECL2B site to effect constitutive activation. This result contrasts
with that obtained with a well studied activating mutation in the
-adrenergic receptor. Mutation of Ala
in the
-receptor's third cytoplasmic loop
to any other amino acid caused constitutive activation, suggesting that
the mechanism of mutational activation is disruption of an interaction
that normally prevents the unliganded receptor from activating G
proteins (21) . By contrast, the observation that multiple and
specific amino acid substitutions are required for the constitutive
activation seen with XECL2B raises the possibility that the XECL2B
mutation causes a specific conformational change in addition to or
instead of simply disrupting interactions that maintain the receptor in
its off-state.
The constitutive activity of the XECL2B mutant receptor was manifest in mammalian cells as well as in the Xenopus oocyte expression system. Expression of XECL2B in COS-7 cells yielded high basal phosphoinositide hydrolysis and induced expression of a luciferase reporter gene under the transcriptional control of the c-fos 5`-regulatory region (Fig. 2). Activation of luciferase expression via this reporter reflects activation of the mitogen-activated protein kinase and other intracellular signaling pathways(20, 22) . Thrombin further stimulated phosphoinositide hydrolysis and luciferase expression in cells expressing XECL2B. The 20% increase in luciferase expression caused by thrombin in cells expressing XECL2B was less than the 2-fold increase for thrombin-induced phosphoinositide hydrolysis presumably because luciferase accumulated between XECL2B transfection and stimulation with thrombin. The fact that the XECL2B receptor could indeed respond to thrombin raised the question, might the constitutive activity seen in cells expressing XECL2B depend in part on tonic activation by residual thrombin from serum or by some other protease? Ablation of the thrombin cleavage site in the XECL2B receptor chimera (XECL2B-S42P) ablated its ability to respond to added thrombin, but did not alter its constitutive activity (Fig. 2). The constitutive activity of XECL2B thus does not require unmasking of the receptor's tethered ligand domain. This is consistent with the hypothesis that the XECL2B mutation increases the receptor's probability of entering an active conformation even in the absence of agonist. Alternatively, one could postulate that unstimulated XECL2B and wild-type receptors enter the active conformation with the same frequency, with the XECL2B mutation interfering with receptor uncoupling and shutoff.
Figure 2:
Constitutive activity of chimeric
receptors expressed in mammalian cells. A, phosphoinositide
hydrolysis. COS-7 cells were transiently transfected with the pBJ1
expression plasmid alone (Vector) or with plasmid directing
expression of the wild-type human thrombin receptor (HTR),
XECL2B, a mutant thrombin receptor lacking potential serine and
threonine phosphorylation sites (C-tail S/T A), or these same
cDNAs with the thrombin cleavage site mutated to a noncleavable form
(S42P). After culture for 36 h, cells were loaded with
[
H]myoinositol (see ``Experimental
Procedures''). At time 0, 20 mM LiCl was added with and
without 10 nM thrombin. Cells were then incubated for 120 min
at 37 °C to allow accumulation of
[
H]myoinositol phosphates, which were quantitated
as described under ``Experimental Procedures.'' Inositol
phosphate data are means ± S.D. (n = 3) of a
representative experiment; this experiment was replicated five times.
Basal signaling by the XECL2B construct differed from that of the
wild-type human thrombin receptor, with p < 0.05 by two-way
analysis of variance and Bonferroni's t test(25) . Surface expression of the wild-type human
thrombin receptor and mutant receptors was determined in parallel
cultures by antibody binding; data shown are the means of duplicate
determinations. B, c-fos promoter activation. COS-7
cells were transfected with vector, the wild-type thrombin receptor, or
XECL2B together with the luciferase reporter construct p2FTL (see
``Experimental Procedures''). 36 h after transfection, they
were incubated for an additional 3 h in the presence or absence of 10
nM thrombin. Luciferase activity in the cell extract was
measured as light emission in a luminometer. Data (mean ± S.D.; n = 3) are representative of five separate experiments.
Basal signaling of the wild-type human thrombin receptor and XECL2B was
different, with p < 0.05. Surface expression was measured
in parallel as described for A with duplicate determinations. C, comparison of concentration responses of XECL2B to the
wild-type receptor and a desensitization-defective mutant thrombin
receptor. Cells were transfected and loaded with
[
H]myoinositol as described for A and
then incubated with the indicated concentrations of thrombin for 120
min in the presence 20 mM LiCl.
[
H]Inositol phosphate accumulation over this
period was measured as described under ``Experimental
Procedures.'' Data shown are means ± S.D. (n = 3); this experiment was replicated twice. Surface
expression was measured as described for A. Note that basal
phosphoinositide hydrolysis in COS cells expressing the same receptors
is shown in A.
The major
uncoupling mechanism for regulating G protein-coupled receptors is
phosphorylation of activated receptors by G protein-coupled receptor
kinases(23, 24) . Previous studies showed that
replacing serines and threonines in the thrombin receptor's
carboxyl-terminal tail with alanines prevented its agonist-dependent
phosphorylation in Rat-1 and COS-7 cells and rendered it insensitive to
inhibition by coexpressed -adrenergic receptor kinase 2 in the Xenopus system(17) . We compared signaling by this
``desensitization-defective'' mutant (C-tail Ser/Thr
Ala) with that of XECL2B in COS-7 cells. Unlike XECL2B, the C-tail
Ser/Thr
Ala mutant did not cause constitutively high
phosphoinositide hydrolysis in the absence of thrombin (Fig. 2A). In the presence of thrombin, the C-tail
Ser/Thr
Ala mutant showed enhanced responses compared with
wild-type and XECL2B receptors, consistent with the hypothesis that
each activated C-tail Ser/Thr
Ala receptor coupled longer to
phosphoinositide hydrolysis before its signaling was terminated (Fig. 2C)(14) . The signaling behavior of
XECL2B is thus distinct from that of a mutant receptor with defective
desensitization. Overall, our data are most consistent with the
hypothesis that the XECL2B mutation increases the probability of the
receptor entering an active conformation.
The XECL2B mutation substitutes native sequence from the Xenopus thrombin receptor at the cognate position in the human thrombin receptor. The XECL2B sequence clearly does not cause constitutive activation in its native context in the Xenopus receptor. How is it that the wild-type human and Xenopus thrombin receptors do not show constitutive activity while the chimera does? A chimera in which the Xenopus thrombin receptor's entire second extracellular loop was substituted for the cognate human loop displayed less constitutive activity than XECL2B, and a chimera in which the Xenopus thrombin receptor's entire extracellular surface was substituted for that of the human receptor (XECL All) showed less still ( Fig. 1and Fig. 3). These chimeras showed robust signaling to thrombin, thus their lack of basal signaling was not due to a general loss of function. The partial complementation of the XECL2B phenotype by addition of other Xenopus extracellular structures implies a direct or indirect interaction of these structures and suggests two alternative mechanisms for XECL2B's gain of function. The XECL2B mutation may effect constitutive activation by interacting with neighboring exodomain structures, disrupting normal interactions among the human receptor's extracellular domains that help constrain the receptor in an off-state, and/or generating novel interactions that cause activation. Alternatively, the receptor's extracellular loops may interact only indirectly by constraining the arrangement of the receptor's transmembrane domains. In this model, the XECL2B mutation would cause activation via linkage of extracellular loop 2 to transmembrane domains. Explaining the complementation phenomenon described above with this second model is more cumbersome that with the first.
Previously identified activating mutations reside in putative transmembrane domains 2, 3, 6, and 7 and in the first and third intracellular loops(1, 2, 3, 4, 5, 6, 7, 8, 9) . In addition, an activating missense mutation at the junction of transmembrane domain 2 and extracellular loop 1 has been uncovered in the melanocyte-stimulating hormone receptor(5) . The location of the XECL2B mutation spans a putative asparagine-linked glycosylation site and is well within the thrombin receptor's predicted second extracellular loop. This mutation clearly shows that changes in an extracellular domain of a G protein-coupled receptor can cause transmembrane signaling and receptor activation. As noted above, two independent approaches identified human thrombin receptor residues 259-268 as important determinants of the receptor's agonist specificity. The XECL2B substitution involves this same region. The observation that the site of the XECL2B activating mutation overlaps a site shown to be important for agonist specificity, a putative agonist-docking site, is provocative. It suggests that the activating mutation may cause a conformational change similar to that caused by agonist docking and supports the hypothesis that agonist interactions with extracellular loops may be important for signal transduction.