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INTRODUCTION |
Members of the superfamily of G protein-coupled receptors
(GPCRs)1 share a heptahelical
transmembrane topology and similar mechanisms for transducing signals
through trimeric G proteins to intracellular effectors. Subfamilies of
GPCRs, distinguished by sequence identity, generally include several
receptor isoforms or subtypes that can be activated by similar ligands.
Although receptor subtypes within a GPCR subfamily often regulate
similar intracellular effectors, they can also signal to distinct
effectors and signaling networks through a process termed receptor
subtype-specific signaling. Using the subfamily of somatostatin
receptors as a model, the objective of this study was to determine a
molecular basis for subtype-specific signaling by GPCRs.
The somatostatin receptor family includes five subtypes (SSTR1 to
SSTR5) (1-5) that belong to the class A group of GPCRs. Each member is
activated by the neuroendocrine peptide somatostatin-14 (SST). SST
inhibits diverse cell functions such as hormone secretion, neurotransmitter release, smooth muscle contractility, and cell proliferation. These cellular effects are mediated by coupling of the
five SSTR subtypes to similar as well as distinct effectors and
signaling networks. All five subtypes couple to the inhibition of
adenylyl cyclase (6) and the activation of tyrosine phosphatases (7-13). Different SSTR subtypes, however, also regulate distinct effectors. SSTR2 to -5, but not SSTR1, activate the GIRK1 inwardly rectifying K+ channel (14), SSTR1 and -2 inhibit
voltage-activated Ca2+ channels (15-17), SSTR2 and -5 stimulate phospholipase C activity (8, 18), and only SSTR4 has been
shown to stimulate phospholipase A2 (19).
SST acting at endogenous SSTRs in enteric endocrine cells (20) and
hepatic cells (21) also inhibits the activity of the ubiquitously
expressed Na-H exchanger NHE1. When heterologously expressed in
fibroblasts, SSTR1, but not SSTR2, mediates this effect (22). Using
chimeric SSTR2/SSTR1 receptors, we found previously that the
collective, but not individual, replacement of intracellular domains 2 (IL2) and 3 (IL3) of SSTR2 with those of SSTR1 is sufficient to confer
inhibition of NHE1 (22). This suggested that receptor subtype-selective
coupling to NHE1 might require an interactive conformation of these two
intracellular domains. From previous studies on GPCR regions critical
for selective signaling, a general principle has emerged that the
relative contribution of different intracellular domains to the
selectivity of G protein recognition, and hence effector coupling,
varies among different classes of GPCRs (reviewed in Ref. 23).
Currently, there is no a priori certainty that related
structural domains have similar functions in different receptors
(24).
In the present study we identified selective amino acid motifs in IL2
and IL3 of SSTRs that confer the inhibition of NHE1. We first extended
the investigation of SSTR inhibition of NHE1 and found that, when
stably expressed in fibroblasts, SSTR3 and -4, but not SSTR5, mediated
SST inhibition of NHE1. Using a FASTA search of the Swiss
Protein Database and aligning amino acid residues of IL2 and IL3 of 16 SSTRs retrieved with PILEUP, we found that consensus motifs of
T(s,p)V in IL2 and QQ(r) in IL3 were present in NHE1-coupled
SSTR1, -3, and -4 but absent in SSTR2 and -5, which do not signal to
NHE1. The collective, but not individual, replacement of cognate amino
acids in SSTR2 with these motifs was sufficient to confer SST
inhibition of NHE1. Moreover, we found that these IL2 and IL3 motifs
are present in selective subtypes of class A GPCRs within the
D2-dopamine,
2-adrenergic, and thromboxane A2 subfamilies. We previously determined that
D2-dopamine receptors couple to the inhibition of NHE1 (25,
26) and now show that activation of the
2B-adrenergic
receptor, which contains IL2-TV and IL3-QQ residues, but not the
2A-adrenergic receptor, which contains IL2-TV, but not
IL3-QQ, also inhibits NHE1. Together these findings provide a molecular
basis for receptor subtype-specific signaling, suggesting that common
motifs shared by distinct subfamilies of GPCRs can be used to regulate
a common effector.
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EXPERIMENTAL PROCEDURES |
Receptor Constructs, Cell Culture, and Transfection--
The
cDNAs for rat SSTR3 and rat SSTR4 were provided by Cyanamid
(Princeton, NJ) and subcloned into pcDNA1 (Invitrogen) at EcoRI/XbaI sites. Mouse fibroblast
Ltk- cells and CHO-K1 cells were transfected with SSTR3,
and Ltk- cells were transfected with SSTR4 by using
Ca2+-phosphate precipitation as previously described (22).
The cells were co-transfected with pRSVneo. G418-resistant clones were
selected and screened for receptor expression by radioligand binding
with [125I-Tyr11]SST. Ltk-R3 and Ltk-R4 cells
were maintained in DME-H21 medium containing 5% fetal bovine serum
(FBS) and 0.2 mg/ml G418, and CHO-R3 cells were maintained in F-12
Hamm's medium containing 10% FBS and 0.2 mg/ml G418. CHO-K1 cells
stably expressing SSTR5 (CHO-R5) were provided by Y. C. Patel
(Royal Victoria Hospital and Montreal Neurological Institute, Montreal,
Canada) (27) and maintained in F-12 Hamm's medium containing 10% FBS
and 0.2 mg/ml G418. hSSTR1 and -2 were obtained from G. Bell
(University of Chicago) and subcloned into the mammalian expression
vector pCMV at HindIII/XbaI sites.
hSSTR2 mutants, hSSTR22TV,
hSSTR23WQQ, and
hSSTR22TV3WQQ, were constructed by replacing
arginine-threonine (RT) in IL2 and
serine-serine-lysine (SSK) in IL3 of hSSTR2 with cognate threonine-valine (TV) and tryptophan-glutamine-glutamine (WQQ)
of hSSTR1, respectively. Collective mutants were constructed by using a QuikChange Site-directed Mutagenesis Kit (Stratagene, Inc.)
with specifically designed primers of
5'-CAAGTGGAGGAGACCCACGGTGGCCAAGATGATCACCATG-3' and
5'-CATGGTGATCATCTTGGCCACCGTGGGTCTCCTCCACTTG-3' for IL2
mutations and 5'-CTCTGGAATCCGAGTGGGCTGGCAGCAGAGGAAGAAGTCTGAGAAGAAG-3'
and 5'-CTTCTTCTCAGACTTCTTCCTCTGCTGCCAGCCCACTCGGATTCCAGAG-3' for
IL3 mutations. DNA sequencing confirmed the indicated
mutations in the hSSTR2 mutant constructs. cDNAs of
hSSTR1, hSSTR2, and hSSTR2 mutants
were co-transfected with pRSVneo into clonal CCL39 fibroblasts, which
do not express endogenous SSTR, using the Transfast transfection reagent (Promega, Inc.). Clones stably expressing wild-type and mutant
SSTRs were selected by G418 resistance (0.6 mg/ml) and screened for
receptor expression by radioligand binding with
[125I-Tyr11]SST. Positive clones were
maintained in DME-H21 medium supplemented with 5% FBS and 0.2 mg/ml
G418. HEK293 cells stably expressing
2A- and
2B-adrenergic receptors were obtained from J. Donello and K. Kedzie (Allergan) and maintained in DME-H21 medium supplemented with 5% FBS.
Radioligand Binding--
Radioligand binding to total cell
membranes was used to determine receptor expression in the cell clones
stably expressing SSTR1 to 5. Cell membranes, prepared by hypotonic
lysis of cells (22), were resuspended in 50 mM Tris/HCl (pH
7.4) containing 5 mM MgCl2, 0.2 mg/ml
bacitracin, 20 µg/ml leupeptin, 1 µg/ml soybean trypsin inhibitor,
and 20 µg/ml aprotinin. Membranes (10 µg) were incubated in the
resuspension buffer containing 0.06 nM
[125I-Tyr11]SST (Amersham Biosciences;
specific activity, 2000 Ci/mol) for 60 min at 30 °C in the absence
(total binding) or presence (nonspecific binding) of 100 nM
SST (Bachem). The dissociation constant (Kd) was
determined by incubating cells in HBSS with
[125I-Tyr11]somatostain-14 and serial
dilutions of unlabeled SST-14. Values for Kd were
determined from displacement curves using GraphPad Prism, version 3.0. The binding reaction was terminated by vacuum filtration over Whatman
GF/F glass filters. Filters were washed three times with resuspension
buffer, and bound radioactivity was subjected to counting in a Packard
-spectrometer. Specific binding was determined by subtracting the
values for nonspecific binding from the values for total binding. Data
represent the mean ± S.E. of three separate membrane preparations.
Intracellular cAMP Accumulation--
SSTR function was confirmed
by the ability of SST to inhibit cAMP accumulation. Cells plated at
0.5 × 105 cells/well in 24-well plates and maintained
for 48 h at 37 °C were incubated with the phosphodiesterase
inhibitor 3-isobutyl-1-methylxanthine (100 µM) for
10 min at 37 °C in the absence or presence of 10 µM
forskolin or forskolin plus 100 nM SST. Cells were washed
with phosphate-buffered saline and lysed with 5% trichloroacetic acid. The acid-solubilized intracellular cAMP in the cell lysate was determined by using a cAMP radioimmunoassay (Biomedical Technologies, Inc.) according to the suggested protocol of the manufacturer. Data
represent the mean ± S.E. of three separate cell preparations.
NHE1 Activity and pHi--
NHE1 activity was
determined by measuring the rate of pHi recovery
(dpHi/dt) from an
NH4Cl-induced acid load in a nominally
HCO3--free Hepes buffer (28). Cells plated on
glass coverslips were maintained in the absence of serum for 16-20 h
before pHi determinations, transferred to a
Hepes buffer, and loaded with the acetoxymethyl ester of the
pH-sensitive fluorescent dye,
2,7-biscarboxyethyl-5-(6)-carboxyfluorescein (BECEF-AM, 1 µM; Molecular Probes, Inc.) for 10 min at 37 °C. The
coverslips were placed in a cuvette maintained in a thermostatically controlled cuvette holder within a Shimadzu RF5000 spectrofluorometer. BCECF fluorescence was measured at 530 nm by exciting the dye alternately at 500 and 440 nm. The emission ratio was calibrated to pH
for each determination by using the high K+/nigericin
(Molecular Probes, Inc.) technique (29). To determine NHE1 activity,
cells were pulsed for 10 min with 30 mM NH4Cl. The rate of pHi recovery
(dpHi/dt) from an acid load induced by the rapid removal of NH4Cl was calculated
at pHi intervals of 0.05 unit by measuring the
slope of the pHi tracing at the indicated
pHi. Steady-state pHi was
determined in a Hepes buffer in the absence of an NH4Cl
prepulse. Changes in quiescent NHE1 activity and
pHi were determined by adding 10% FBS in the
absence or presence of 100 nM SST during the
NH4Cl prepulse and recovery periods. Data represent the
mean ± S.E. of four to six separate cell preparations.
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RESULTS |
Subtype-specific Coupling of SSTR to the Inhibition of
NHE1--
We previously reported that, when stably expressed in
Ltk- cells or transiently expressed in HEK293 cells,
SSTR1, but not SSTR2, mediates SST inhibition of NHE1 activity (22). To
investigate whether the inhibition of NHE1 is shared by other SSTR
subtypes, we stably expressed SSTR3 and -4 in Ltk- cells.
After three separate transfections, we were unable to obtain
Ltk- cells stably expressing SSTR5. We therefore used
CHO-K1 cells stably expressing SSTR5 (27) and established CHO-K1 cells
stably expressing SSTR3. Receptor expression was determined by
radioligand binding using [125I-Tyr11]SST,
and receptor function was confirmed by the ability to mediate SST
inhibition of forskolin-induced cAMP accumulation (Fig.
1). All three SSTRs coupled to the
inhibition of cAMP accumulation, although this was more effective in
Ltk- cells than in CHO cells perhaps because of higher
receptor expression. These data are consistent with previous findings
that all five SSTRs mediate SST inhibition of adenylyl cyclase (6).

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Fig. 1.
Heterologously expressed SSTR subtypes
inhibit cAMP accumulation. The effect of SST (100 nM)
on forskolin (10 µM)-induced increases in cAMP
accumulation was determined in Ltk cells stably
expressing SSTR3 (Ltk-R3) and SSTR4 (Ltk-R4) and
in CHO-K1 cells stably expressing SSTR3 (CHO-R3) and SSTR5
(CHO-R5). Data are expressed as a percentage of forskolin
stimulation and represent the means ± S.E. of three separate cell
preparations. Also indicated is the expression of SSTR subtypes
(fmol/mg protein), determined by radioligand binding of
[125I-Tyr11]SST to cell membranes.
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We found that SSTR3 and -4, but not SSTR5, mediate SST inhibition of
NHE1 activity. NHE1 activity was determined in a nominally HCO
-free Hepes buffer and expressed as the rate of
recovery of intracellular pH (pHi) from an
NH4Cl-induced acid load. SST (100 nM) had no
effect on quiescent NHE1 activity in any of the SSTR-expressing cell
lines (n = 3; data not shown), in accordance with its
inability to inhibit quiescent cAMP accumulation. We found, however,
that SST inhibited serum-stimulated NHE1 activity in Ltk-
cells expressing SSTR3 (Ltk-R3) or SSTR4 (Ltk-R4) and in CHO cells
expressing SSTR3 (CHO-R3) but not in CHO cells expressing SSTR5
(CHO-R5) (Fig. 2, A-D). At
pHi 6.70, SST decreased the rate of
serum-stimulated pHi recovery
(dpHi/dt) from 26.7 ± 2.1 × 10
4 pH/s to 15.1 ± 1.4 × 10
4 pH/s in Ltk-R3 cells (mean ± S.E.;
n = 6), from 25.2 ± 1.8 × 10
4
pH/s to 15.2 ± 1.2 × 10
4 pH/s in Ltk-R4 cells
(n = 4), and from 29.1 ± 2.8 × 10
4 pH/s to 15.6 ± 1.5 10
4 pH/s in
CHO-R3 cells (n = 5). In contrast, in CHO-R5 cells,
serum-stimulated NHE1 activity was not significantly different in the
absence (27.6 ± 1.9 × 10
4 pH/s) or presence
(26.3 ± 2.1 × 10
4 pH/s) of SST
(n = 4; p > 0.1). SST also decreased
serum-induced steady-state pHi in Ltk-R3,
Ltk-R4, and CHO-R3 cells but not in CHO-R5 cells (Fig. 2E).
These new data, together with our previous findings (22), indicate that
SSTR1, -3, and -4, but not SSTR2 and -5, couple to the inhibition of
NHE1.

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Fig. 2.
SSTR3 and -4, but not SSTR5, couple to the
inhibition of NHE1 activity. The rates of
pHi recovery from an NH4Cl-induced
acid load were determined in cells stably expressing the indicated SSTR
subtypes and expressed as
dpHi/dt × 10 4 pH/s. A-D, the means ± S.E. of
pHi recovery from four to six separate cell
preparations are indicated in response to serum (10%) in the absence
or presence of SST (100 nM). E, steady-state
pHi in response to 10% serum was determined in
the absence or presence of SST (100 nM) and expressed as
the means ± S.E. of four to six separate cell preparations.
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SSTR Motifs Conferring Inhibition of NHE1--
Using chimeric
SSTR2/SSTR1 receptors, we previously determined that collective, but
not individual, replacement of IL2 and IL3 of SSTR2 with those of SSTR1
confers inhibition of NHE1 by SST (22). These findings indicate that an
interaction between the second and third cytoplasmic domains of SSTR1
might be critical for this effect. We therefore examined the sequence
of all five receptor subtypes by performing a FASTA search of the Swiss
Protein Database and aligning amino acid residues of IL2 and IL3 of 16 SSTRs retrieved with PILEUP. Comparing the amino acid sequences of
SSTR1, -3, and -4 with those of SSTR2 and -5 revealed a number of
potentially important changes in both IL2 and IL3 (Fig.
3A). All species of SSTR2 and
SSTR5 have an arginine in IL2 (Arg-16) that is absent in SSTR1,
-3, and -4. This difference in charge could possibly restrict an
IL2-IL3 interaction of the receptor, which is critical for NHE1
coupling. The cognate position in IL2 of SSTR1, -3, and -4 contains a
consensus motif of T(s,p)V. In IL3, SSTR1 and -4 have two polar
glutamines, QQ, that are not present in SSTR2 or -5. In SSTR3,
the second Gln varies to Arg. Homology modeling of SSTR1 based
on the recently determined structure of rhodopsin (30) suggests that
the TV motif is at the C-terminal end of IL2 near the cytoplasmic tip
of transmembrane (TM) IV, whereas QQ is squarely within IL3 (Fig.
3B).

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Fig. 3.
Subtype-specific SSTR amino acid motifs.
A, sequence alignment of segments of intracellular
domains IL2 and IL3 for SSTRs. Conserved residues shared by all five
subtypes are indicated by blue boxes. Residues common to
SSTR1, -3, and -4 or to SSTR2 and -5 are indicated by red
boxes. B, intracellular end view from the cytoplasm of
the predicted structure of SSTR1 based on the crystal structure of
bovine rhodopsin (30) (molecule A of code 1F88 in the Protein Data
Bank). The TM helices are rainbow color-coded: I,
red; II, orange; III, yellow; IV,
green; V, light blue; VI, dark blue;
and VII, magenta. Helix VIII (non-transmembrane) is
pink, and the termini and loops are gray. The gap
in IL3 represents missing segments 236-239, and the extreme C-terminal
tail (segments 334-348) is omitted for clarity. Black
spheres represent the positions of IL2-TV, and red
spheres represent the positions of IL3-QQ.
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To determine whether these selective amino acid residues in SSTR1, -3, and -4 were sufficient to confer coupling to the inhibition of NHE1, we
made site-directed substitutions in hSSTR2 by replacing the
arginine-threonine (RT) residues in IL2 with threonine-valine (TV)
found in the cognate position of hSSTR1 and replacing the serine-serine-lysine (SSK) motif in IL3 with
tryptophan-glutamine-glutamine (WQQ) found in hSSTR1 (Fig.
3A). Substitutions in IL2 and IL3 of SSTR2 were made
individually (SSTR22TV and SSTR23WQQ) and
collectively (SSTR22TV3WQQ). Because we were unable to
stably express high levels of mutant SSTR2 in Ltk- cells,
we used CCL39 hamster lung fibroblasts, which express only the NHE1
Na-H exchanger subtype (31), and which we previously used to study the
regulation of NHE1 activity (28, 59). To compare the action of
mutant SSTR2 with that of wild-type receptors, we also stably expressed
wild-type SSTR1 and SSTR2 in CCL39 cells. Receptor expression was
determined by radioligand binding using [125I-Tyr11]SST (Fig.
4). Competition binding assays were used
to confirm that receptor affinities between wild-type and mutant SSTR
were similar. The dissociation constant (Kd) was
determined to be 3.39 nM for SSTR1, 7.65 nM for
SSTR2, 2.88 nM for SSTR22TV, 3.31 nM for SSTR23WQQ, and 1.32 nM for
SSTR22TV3WQQ. The Kd of SSTR2 mutants
was more similar to the Kd of SSTR1 than to the
Kd of SSTR2, suggesting that the mutations might
alter receptor conformation, possibly through a change in receptor
domains or a change in G protein coupling. The function of wild-type
and mutant receptors was confirmed by their ability to mediate SST
inhibition of forskolin-induced cAMP accumulation (Fig. 4). The
relatively greater attenuation of cAMP by wild-type SSTR2, compared
with wild-type SSTR1, was previously found in Ltk- and
HEK293 cells (22). CCL39 cells transfected with pRSVneo in the absence
of SSTR complementary DNA (cDNA) had no detectable specific binding
of [125I-Tyr11]SST and had no change in
forskolin-stimulated cAMP accumulation in the presence of SST (Fig. 4,
CCL-Neo).

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Fig. 4.
Inhibition of cAMP accumulation by wild-type
and mutant SSTR. The effect of SST (100 nM) on
forskolin (10 µM)-induced increases in cAMP accumulation
was determined in CCL39 cells stably expressing wild-type SSTR1
(CCL-R1) or SSTR2 (CCL-R2), cells transfected
with pRSVneo (CCL-Neo) (A), or cells expressing
SSTR mutants with the indicated substitutions in IL2 or IL3
(B). Data are expressed as a percentage of forskolin
stimulation and represent the means ± S.E. of three separate cell
preparations. Also indicated is the expression of wild-type and mutant
SSTR (fmol/mg protein) determined by radioligand binding of
[125I-Tyr11]SST to cell membranes.
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To study NHE1 activity, we first confirmed our previous findings in
Ltk- and HEK293 cells (22) that wild-type SSTR1, but not
SSTR2, mediates SST inhibition of NHE1. In both CCL-R1 and CCL-R2
cells, the addition of 10% serum increased quiescent NHE1
activity; however, in the presence of SST, serum-stimulated activity
was attenuated in CCL-R1 but not CCL-R2, cells (Fig.
5). Serum also increased quiescent NHE1
activity in CCL39 cells expressing mutant SSTR2; however, SST
attenuated serum-stimulated activity only in cells containing
collective IL2 and IL3 substitutions in SSTR2
(CCL-R22TV3WQQ) (Fig.
6C). In contrast, SST had no
effect on serum-stimulated NHE1 activity in cells expressing individual
IL2 and IL3 substitutions in SSTR2 (CCL-R22TV and
CCL-R23WQQ) (Fig. 6, A and B).
Consistent with its ability to mediate inhibition of NHE1 activity, SST
attenuated serum-induced steady-state pHi in
CCL-R22TV3WQQ cells but not in CCL-R22TV or
CCL-R23WQQ cells (Fig. 6D). To determine whether
there were clonal variations in the ability of mutant SSTR2 to regulate
NHE1 activity, we studied additional CCL39 clones stably expressing
each of the mutant receptors. In three separate cell preparations, we
consistently found that SST inhibited serum-stimulated NHE1 activity in
cells expressing SSTR22TV3WQQ but not in cells expressing
SSTR22TV or SSTR23WQQ (data not shown). Hence,
the ability to inhibit NHE1 was conferred by the collective
substitutions of cognate amino acids in IL2 and IL3 of
hSSTR2 with TV and WQQ of hSSTR1 but not
individual substitutions in each of the domains.

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Fig. 5.
SSTR1, but not SSTR2, couples to the
inhibition of NHE1 activity. The rates of
pHi recovery
(dpHi/dt × 10 4 pH/s) from an NH4Cl-induced acid load
were determined in CCL39 cells expressing wild-type SSTR1
(A) or SSTR2 (B). Data were obtained from
quiescent cells (Control) and from cells treated with serum
(10%) in the absence or presence of SST (100 nM) and are
representative of three separate cell preparations.
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Fig. 6.
Collective but not individual substitution of
IL2 and IL3 motifs confers inhibition of NHE1 activity.
A-C, the rates of pHi recovery
(dpHi/dt × 10 4 pH/s) from an NH4Cl-induced acid load
were determined in cells stably expressing SSTR2 containing the
indicated substitutions in IL2 and IL3. Data were obtained from
quiescent cells (Control) and from cells treated with serum
(10%) in the absence or presence of SST (100 nM) and are
representative of four to six separate cell preparations. D,
steady-state pHi in quiescent cells and in cells
treated with serum in the absence or presence of SST is expressed as
the means ± S.E. of four to six separate cell preparations.
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Shared Consensus Motifs in Class A GPCRs for Inhibition of
NHE1--
Because TV in IL2 and WQQ in IL3 act in concert to provide a
molecular basis for SSTR inhibition of NHE1 activity, we investigated whether these motifs occur in other class A GPCRs. We previously determined that endogenously expressed D2-dopamine
receptors in GH3 cells (25) and heterologously expressed
D2-dopamine receptors in Ltk- cells (26)
couple to the inhibition of NHE1. Using the tGRAP alignment data base
(32), we found that both motifs are present in the
D2-dopamine receptor but not in the D1- or
D3-dopamine receptors (Fig.
7A). We therefore investigated
whether amino acid motifs T(s,p)V near or within IL2 and QQ(r) near or
within IL3 were found in other class A GPCRs, as defined by the GPCR
data base (33). SSTR and D2-dopamine family sequences were
added when analyzing each family to help identify the exact location of
the respective motifs. Alignment of loop regions and their immediate TM
extensions using CLUSTALW failed to align either the T(s,p)V or QQ(r)
motifs between families. Alignment of IL3 was more difficult than that
of IL2 because of large insertions in some families, which made it
impossible to use an alignment tool. These loops were therefore aligned
manually. There were many instances of these motifs occurring
independently, especially the T(s,p)V motif, which is present in
classes of
-adrenergic and C-C-type chemokine receptors (data not
shown). Co-occurrence of TV in IL2 and QQ in IL3, however, was rare and
was found selectively in
2B-adrenergic and thromboxane
A2 receptors (Fig. 7A); however, there was no
detectable pattern in the distances of these motifs from TM helices in
these receptors compared with SSTR. Although a TV motif is present in
2A- and
2B-adrenergic receptors, the QQ
motif in the 2B subtype is not found in the 2A subtype (Fig. 7A). Moreover, as in SSTR1, the co-presence of an IL2-TV and
an IL3-QQ in D2-dopamine,
2B-adrenergic, and
thromboxane A2 receptors is nearly invariant across species
(Fig. 7, A and B). These data suggest that the
presence of T(s,p)V near the TM IV-IL2 transition and the presence of
QQ(r) in IL3 might represent conserved motifs that collectively confer
coupling to the inhibition of NHE1.

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Fig. 7.
Co-occurrence of TV and QQ motifs is uniquely
conserved in intracellular IL2 and IL3 of SSTR,
D2-dopamine,
2-adrenergic, and thromboxane
A2 receptors. A, global
alignment of IL2 and IL3 in D2-dopamine,
2-adrenergic, and thromboxane A2 receptors
shows that the motifs (red) are invariant in most species.
B, alignment of IL2 and IL3 in receptor families reveals
that only select subtypes within each family have both motifs
(red).
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Further confirming the validity of this hypothesis, we found that the
2B-adrenergic receptor, which contains both motifs, but
not the
2A-adrenergic receptor, which contains an IL2-TV but not an IL3-QQ, couples to the inhibition of NHE1 activity. Both
receptor subtypes were stably expressed in HEK293 cells. Receptor
expression was 2500 fmol/mg protein for
2A and 3500 fmol/mg protein for
2B, and receptor function was
confirmed by the ability of both receptor subtypes to inhibit cAMP
accumulation (data not shown). Although epinephrine (1 µM) had no effect on quiescent NHE1 in cells expressing
either receptor (data not shown), it markedly attenuated
serum-stimulated NHE1 activity in cells expressing
2B-adrenergic receptors but not in cells expressing
2A-adrenergic receptors (Fig.
8). Together with our findings on SSTR
and D2-dopamine receptors, these data suggest that the co-presence of TV at the TM IV-IL2 transition and IL3-QQ is a determinant for coupling to the inhibition of NHE1.

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Fig. 8.
A2B-Adrenergic but
not 2A-adrenergic receptors couple
to the inhibition of NHE1 activity. NHE1 activity (A
and B), expressed as the rate of pHi
recovery (dpHi/dt × 10 4 pH/s) from an NH4Cl-induced acid load,
and steady-state pHi (C) were
determined in HEK293 cells stably expressing 2A- or
2B-adrenergic receptors. Data were obtained from
quiescent cells treated with serum (10%) in the absence or presence of
epinephrine (Epi) (1 µM) and are
representative of three separate cell preparations.
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DISCUSSION |
Previous structure-function studies on GPCRs suggest that there is
no a priori certainty that related structural domains have similar functions in different receptors (24). We have now identified patterns of amino acid residues that are shared by different
subfamilies, but not necessarily by similar subtypes within a
subfamily, which confer coupling to a common effector. Selective
residues near the TM IV-IL2 transition and in IL3 of three SSTR
subtypes, SSTR1, -3, and -4, that signal to the inhibition of NHE1 are
not present in SSTR2 and -5 subtypes, which are not coupled to NHE1
regulation. The collective substitution of these residues in SSTR2,
however, was sufficient to switch signaling to the inhibition of NHE1. Moreover, we found that these IL2 and IL3 residues are present in the
D2-dopamine,
2B-adrenergic, and
thromboxane A2 receptors, and we confirmed that activation
of D2-dopamine and
2B-adrenergic receptors
inhibits NHE1 activity.
Studies using site-directed mutagenesis, chimeric receptors, and
synthetic peptides have identified residues within the IL2 (34-36) and
IL3 (37-40) regions that are critical for G protein and effector
coupling. Although selective regions of IL2 and IL3 are essential for G
protein recognition, the cooperative interaction of these domains has
also been shown to be critical for conferring coupling specificity
(41-44). Our finding that the combined, but not individual,
replacement of IL2 and IL3 motifs is required to switch SSTR2 coupling
to NHE1 supports the requirement for a cooperative interaction between
these two domains.
Conservation of sequences within the seven TM bundle of GPCR suggests
that the three-dimensional structure recently identified for rhodopsin
(30) might be shared by most GPCRs. Residues within IL2 and IL3,
however, are not highly conserved, which suggests that intracellular
surfaces may be structurally distinct. Recent findings by Chung
et al. (45) on the NMR structure of the IL2 region of the
2A-adrenergic receptor indicate that it is predominantly helical, possibly extending part of the TM IV helix, in contrast to its
L-shaped structure in rhodopsin (30). In SSTR1 the TV is in IL2
connecting TM III and IV, and QQ is adjacent to TM VI (Fig.
3B); the current activation model of GPCR suggests that conformational changes include a separation of TM III and TM VI (46).
As viewed from the cytoplasmic face, a change in the orientation of
these two TM domains is predicted to occur by a clockwise rotation of
TM VI relative to TM III. Moreover, signaling is impaired by mutations
that conformationally constrain TM III and TM VI of rhodopsin either by
engineered disulfide bridges (47, 48) or by Zn2+-activated
metal ion-binding sites (49). If this is correct, a plausible model is
that receptor activation would have to change the relative orientation
and accessibility from the cytoplasm of both TV, connected to TM III by
the IL2 loop, and QQ, connected to TM VI through the IL3 loop. In this
scenario, the variable location of TV either on the loop side or on the
TM IV side is consistent with penetration of the C terminus of G
-subunits into the membrane to allow for direct interaction with the
TM residues.
Divergent signaling by a GPCR to distinct signaling networks can occur
at the receptor-G protein interface or at the G protein-effector interface. Divergent signaling by SSTR1 occurs by both mechanisms. SSTR
inhibition of adenylyl cyclase and activation of tyrosine phosphatases
involve divergent signaling at the G protein-effector interface because
both actions are mediated by pertussis toxin-sensitive
i
subunits. Divergent signaling by SSTR1, -3, and -4 to adenylyl cyclase
and NHE1, however, must occur at the receptor-G protein interface
because inhibition of adenylyl cyclase, but not NHE1, is blocked by
pertussis toxin (20, 22). Distinct receptor contact sites for
activating different G proteins have been identified in juxtamembrane
regions of IL2 and IL3 of
-adrenergic (50, 51) and vasopressin (36)
receptors. This is analogous to our findings that these regions
selectively confer a Gi-independent inhibition of NHE1.
Although the identity of the trimeric G protein coupling SSTR to NHE1
remains to be determined, it is unlikely to be a member of the
Gs, Gi, or Gq families. Expression
of mutationally active
s and
i subunits
has no effect on NHE1 activity (52), and activation of
q, like the effect of
13, stimulates NHE1 (52,
53). The only
-subunit shown to inhibit NHE1 is
12 (31).
Expression of mutationally active
12 inhibits only stimulated NHE1
and has no effect on quiescent activity, as we found with SSTR
subtypes. If G12 does couple SSTR to the inhibition of NHE1, it might
act by inhibiting signaling by G13 or Gq, possibly by sequestering a common guanine nucleotide exchange factor (GEF). Stimulation of NHE1 activity by G13 is mediated by the low molecular weight GTPase Rho (28, 54), and G13 activates Rho by stimulating p115RhoGEF (55). Although p115RhoGEF binds to both G12 and G13, its
activation of Rho is stimulated by G13, but not G12, and activation of
G12 inhibits G13 stimulation of p115RhoGEF (55). Hence, one possible
mechanism whereby activated NHE1 is inhibited might be through G12
blocking the G13-p115RhoGEF-Rho signal. Consistent with this
possibility is the finding that thromboxane A2 receptors, which contain the IL2 and IL3 motifs coupling to the inhibition of
NHE1, activate G12 (56).
Most GPCR subtypes that are activated by similar ligands share similar
sequences and are predicted to have evolved by duplications of a common
ancestral gene. We found, however, that the conserved T(s,p)V and QQ(r)
motifs are shared by distinct GPCR subfamilies but not by all subtypes
within a given subfamily. This suggests that these subtypes diverged
from a common ancestor that coupled to NHE1 through the T(s,p)V and
QQ(r) motifs and that these motifs were mostly lost in subsequent
divergences that gave rise to families. Alternatively, the appearance
of the same motif in some members of different subfamilies may
represent convergent evolution to a motif that is linked to NHE1
inhibition. There are not many examples of convergent evolution to a
common functional architecture and activity, even in proteins with
entirely different structures (57). If the shared "NHE1 motifs"
represent convergent evolution, would it necessarily indicate the
regulation of a common NHE1-dependent cell function by
different GPCR subclasses? Inhibition of cell proliferation is an
action shared by decreased NHE1 activity (58, 59), SSTR1 (7, 60), and
D2-dopamine receptors (61, 62), but not by
2B-adrenergic receptors (63). Activation of
phospholipase A2 is shared by SSTR4 (19),
D2-dopamine receptors (64), and
2B-adrenergic receptors (65). However, inhibition of
NHE1 is associated with both increases (66) and decreases (67) in phospholipase A2 activity. One conserved function shared by
all three receptor types is the inhibition of endocrine secretion and
neurotransmitter release (68-71). Although a specific role for NHE1
activity in secretion has not been determined, remodeling of the
actin-based cytoskeleton, which is impaired by inhibition of NHE1 (28,
59, 72, 73), is a critical determinant in the secretory process (74,
75).
In summary, we have identified specific motifs in the IL2 and IL3
domains of GPCRs from distinct receptor families that confer a common
action of inhibiting NHE1 activity. The coordinate requirement of these
motifs for inhibiting NHE1 suggests a cooperative interaction of IL2
and IL3 domains for effector coupling. Moreover, our findings identify
a molecular basis for selective signaling by distinct receptor subtypes
that are activated by common ligands.