Conserved Motifs in Somatostatin, D2-dopamine, and alpha 2B-Adrenergic Receptors for Inhibiting the Na-H Exchanger, NHE1*

Chin-Yu LinDagger , Madhulika G. VarmaDagger , Anita JoubelDagger , Srinivasan Madabushi§, Olivier Lichtarge§, and Diane L. BarberDagger

From the Dagger  University of California, San Francisco, San Francisco, California 94143 and § Baylor College of Medicine, Houston, Texas 77030

Received for publication, December 4, 2002, and in revised form, January 29, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Receptor subtypes within families of G protein-coupled receptors that are activated by similar ligands can regulate distinct intracellular effectors. We identified conserved motifs within intracellular domains 2 and 3 of selective subtypes of several G protein-coupled receptor families that confer coupling to the Na-H exchanger, NHE1. A T(s,p)V motif within intracellular domain 2 and a QQ(r) motif within intracellular domain 3 are shared by the somatostatin receptor subtypes SSTR1, -3, and -4, which couple to the inhibition of NHE1, but not by SSTR2 and -5, which do not signal to NHE1. Only the collective substitution of cognate SSTR2 residues with these two motifs conferred the ability of mutant SSTR2 to inhibit NHE1. Both motifs are present in D2-dopamine receptors, which inhibit NHE1, and in alpha 2B-adrenergic receptors, which couple to the inhibition of NHE1, but not in alpha 2A-adrenergic receptors, which do not regulate NHE1. These findings indicate that motifs shared by different subfamilies of G protein-coupled receptors, but not necessarily by receptor subtypes within a subfamily, can confer coupling to a common effector.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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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, alpha 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 alpha 2B-adrenergic receptor, which contains IL2-TV and IL3-QQ residues, but not the alpha 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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INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 alpha 2A- and alpha 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 gamma -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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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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.

We found that SSTR3 and -4, but not SSTR5, mediate SST inhibition of NHE1 activity. NHE1 activity was determined in a nominally HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-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.

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.

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.

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.

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 alpha -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 alpha 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 alpha 2A- and alpha 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, alpha 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, alpha 2-adrenergic, and thromboxane A2 receptors. A, global alignment of IL2 and IL3 in D2-dopamine, alpha 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).

Further confirming the validity of this hypothesis, we found that the alpha 2B-adrenergic receptor, which contains both motifs, but not the alpha 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 alpha 2A and 3500 fmol/mg protein for alpha 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 alpha 2B-adrenergic receptors but not in cells expressing alpha 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 alpha 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 alpha 2A- or alpha 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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, alpha 2B-adrenergic, and thromboxane A2 receptors, and we confirmed that activation of D2-dopamine and alpha 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 alpha 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 alpha -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 alpha 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 alpha -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 alpha s and alpha i subunits has no effect on NHE1 activity (52), and activation of alpha q, like the effect of alpha 13, stimulates NHE1 (52, 53). The only alpha -subunit shown to inhibit NHE1 is alpha 12 (31). Expression of mutationally active alpha 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 alpha 2B-adrenergic receptors (63). Activation of phospholipase A2 is shared by SSTR4 (19), D2-dopamine receptors (64), and alpha 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.

    ACKNOWLEDGEMENTS

We thank E. Menga and H. Bourne for comments and suggestions regarding GPCR structures and M. McKenney for editing the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants DK40259 (to D. L. B.) and T32DE07204 (to C.-Y. L.).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: Box 0512, 513 Parnassus Ave., University of California, San Francisco, CA 94143-0512; Tel.: 415-476-3764; Fax: 415-502-7338; E-mail: barber@itsa.ucsf.edu.

Published, JBC Papers in Press, February 3, 2003, DOI 10.1074/jbc.M212315200

    ABBREVIATIONS

The abbreviations used are: GPCR, G protein-coupled receptor; SSTR, somatostatin receptor; IL2 and IL3, intracellular domains 2 and 3, respectively; NHE1, Na-H exchanger; CHO, Chinese hamster ovary; FBS, fetal bovine serum; DME medium, Dulbecco's modified Eagle's medium; TM, transmembrane; GEF, guanine nucleotide exchange factor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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