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J Biol Chem, Vol. 274, Issue 40, 28682-28689, October 1, 1999

The Proximal Portion of the COOH Terminus of the Oxytocin Receptor Is Required for Coupling to G_q, but Not G_i
INDEPENDENT MECHANISMS FOR ELEVATING INTRACELLULAR CALCIUM CONCENTRATIONS FROM INTRACELLULAR STORES^*

Sarasija Hoare, John A. Copland, Zuzana Strakova, Kirk Ives^§, Yow-Jiun Jeng, Mark R. Hellmich^§, and Melvyn S. Soloff^¶

From the Departments of  Obstetrics and Gynecology, ^§ Surgery, and the ^¶ Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1062

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

As the oxytocin receptor plays a key role in parturition and lactation, there is considerable interest in defining its structure/functional relationships. We previously showed that the rat oxytocin receptor transfected into Chinese hamster ovary cells was coupled to both G_q/11 and G_i/o, and that oxytocin stimulated ERK-2 phosphorylation and prostaglandin E₂ synthesis via protein kinase C activity. In this study, we show that deletion of 51 amino acid residues from the carboxyl terminus resulted in reduced affinity for oxytocin and a corresponding rightward shift in the dose-response curve for oxytocin-stimulated [Ca²⁺]_i. However, oxytocin-stimulated ERK-2 phosphorylation and prostaglandin E₂ synthesis did not occur in cells expressing the truncated receptor. Oxytocin also failed to increase phospholipase A activity or activate protein kinase C, indicating that the mutant receptor is uncoupled from G_q-mediated pathways. The 51 receptor is coupled to G_i, as oxytocin-stimulated Ca²⁺ transients were inhibited by pertussis toxin, and a G sequestrant. Preincubation of 51 cells with the tyrosine kinase inhibitor, genistein, also blocked the oxytocin effect. A 39 mutant had all the activities of the wild type oxytocin receptor. These results show that the portion between 39 and 51 residues from the COOH terminus of the rat oxytocin receptor is required for interaction with G_q/11, but not G_i/o. Furthermore, an increase in intracellular calcium was generated via a G_i-tyrosine kinase pathway from intracellular stores that are distinct from G_q-mediated inositol trisphosphate-regulated stores.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Oxytocin (OT)¹ is a nine-amino acid peptide that stimulates uterine smooth muscle and mammary myoepithelial cell contraction, and prostaglandin production by uterine endometrial and amnion cells. Nucleic acid sequencing of cDNA clones of the oxytocin receptor (OTR) indicated that it is a member of the G protein-coupled receptor (GPCR) superfamily (1). As OT plays a pivotal role in parturition and lactation, there is considerable interest in defining the structure of the OTR. It has been shown for a number of GPCRs that several regions in the cytoplasmic domains contribute directly or indirectly to G protein coupling (see Ref. 2 for a review). The juxtamembrane portions of cytoplasmic loop 3 and cytoplasmic loop 2 of several family members have been implicated in receptor-G protein interactions. In addition, the COOH-terminal region of adrenergic receptors (3, 4) and other receptor types (5-7) is also required for G protein interactions, but this domain does not appear to be important for receptor function of all GPCR family members (8-10).

The COOH-terminal domain of some GPCRs plays an important role in G protein isotype selectivity (11, 12). At least four isoforms of the prostaglandin EP3 receptor, differing only at their COOH-terminal tails (produced by alternative splicing), couple to different G proteins to activate different second messenger systems (13, 14). The COOH terminus of the human parathyroid hormone receptor directs the receptor toward an interaction with G_s, whereas a core region composed of the first, second, and third intracellular loops can interact promiscuously with different G proteins (11). A truncated human AT1 receptor mutant lacking the carboxyl-terminal 50 residues is deficient in coupling to G_i, but it retains full ability to bind to G_q (12).

The COOH terminus of some GPCRs has also been shown to be important for desensitization, which is manifested as a diminution in responsiveness for some period of time following agonist stimulation. COOH-terminal truncation of the ₂-adrenergic, _1B-adrenergic, lutropin/choriogonadotropin, platelet-activating factor, and neurokinin-2 receptors has been shown to impair homologous desensitization (15-20). Work on the -adrenergic receptor, in particular, has indicated that one mechanism of desensitization involves the rapid internalization of membrane-bound receptors following agonist stimulation (15). Removal of the COOH-terminal tail of some GPCRs has been shown to greatly reduce agonist-induced internalization of the receptor while having little or no effect on signal transduction (21-25).

Most GPCRs have a conserved cysteine in the COOH-terminal cytoplasmic tail near the seventh transmembrane-spanning region. This cysteine is known to be palmitoylated in rhodopsin (26), the ₂-adrenergic receptor (27), and the _2A-adrenergic receptor (28). In addition to the three intracellular loops delimited by transmembrane domains, a putative fourth cytoplasmic loop is formed in many GPCRs by insertion of palmitoylcysteines into the membrane lipid bilayer (29). It has been suggested that the fourth intracellular loop is important for G protein coupling (29), and mutation of Cys-341 in the carboxyl tail of the human ₂-adrenergic receptor leads to an uncoupled, nonpalmitoylated form of the receptor (27). Studies with m2 receptors indicate that palmitoylation is not an absolute requirement for receptor interaction with G proteins, but it enhances the ability of the receptors to interact with G proteins (30). In other cases, elimination of palmitoylation sites does not affect receptor-G protein interactions (31-35). However, down-regulation of the receptor number after prolonged agonist exposure was completely abolished by this mutation (32). Depalmitoylation also has been shown to increase the rate of the luteinizing hormone/human chorionic gonadotropin receptor internalization (33). In other instances, replacement of Cys results in the specific loss of coupling to one G protein isotype but not another, namely mutation of the human endothelin receptor A resulted in no effect on the ability of endothelin to activate adenylyl cyclase, but inhibited activation of PLC (36).

Previous work from our laboratory, using a CHO cell line that was stably transfected with the rat OTR, showed that OT stimulated rapid increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i), extracellular signal-related kinase-2 (ERK-2) phosphorylation, and PGE₂ synthesis (37, 38). Furthermore, the OTR was coupled to both G_q/11 and G_i in transfected CHO cells (CHO-OTR cells) (38), and in pregnant rat myometrium (39). In the present studies, we have systematically analyzed the importance of the COOH-terminal domain of the OTR on several receptor-associated processes: ligand affinity, G protein coupling via specific signal pathways, receptor desensitization, and selectivity of G-protein coupling. With the exception of a few amino acid residues, the COOH-terminal domain of the OTR is highly conserved between species (Fig. 1). This observation argues in favor of the functional importance of the COOH-terminal domain of the OTR. Previous work with the rat V_1a vasopressin receptor indicated that the COOH-terminal region of the receptor is inaccessible to antibodies directed against a COOH-terminal peptide when the receptor is coupled to G proteins, but is accessible when receptor-G protein complexes are dissociated (40). As the V_1a vasopressin receptor is closely related to the OTR, we have examined the importance of the COOH-terminal domain of the OTR in G protein coupling by creating COOH-terminal deletion mutants. Our approach has been to create COOH-terminal truncations of 22, 39, and 51 residues. The OTR has two adjacent Cys residues at positions 351 and 352, which are potential palmitoylation sites (Fig. 2). To determine the importance of these sites in OT action in the present studies, both these residues were replaced by Ser in one of the mutant OTRs analyzed.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Amino acid sequence comparison of the COOH-terminal region of OTRs from several species, showing highly conserved residues. Residues that are identical to the sequence of the rat OTR are indicated in boxes.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Minimal essential medium, fetal calf serum, Geneticin (G418 sulfate), and other antibiotics were purchased from Life Technologies, Inc. Chinese hamster ovary cells (CHO-K1) were purchased from the American Type Culture Collection (CCL61). Antibodies to ERK-1/2, Y/S phosphorylated ERK-1/2, and PKC (cross-reactive with , , and  forms) were purchased from Santa Cruz Biotechnology. DL-Threo-dihydrosphingosine and thapsigargin were purchased from Sigma. Genistein was obtained from Calbiochem. OT antagonist (OTA = [d(CH₂)₅,Tyr(Me)²,Thr⁴,Tyr-NH₂9]OVT) was purchased from Peninsula Laboratories.

DNA Constructs and Transfections-- Rat OTR cDNA was provided by Dr. Stephen J. Lolait. Full-length and carboxyl-terminal mutants, lacking 22, 39, and 51 residues (Fig. 2), were generated from rat OTR cDNA by polymerase chain reaction, using primers sets containing an EcoRI and XhoI end. The amplified DNAs were ligated with the expression vector pcDNA3.1 Myc/His A (Invitrogen, San Diego, CA) in frame with the Myc/His epitopes at the 3'-terminus. Mutation of two Cys residues at positions 343 and 344 to Ser was carried out by the method of Higuchi et al. (41). The sequences of all the DNA constructs were verified by DNA sequence analysis. The primer pairs for the full-length cDNA were primer 1 (5'-GAATTCCTGAGTCGCGTCGCGTCG-3') and primer 2 (5'-CTCGAGTGCTGAAGATGGCTGAGAGC-3'). Each truncation mutant was generated with primer 1 and a unique primer 2. The primers 2 for 22, 39, and 51 were 5'-CTCGAGGTTGCTCTTCTTGCTGACAC-3', 5'-CTCGAGACGAGCAGAGCAGCAGAAGA-3', and 5'-CTCGAGGTGGAAGAGGTGACCTGTGA-3', respectively. For construction of the Cys to Ser replacement mutants, full-length primers 1 and 2 were used along with 5'-GTGCAGCGCTTCTTCTCCTCCTCTGCTCGT-3' and 5'-ACGAGCAGAGGAGGAGAAGAAGCGCTGCAC-3'.

CHO-K1 cells were grown in -minimal essential medium containing 5% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. The expression plasmids were introduced into these cells by calcium phosphate-mediated transfection, and clonal cell lines that stably expressed the cDNAs were obtained by Geneticin (400 µg/ml) selection. The cells were maintained under an atmosphere of 5% CO₂.

Receptor Binding Assay-- Determination of the apparent K_d and B_max values of each of the cell lines was carried out with cells in six-well plates as described previously (42), using increasing concentrations of ¹²⁵I-OTA (0.14 to 100 pM). The cells were incubated for 1 h at 22 °C in 1 ml of Tyrode's solution, pH 7.5, containing 0.1% bovine serum albumin. The binding data were examined by nonlinear regression analysis (GraphPad Software Inc., San Diego, CA), and binding constants were determined by assuming a single class of independent binding sites. Oxytocin competition studies were carried out using a fixed concentration of ¹²⁵I-OTA and increasing concentrations of OT (0.1-100 nM) in 24-well plates (0.3 ml/plate). The cells were then rinsed (3 × 1 ml) in assay buffer and solubilized in 1 M NaOH; radioactivity was determined with a  counter.

Measurement of Intracellular Free Calcium Concentrations, Inositol Phosphates, and PKC Activation-- Real-time recordings of intracellular calcium concentrations ([Ca²⁺]_i) were performed on single cells, as described previously (37). Each point in the figures represents the mean ± S.E. values from 35 cells. The concentration of OT-stimulated synthesis of inositol phosphates was measured as described previously (37). Translocation of PKC was determined by immunoblot analysis of the cytosol and Triton X-100-solubilized cell fractions that were prepared according to Ogiwara et al. (43).

PGE₂ Synthesis and MAP Kinase Phosphorylation-- PGE₂ synthesis was determined using a PGE₂ enzyme immunoassay system from Amersham Pharmacia Biotech, as described previously (37). The phosphorylation of ERK-2 MAP kinase and the effects of pertussis toxin were determined by immunoblotting, as described previously (38). For analysis of phosphorylated p38 MAP kinase, immunoblots were incubated with antibody directed against dually phosphorylated p38 (New England Biolabs), followed by stripping of the blots and reprobing with an antibody to p38 (Santa Cruz Laboratories).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression of Wild Type and Deletion Mutant Constructs-- The COOH-terminal truncation and replacement sites are shown in Fig. 2. All of the cDNAs were expressed in CHO cells, as shown by ¹²⁵I-OTA binding to cell surface OTR on intact cells (Table I). The apparent K_d values of binding to the mutant receptors were comparable, but the number of receptors sites expressed per cell varied between mutants. The concentration of binding sites for the 51 mutant was about 10% that of the wild type. Efforts to obtain 51 clones with a greater number of binding sites were unsuccessful. We also determined the concentrations of OT reducing ¹²⁵I-OTA binding by 50% (IC₅₀). IC₅₀ values for the 22, 39, and C351S,C352S mutants were comparable to that of the wild type, while the 51 mutant had an IC₅₀ value that was about 3-7 times greater than the others (Fig. 3).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Truncation and replacement sites in the COOH-terminal portion of the rat OTR. The two cysteine sites at positions 351 and 352 were converted to serines.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Competition by increasing concentrations of OT for 125I-OTA binding sites on wild type and mutant OTRs expressed in CHO cells. IC50 values and 95% confidence limits are shown in the inset.

Effect of Truncation on the Ca²⁺ Response to OT-- We have shown previously that stimulation of CHO-OTR cells with OT results in a rapid, transient increase in intracellular Ca²⁺ concentrations (37). Both intra and extracellular sources of Ca²⁺ were involved (37). In the present studies, OT stimulated intracellular Ca²⁺ in all of the mutant lines (Fig. 4A). However, the 51 mutant required 175 nM OT for stimulation (Fig. 4B), while the other mutants and wild type CHO-OTR cells responded to 10 nM OT or less (data not shown). The reduced sensitivity to OT by the 51 mutant is consistent with the greater IC₅₀ value of OT displacement of ¹²⁵I-OTA (Fig. 3), and suggests that higher concentrations of OT are required for stimulation because of lower affinity for the peptide. Removal of extracellular Ca²⁺ with EGTA eliminated the sustained [Ca²⁺]_i phase in both 51 and wild type cells, indicating that the mutation had no effect on the relative intracellular and extracellular contributions to [Ca²⁺]_i (data not shown). These results further indicate that the major source of [Ca²⁺]_i arises from intracellular stores. Treatment of CHO cells, lacking OTRs, with up to and including 1 mM OT had no effect on [Ca²⁺]_i (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   A, oxytocin-stimulated increases in intracellular Ca2+ concentration ([Ca2+]i) in cells expressing wild type and mutant OTRs. To determine whether prior exposure of the cells to OT caused desensitization to a subsequent challenge, the cells were treated at 37 °C with a high dose of OT (200 nM) to stimulate an intracellular Ca2+ transient and, after decay of the signal, the cells were rinsed at 37 °C for 10 min to dissociate bound OT. The cells were then exposed to 20 nM OT (except in the case of the 51 mutant, which required 200 nM), and [Ca2+]i was measured. There was no evidence of desensitization with either the wild type or mutant cells. Each point is the mean ± S.E. of 35 determinations. B, oxytocin dose-response curve using 51 cells. The lowest dose of OT stimulating an increase in [Ca2+]i was about 175 nM, as opposed to the other cell lines, which responded to 10 nM OT (data not shown). Each point is the mean ± S.E. of 35 determinations.

Following OT-stimulated elevation in [Ca²⁺]_i, using relatively high OT concentrations (200 nM), Ca²⁺ levels returned to near-base-line levels and the cells were refractory to further stimulation by OT in the medium. The cells were rinsed over a 10-min period to dissociate OT from OTRs, and were challenged with 10% of the OT dose to determine whether they became desensitized. In the case of the 51 cells, the second dose was the same as the first because of the lesser initial sensitivity of this mutant to OT (Fig. 4B). Neither the wild type or any of the mutants exhibited a significantly diminished response to OT after the second dose of OT, indicating that the COOH-terminal region of the rat OTR is not involved in desensitization (Fig. 4A). The lack of desensitization was observed over a 10-fold range in OTR concentration (cells expressing wild type OTR versus those expressing the 51 mutation; Table I). Cells expressing wild type OTR in concentrations that were comparable to that of the 51 mutant also showed no evidence of homologous desensitization (data not shown).

OT-stimulated PGE₂ Synthesis-- The addition of increasing concentrations of OT to CHO cells expressing wild type, and the 22, 39 mutant OTRs resulted in the release of PGE₂ in a dose-dependent fashion, showing further that these receptors are functionally coupled to signal transduction pathways (Fig. 5). However, cells expressing the 51 mutant did not release PGE₂ in response to OT (Fig. 5). Increasing the concentration of OT to 1 µM had no effect (data not shown). It would appear from these results that the region between 39 and 51 residues from the COOH terminus is involved in the ultimate generation of a PGE₂ response. Within this region are two adjacent Cys residues (positions 351 and 352), which have been implicated in G protein interactions via palmitoylation (26-28). However, mutation of the two Cys residues to Ser had no effect on the ability of OT to stimulate PGE₂ synthesis (Fig. 5). From estimations of EC₅₀ values (Fig. 5, inset), the potencies of OT in the active mutant lines were generally indistinguishable, in agreement with the IC₅₀ results. The maximal responses were generally proportional to the B_max values, as estimated by ¹²⁵I-OTA binding (Table I).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Oxytocin-stimulated PGE2 synthesis by cells expressing the wild type and mutant OTRs. Each point is the mean ± S.E. of triplicate determinations.

OT-stimulated ERK-2 Phosphorylation-- OT stimulation of PGE₂ synthesis in CHO cells transfected with the full-length rat OTR is thought to involve both the phosphorylation of cytosolic phospholipase A₂ by ERK-2/1, and Ca²⁺-mediated translocation of cytosolic phospholipase A₂ from the cytosolic to membrane fractions (see Ref. 38 for references). Because OT stimulated an increase in [Ca²⁺]_i in 51 cells, we determined whether the inability of OT to stimulate PGE₂ synthesis in these cells was the result of deficient ERK-2 phosphorylation. As shown previously (38), OT (50 nM) causes the rapid (2 min) phosphorylation of ERK-2 in CHO-OTR cells, as evidenced by the electrophoretic mobility shift of a fraction of total ERK-2 on immunoblots (pp42, Fig. 6A). Comparable results were obtained with each of the mutant OTRs after stimulation with 50 nM OT, with the exception of the 51 mutant, which demonstrated only base-line phosphorylation (like OTR-negative CHO cells, Fig. 6A). To take into account the lower affinity of 51 cells for OT, we used increasing concentrations of OT and a more sensitive antibody assay that measures dually phosphorylated (S/T and Y) ERK-2. Concentrations of OT up to 1 µM had no effect on ERK-2 phosphorylation in 51 mutant cells after 5 min of treatment (Fig. 6B). In contrast, 100 nM phorbol 12-myristate 13-acetate (PMA) was effective in stimulating ERK-2 phosphorylation in these cells (Fig. 6B). Treatment of the wild type and mutant cell lines and CHO cells with basic fibroblast growth factor (bFGF), 100 ng/ml for 5 min, resulted in ERK-2 phosphorylation in all of the cell lines (Fig. 6C), showing (along with the PMA results in Fig. 7B) that there is no impairment in ERK-2 phosphorylation in 51 cells. bFGF stimulates ERK-2 phosphorylation by a G protein-independent mechanism (44).

View larger version (49K): [in this window] [in a new window]   Fig. 6.   A, oxytocin-stimulated ERK-2 phosphorylation, as measured by an electrophoretic mobility shift and immunoblotting of ERK. Antibody reacting to both phosphorylated (pp42) and nonphosphorylated (p42) ERK was used. The antibody also cross-reacted slightly with ERK-1 (p44). B, lack of effect of increasing concentrations of OT on ERK-2 phosphorylation in 51 mutant cells, as measured by immunoblotting. Antibody reacting to dually phosphorylated ERK-2 was used. PMA (100 ng/ml) activation of ERK-2 phosphorylation in 51 mutant cells indicates no impairment in the ability of ERK-2 to be phosphorylated in these cells. C, effect of bFGF (100 ng/ml) on ERK-2 phosphorylation in wild type and mutant cells. The length of time of stimulation was either 2 min (A) or 5 min (B and C).

View larger version (10K): [in this window] [in a new window]   Fig. 7.   Lack of stimulation of InsP production by OT in 51 cells. The nonspecific G protein activator, sodium fluoroaluminate (AlF), was stimulatory in 51 cells. The number of OTR binding sites in the wild type cells was matched approximately to that expressed by 51 cells, by selecting the appropriate clone from a pool of clones with different numbers of binding sites. *, p < 0.05.

Evidence for OT-stimulated Ca²⁺ Transients That Are G_q-independent-- G_q-mediated PLC activation in 51 cells is not consistent with the ability of OT to elicit increases in [Ca²⁺]_i from intracellular stores on the one hand, and the inability of OT to stimulate PGE₂ synthesis and ERK-2 phosphorylation on the other (38). To determine whether OT activates PLC in 51 cells via G_q mediation, we measured two sequelae of G_q/PLC activity: inositol phosphates production and PKC activation. In these and subsequent experiments, a wild type clonal cell line expressing about the same number of OTRs as the 51 cells was used. Treatment of cells expressing the full-length receptor with 500 nM OT for 30 min resulted in about a 5-fold increase in inositol phosphate (InsP) production (Fig. 7). In contrast, InsP production by 51 cells was unchanged after OT treatment (Fig. 7). However, these cells were capable of being stimulated because sodium fluoroaluminate, a nonspecific G-protein activator, induced a significant increase in InsP production. Treatment of cells expressing full-length OTR with OT for 5 and 15 min resulted in the activation of PKC, as measured by an increase in the amount of PKC associated with the membrane fraction (Fig. 8A). There was a barely detectable level of PKC associated with the membrane fraction in 51 cells in the basal state, but no increase following OT treatment (Fig. 8A). PKC was translocated from the cytosol to membrane fractions in 51 cells after treatment with 100 nM PMA, indicating that the lack of effects of OT were not due to impaired PKC activation (Fig. 8B). The lack of OT activation of PKC in the 51 cells is also consistent with the lack of effect of OT on ERK-2 phosphorylation and PGE₂ synthesis, both of which are mediated by PKC (38).

View larger version (31K): [in this window] [in a new window]   Fig. 8.   A, lack of PKC activation in 51 cells by OT, as measured by an increase in PKC immunoactivity associated with the membrane fraction. In contrast, OT stimulation caused an increase in PKC concentration in the membrane fraction of cells expressing the wild type OTR. A nonspecific immunoreactive band below the PKC band serves as a convenient indicator of uniform protein loading on the SDS-PAGE gel. The wild type OTR and 51 clones were matched for approximately the same number of OTA binding sites. B, PMA-induced translocation of PKC from the cytosol (CYT) to membrane (MEM) fractions in 51 cells shows that these cells have PKC that is capable of being activated.

The Presence of an Intracellular, InsP-independent Pathway in OT-stimulated Ca²⁺_i Release That Is Mediated by Pertussis Toxin-sensitive G and Tyrosine Kinase Activity-- Treatment of 51 and wild type OTR expressing cells with thapsigargin (250 nM) depleted intracellular stores of Ca²⁺ in the absence of extracellular Ca²⁺, and inhibited the effects of OT on further increases in [Ca²⁺]_i (Fig. 9A). The absence of InsP formation in the 51 cells indicates that InsP₃-independent stores of Ca²⁺ must be responsible for the OT-induced rise in [Ca²⁺]_i. Cells expressing 51 and wild type OTRs were pretreated with selective inhibitors to determine whether the mutant G_i-mediated pathways effect an intracellular Ca²⁺ response to OT stimulation, and whether this process is mediated by tyrosine kinase activation. Pertussis toxin treatment (500 ng/ml for 16-20 h) completely obliterated the Ca²⁺ response to OT in 51 expressing cells and reduced the OT-stimulated increase in wild type cells by more than 60% (Fig. 9B). Transfection of the cells with a plasmid expressing the G sequestrant ARK1ct (10 µg of DNA/6-cm dish, 24 h before addition of OT), completely blocked the effects of OT in 51 cells, as compared with cells transfected with the empty vector (Fig. 9C). However, the G sequestrant had no effect on OT-stimulated [Ca²⁺]_i in wild type cells (Fig. 9C). Preincubation of cells with increasing concentrations (1, 10, 100 µM) of the tyrosine kinase inhibitor, genistein, for 1 h before stimulation with OT resulted in the complete inhibition of the OT-induced [Ca²⁺]_i transient in 51 cells, even at the lowest concentration (Fig. 9D). The OT response in wild type cells was reduced by more than 35% by treatment with 1 µM genistein). The inhibition was progressively greater with 10 and 100 µM genistein (Fig. 9D).

View larger version (29K): [in this window] [in a new window]   Fig. 9.   A, depletion of intracellular stores by thapsigargin in 51 cells, and elimination of Ca2+ from the medium, results in the lack of any further increases in [Ca2+]i after addition of OT, as is also the case with cells expressing the wild type OTR. These findings indicate that OT stimulates the release of Ca2+ from intracellular stores in both 51 and wild type cells. B, OT-stimulated [Ca2+]i transients in 51 cells is almost completely inhibited by preincubation with pertussis toxin, 500 ng/ml for 16-20 h. Pertussis toxin also partially inhibits the response to OT in cells expressing the wild type OTR. C, transfection of 51 cells with the sequestrant, ARK1ct, blocked OT-stimulated increases in [Ca2+]i. The empty vector was used as a control. In contrast, ARK1ct had no effect on OT stimulation in the wild type OTR cells. D, increasing concentrations of genistein inhibited OT-stimulated increases in [Ca2+]i in 51 and wild type OTR expressing cells. The lowest concentration of genistein tested, 1 µM, completely inhibited the effects of OT in 51 cells. Each point in each figure is the mean ± S.E. of 35 determinations.

Phosphorylation of p38 MAP Kinase-- To further analyze G protein-coupled pathways present in the 51 mutant, we examined p38 MAP kinase phosphorylation. Treatment of both wild type and 51 cells with 250 nM OT for 5 and 10 min resulted in increased phosphorylation of p38, as measured by immunoblotting with an antibody to dually phosphorylated p38 (Fig. 10). Although basal levels of p38 phosphorylation were elevated, the addition of OT further stimulated phosphorylation after 5 min. OT also caused an increase in p38 phosphorylation in cells expressing the wild type OTR after both 5 and 10 min (Fig. 10). Pretreatment of both mutant and wild type cell lines with oxytocin antagonist or pertussis toxin inhibited p38 phosphorylation (Fig. 10). Treatment of either cell type with PMA resulted in increased p38 phosphorylation (Fig. 10), indicating that both G_i (pertussis toxin-sensitive) and G_q (PKC-mediated) pathways are involved in activation of p38. G_q is likely dissociated from the 51 OTR, but direct activation of PKC by PMA occurs downstream from OTR-G_q coupling.

View larger version (23K): [in this window] [in a new window]   Fig. 10.   A, oxytocin-stimulated p38 phosphorylation in CHO cells expressing the 51 mutant and wild type OTRs. The immunoblots were first probed with antibody recognizing dually phosphorylated (pT180, pY182) p38, stripped, and then probed with antibody to p38 to show uniformity of protein loading. The cells were treated with OT for 5 or 10 min, or were pretreated with OTA (1 µM, 5 min) or pertussis toxin (500 ng/ml, 16-20 h) before treatment with OT for 5 min. Alternatively, cells were treated with PMA (20 ng/ml) for 5 min.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The COOH-terminal portion of GPCRs has been shown to be involved in a number of essential activities. Depending on the particular GPCR, these activities include G protein coupling (3-7), selectivity of G protein isotype (11, 11-14, 45), and homologous desensitization or internalization (15-25). Whereas truncation of some GPCRs has been shown to modify any one or more of its functions, shortening of the COOH terminus of other family members has no apparent effects (8-10). Thus, no uniform hypothesis regarding the importance of the COOH-terminal region can be applied a priori to the OTR, which has not been previously examined in detail (46, 47). In view of the high degree of homology of the COOH-terminal domain of OTRs from several species, we reasoned that conservation would be associated with some important function(s). However, truncation of 22 and 39 residues from the COOH terminus or replacement of the Cys residues, which have been thought to be palmitoylation sites for the formation of a fourth intracellular loop, had no effect on OTR functions. These include ligand affinity, OT stimulation of increases in intracellular Ca²⁺, ERK-2 phosphorylation, and PGE₂ synthesis. The mutants were also indistinguishable from the wild type OTR with respect to homologous desensitization.

Treatment of 51 cells with OT caused a Ca²⁺_i transient, although higher concentrations of OT were required than with the other mutants or wild type receptor. This lower sensitivity of 51 cells is consistent with the lower affinity of the 51 receptor for OT. In contrast, no dose of OT (up to 1 µM) stimulated ERK-2 phosphorylation or PGE₂ synthesis in the 51 cells. As we have shown previously, OT-stimulated ERK-2 phosphorylation and PGE₂ synthesis are mediated by PKC (38). PKC activation occurs as a result of increased diacylglycerol synthesis, which is a product of the G_q/PLC pathway. Upon activation of cell surface receptors, PKCs translocate from the cytoplasm to membrane surfaces (48). We found that OT failed to activate PKC- translocation in the 51 mutant cells, but not in cells expressing the wild type OTR. The lack of PKC activation in 51 cells would account for the absence of OT-stimulated ERK-2 phosphorylation and PGE₂ synthesis. The absence of PLC activation in the 51 cells accounts for the inability of OT to increase total InsP synthesis. Thus, the Ca²⁺ mobilizing effect of OT in 51 cells occurred independently of OTR-mediated G_q/PLC stimulation.

Our previous work, showing that wild type OTRs transfected into CHO cells are coupled to G_i as well as G_q (38), led us to consider whether the OT-stimulated increase in [Ca²⁺]_i in 51 cells is mediated by G_i instead of G_q. In Rat-1 and COS-7 cells, GPCRs coupled to pertussis toxin-sensitive G proteins mediate ERK-1/2 activation via a G subunit complex signal pathway that is dependent upon tyrosine phosphorylation and p21^ras activation (49, 50). Cellular expression of a specific G subunit sequestrant peptide derived from the carboxyl-terminal G subunit binding domain of the -adrenergic receptor kinase 1, ARK1ct, was shown to inhibit LPA and _2A adrenergic receptor-mediated Shc phosphorylation in COS-7 cells (49). 51 cells were pretreated with either a G_i inhibitor (pertussis toxin), the G sequestrant (ARK1ct), or a tyrosine kinase inhibitor (genistein). All three agents inhibited OT-stimulated increases in [Ca²⁺]_i in 51 expressing cells; pertussis toxin and genistein also reduced the effects of OT in wild type cells. Therefore, based on our findings, it would appear that the 51 mutant OTR is functionally coupled to G_i, but lacks the coupling to G_q. Both G_i and G_q coupling to the OTR occurs in cells expressing the wild type OTR. We would extrapolate from the results that truncation of the COOH-terminal tail by 22 or 39 residues, and mutating residues 351 and 352 (Cys  Ser) do not affect G_q coupling.

Because OT-stimulated ERK-2 phosphorylation in CHO-OTR cells is largely mediated by G_q, we examined pertussis toxin-sensitive p38 MAP kinase phosphorylation as an indicator of a G_i-mediated process. This pathway has been shown to be activated upon stimulation of both G_q/11-coupled m1 and G_i-coupled m2 muscarinic-acetylcholine receptors (51). Overexpression of G or a constitutively activated mutant of G₁₁, but not G_i, also stimulated p38 kinase activity (51). p38 kinase phosphorylation was stimulated by OT in 51-expressing cells by a process that was completely inhibited by pertussis toxin. Because PMA also stimulated p38 phosphorylation in these cells, it is apparent that both G_q and G_i mediate p38 kinase activity. Nagao et al. (52) showed that G_q/11 stimulates p38 MAP kinase activity through PKC and Src family kinase-dependent pathways. These findings suggest that PMA stimulation of p38 phosphorylation in 51 cells occurs through PKC activation of tyrosine kinase-regulated steps. Parenthetically, the present findings are the first observations of an effect of OT on p38 phosphorylation.

Pathways connecting G_i activation and intracellular Ca²⁺ transients in 51 cells are not currently known. Depletion of intracellular Ca²⁺ stores with thapsigargin, an inhibitor of endoplasmic reticulum Ca²⁺-ATPase activity, resulted in the loss of the Ca²⁺_i transient following OT stimulation of 51 cells. ₁-Adenoreceptors utilize two different G subunits to increase [Ca²⁺]_i in rat myocytes (53). G_q appears to activate InsP production and induce the release of Ca²⁺ from intracellular stores, while G₁₁ may enhance the Ca²⁺-activated Ca²⁺ influx that replenishes intracellular Ca²⁺ stores (53). This mechanism does not appear to occur with the OTR, as both the pertussin toxin-sensitive and insensitive pathways involved intracellular Ca²⁺ stores. If G-stimulated tyrosine kinases resulted in activation of PLC-, we would have expected to find a rise in inositol phosphates in the 51 cells after OT treatment. G activation of PLC- isoforms to stimulate PtdInsP2 hydrolysis independent of protein tyrosine kinases (54) also appears to be absent in 51 cells. Previous work from this laboratory demonstrated that OT inhibited (Ca²⁺ + Mg²⁺) ATPase activity in sarcolemmal membranes from the rat uterine myometrium (55). It was postulated that inhibition of the calcium pump would hinder the extrusion of intracellular Ca²⁺, thus allowing OT-stimulated elevations in [Ca²⁺]_i to be maintained. Although mechanisms coupling the OTR and (Ca²⁺ + Mg²⁺)ATPase have not been described, it is unlikely that this pathway accounts for [Ca²⁺]_i increases in 51 cells because the source of Ca²⁺ in 51 cells was thapsigargin-sensitive.

Mobilization of Ca²⁺ from intracellular stores is mediated by three major receptors on the endoplasmic reticulum, the InsP₃, ryanodine, and sphingosine-1-phosphate systems. Although we measured total InsPs to ensure that transient increases in InsP₃ would not go undetected; we were unable to detect any increase after OT treatment of 51 cells. These results indicate that the increase in Ca²⁺_i in 51 cells caused by OT is not mediated by the InsP3 receptor. We did not study the effects of ryanodine on [Ca²⁺]_i release, as the ryanodine receptor/Ca²⁺ release channel, which is an essential component of excitation-contraction coupling in striated muscle cells, is not found in any significant concentration in CHO cells (56, 57). Sphingosine 1-phosphate has been shown to release Ca²⁺ from the endoplasmic reticulum in several cell types in conjunction with occupancy of surface IgG receptors (58-61). Sphingosine-1-phosphate biosynthesis is catalyzed by sphingosine kinase (62, 63), a ubiquitous enzyme found in the cytosol (64, 65) and the endoplasmic reticulum (58). However, addition of the sphingosine analogue, DL-threo-dihydrosphingosine, which is a competitive inhibitor of sphingosine kinase activity (60), had no effect on OT-induced Ca²⁺_i transients (data not shown). Thus, the signals mediating the release of Ca²⁺ from intracellular stores are not known at the present time.

Residues in the N-terminal part of the COOH terminus of the human V₂ vasopressin receptor have been shown to be necessary for correct folding; the COOH-terminal residues are also important for efficient cell surface expression (66). Although the reduced affinity of the 51 mutant for OT could be a result of its dissociation from G_q, it might be due instead to modified protein folding as has been indicated for the V₂ vasopressin receptor. The distinction is important because if G_q-coupled OTR has a higher affinity for OT than the G_i-coupled form, the preferred pathway for OT-stimulated increases in [Ca²⁺]_i with low concentrations of OT would be through PLC activation and InsP₃-mediated stimulation of Ca²⁺ release from intracellular stores. However, because pertussis toxin and genistein substantially inhibited the OT-stimulated increase in [Ca²⁺]_i in wild type cells, the OTR-associated G/protein tyrosine kinase pathway appears to be as important as the G_q/PLC pathway for increasing [Ca²⁺]_i. Of about 30 stable clones of 51 examined, only the one used in these studies had ¹²⁵I-OTA binding activity. These results suggest that processing of the 51 mutant protein is impaired.

The results of our studies are summarized in Fig. 11. The model postulates that, in addition to elements of the third intracellular loop, the proximal portion of the COOH-terminal domain of the OTR is required for coupling to G_q. In contrast, coupling to G_i occurs without participation of the COOH-terminal region. The loss of association of the 51 mutant with G_q leads to a loss of OT/PLC-mediated events, which include InsP production, PKC activation, ERK-2 phosphorylation, and PGE₂ synthesis (38). Phosphorylation of p38 MAP kinase occurred through a pertussis toxin-sensitive pathway both in the 51 and wild type cells. Although p38 phosphorylation is also G_q (PKC)-dependent through a process that likely involves PKC activation of p21^ras, pertussis toxin-sensitive phosphorylation of p38 serves as a useful indicator of G_i coupling to 51. We have also shown that the G_i-tyrosine kinase-mediated pathway is intact in 51 cells, as indicated by the pertussis toxin and genistein sensitivity of OT-stimulated increases in [Ca²⁺]_i. Inositol trisphosphate arising from the G_q-initiated pathway mediates the release of Ca²⁺ from intracellular stores via InsP₃ receptors. Endoplasmic reticulum receptors mediating intracellular Ca²⁺ release from G_i/protein tyrosine kinase pathways are presently unknown. Understanding why G_i and G_q contribute about equally to OT-stimulated rises in [Ca²⁺]_i remains to be established.

View larger version (24K): [in this window] [in a new window]   Fig. 11.   Postulated bifurcating pathways mediating OT-stimulated increases in [Ca2+]i. The 51 mutant, lacking the COOH-terminal domain of the OTR is coupled to Gi, but not Gq. As a result, 51 cells respond to OT with an increase in [Ca2+]i and p38 phosphorylation, but lack InsP formation, PKC translocation, ERK-2 phosphorylation, and PGE2 synthesis.

	ACKNOWLEDGEMENTS

We thank Solweig Soloff and Dan Liebenthal for technical assistance, Dr. Marya Zlatnik for assistance with the ¹²⁵I-OTA binding studies, Dr. Robert Lefkowitz for providing the ARK1ct construct, and Dr. Stephen Lolait for rat OTR cDNA.

	FOOTNOTES

^* This work was supported in part by Grant HD26168 from the National Institutes of Health (to M. S. S.).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: Dept. of Obstetrics and Gynecology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1062. Tel.: 409-772-0715; Fax: 409-772-2261; E-mail: msoloff@utmb.edu.

	ABBREVIATIONS

The abbreviations used are: OT, oxytocin; bFGF, basic fibroblast growth factor; CHO, Chinese hamster ovary; ERK-2, extracellular signal-related kinase-2; GPCR, G protein-coupled receptor; [Ca²⁺]_i, intracellular Ca²⁺ concentration; InsP, inositol phosphate; OTA, oxytocin antagonist; OTR, oxytocin receptor; PGE₂, prostaglandin E₂; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; MAP, mitogen-activated protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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