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Am J Physiol Endocrinol Metab 292: E977-E984, 2007. First published December 5, 2006; doi:10.1152/ajpendo.00531.2005
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Amino acids in the COOH-terminal region of the oxytocin receptor third intracellular domain are important for receptor function

Miao Zhong,1 Bridgette Parish,2 Dilyara A. Murtazina,1 Chun-Ying Ku,1 and Barbara M. Sanborn1,2

1Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado; and 2Department of Biochemistry, University of Texas Medical School, Houston, Texas

Submitted 1 November 2005 ; accepted in final form 19 November 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Previously, residue K6.30 in the COOH-terminal region of the third intracellular domain (3iC) of the oxytocin (OT) receptor (OTR) was identified as important for receptor function leading to phospholipase C activation in both OTR and the vasopressin V2 receptor (V2R) chimera V2ROTR3iC. Substitution of either A6.28K or V6.30K in wild-type V2R did not recapitulate the increase in phosphatidylinositide (PI) turnover observed in V2ROTR3iC. Hence, the role of K6.30 may be context-specific. Deletion of two NH2-terminal OTR3iC segments in the V2ROTR3iC chimera did not diminish vasopressin-stimulated PI turnover, whereas deletion of RVSSVKL (residues 6.19–6.25) reduced receptor expression. Deletion of this sequence in wild-type OTR reduced expression by 50% without affecting affinity for [3H]OT. This OTR mutant was unable to activate PI turnover or extracellular signal-regulated kinase 1/2 phosphorylation. The effects of alanine substitution for individual residues in RVSSVKL indicated differential importance for OTR function. The R6.19A substitution lost high-affinity sites for [3H]OT and the ability to stimulate PI turnover. Affinity for [3H]OT and membrane expression was not affected by any other substitutions. OTR-V6.20A and OTR-K6.24A mutants functioned as well as wild-type OTR, whereas OTR S6.21A, S6.22A, and V6.23A mutants exhibited impaired abilities to activate PI turnover (20–40% of OTR), and the OTR-L6.25A mutant exhibited constitutive activity. In conclusion, specific amino acids in the RVSSVKL segment in the COOH-terminal region of the third intracellular domain of OTR influence the ability of OTR to activate G protein-mediated actions.

vasopressin type II receptor; G protein; phospholipase C; extracellular signal-regulated protein kinase-1/2

G PROTEIN-COUPLED RECEPTORS (GPCRs) are integral membrane proteins composed of seven transmembrane (TM) {alpha}-helices connected by alternating extracellular and intracellular loops. The NH2-terminal regions of GPCRs are extracellular and the COOH-terminal tails are in the cytoplasm. Some GPCRs couple to a restricted subset of G{alpha} proteins, while others couple simultaneously with different types of G{alpha} proteins (41, 42). Examination of the properties of receptor chimeras has led to the identification of structural motifs in a number of GPCRs (44). Although primary sequences vary widely among different classes of GPCRs, the second (2i) and third (3i) intracellular domains and associated juxtamembrane regions harbor critical motifs that enable GPCRs to recognize and activate their cognate G{alpha} protein partners (41, 42). It has been proposed that at least two functional domains may be present on the cytoplasmic face of a GPCR (42). The "activation domain" of the receptor, normally composed of a cationic amphipathic {alpha}-helix, stimulates the guanine nucleotide exchange activity of G{alpha} subunits, whereas the "selectivity domain" directs receptor coupling to specific G proteins.

A conserved interhelical movement of TM6 relative to TM3 and TM5 accompanies the activation of rhodopsin (8, 24, 38), the beta2-adrenergic receptor (32), and the M3 muscarinic receptor (40). The high-resolution crystal structure of bovine rhodopsin shows that this molecule is stabilized by a panel of interhelical hydrogen bonds and hydrophobic interactions, most of which are contributed by the side chains of highly conserved GPCR residues (30). For example, the hydrogen bond network between R3.50 (GPCR consensus numbering scheme; see Ref. 3) and residues E3.49, E6.30, and T6.34 in rhodopsin is theorized to constrain the molecule in the inactive conformation (30). Compared with the highly conserved E/DRY motif at the cytoplasmic end of TM3 in rhodopsin-like GPCRs, the nature of residues at positions 6.30 and 6.34 varies among different classes of this family (www.gpcr.org). Therefore, the potential ionic interaction that would constrain the movement of TM3 and TM6 is present only in some GPCRs. Substitution of other amino acids for E6.30 to disrupt interactions between D3.50 and E6.30 led to constitutive activation in both {alpha}1B- and beta2-adrenergic receptors (1, 13). Interestingly, substitution of E for L6.30 in the µ-opioid receptor resulted in a loss-of-function mutant receptor that failed to respond to agonist stimulation (21). In addition to these potential interactions, interhelical constraints between TM6 and other TMs have been shown to stabilize the inactive conformation of many GPCRs (10).

The four GPCRs that are targets for oxytocin (OT) or vasopressin (AVP) belong to a rhodopsin-like GPCR subfamily. The AVP type 1a (V1aR), type 1b (V1bR), and OT (OTR) receptors activate the GTP-binding protein Gq{alpha} to stimulate phospholipase C (PLC)-beta, whereas the AVP type 2 receptor (V2R) preferentially couples to Gs{alpha} to stimulate cAMP accumulation (4). The functional importance of the E/DRY motif at the end of TM3 has been well studied in OTR. Substitution of A or Q for D3.49 resulted in loss-of-function mutants that failed to express on the cell surface, whereas the R3.50A mutation acquired constitutive activity (7). In another study, substitution of N- for D3.49 created a constitutively active OTR mutant, whereas the OTR-R3.50A mutant was poorly expressed in the membrane and exhibited impaired OT-induced phosphatidylinositol (PI) turnover (9). The D3.49N mutant was no longer pertussis toxin-sensitive with respect to extracellular signal-regulated kinase (ERK) stimulation, suggesting a possible loss of the Gi{alpha}-mediated pathway (9). Despite the apparent discrepancy in OTR-R3.50A phenotype, these studies implicate the E/DRY motif in maintaining intramolecular constraints that affect the active/inactive conformation state of OTR.

Truncation of the COOH-terminal tail of OTR implicated its membrane-proximal region as a determinant for selective coupling to Gq{alpha} (19). Alanine-scanning substitution of the residues in this region has indicated the presence of a putative Helix-8 (45). Part of the hydrophilic face of this helix, composed of H7.59, E7.63, and R7.67, was found to be critical for the coupling function of OTR (45). Furthermore, we found in a previous study that the OTR cytoplasmic extension of TM6, and particularly residue K6.30, was important for coupling to Gq{alpha}/PLC in OTR, as well as in a V2R chimera containing the COOH-terminal region of the OTR third intracellular domain (V2ROTR3iC; see Ref. 43).

Because the nature of the residue at 6.30 is important in a number of rhodopsin-like GPCRs (2, 13, 21, 36, 37), we hypothesized that placing a K at this position in V2R might be sufficient to enhance PI turnover but found that this is not the case. Therefore, there must be other residues/structures in the 3i domain that are required for OTR signaling. We report that residues in the RVSSVKL sequence in the 3i domain have important roles in OTR coupling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals and plasmids. myo-[3H]inositol (22.3 Ci/mmol), [3H]AVP (73 Ci/mmol), and [3H]OT (33 Ci/mmol) were obtained from Perkin-Elmer Life Science (Boston, MA), and BSA (fatty acid free, fraction V) was from ICN Biomedicals (Irvine, CA). Human OT and AVP and 3-isobutyl-1-methylxanthine (IBMX) were obtained from Sigma (St. Louis, MO). Cell culture reagents were obtained from Invitrogen (Gaithersburg, MD), and the cAMP ELISA kit was obtained from Assay Designs (Ann Arbor, MI). The GeneEditor site-directed mutagenesis kit and Transfast transfection reagent were obtained from Promega (Madison, WI), and Fugene 6 transfection reagent was from Roche Diagnostics (Indianapolis, IN). AG 1-X8 resin was obtained from Bio-Rad Laboratories (Hercules, CA), and nitrocellulose membranes were from Schleicher & Schuell (Keene, NH). MultiScreen filter plates were purchased from Millipore (Bedford, MA).

The cDNA clone of human OTR was originally obtained from Dr. M. J. Brownstein (National Institutes of Health, Bethesda, MD) and was cloned into pcDNA6/V5-His C (Invitrogen, Carlsbad, CA; see Ref. 43). The cDNA clone for Gq{alpha} was obtained from Dr. M. I. Simon (California Institute of Technology, Pasadena, CA). The cDNA clone for human V2R was obtained from Dr. M. Birnbaumer (National Institute of Environmental Health Sciences, Research Triangle Park, NC) and subcloned into the EcoR1 and Apa 1 sites of pcDNA6/V5-His B. In all cases, the insert was designed so that the V5-His tag was not expressed.

Mutagenesis and nomenclature. The V2R chimera containing the COOH-terminal region of the OTR3i was constructed previously (43). Mutated receptors V2R-A264K, V2R-V266K, V2ROTR3iC-{Delta}1 (sequence deleted: AAEAPEGAAA), V2ROTR3iC-{Delta}3 (sequence deleted: RVSSVKL), OTR-{Delta}3 (sequence deleted: RVSSVKL), OTR-R259A, OTR-V260A, OTR-S261A, OTR-S262A, OTR-V263A, OTR-K264A, and OTR-L265A were constructed using GeneEditor, and the changes were verified by sequencing. To facilitate comparison of comparable residues between V2R and OTR, we used a GPCR consensus numbering scheme (3). In this system, the highly conserved proline within TM6 of both OTR and V2R is designated as 6.50. OTR residues R259, V260, S261, S262, V263, K264, and L265 are designated as R6.19, V6.20, S6.21, S6.22, V6.23, K6.24, and L6.25, respectively. V2R residues A264 and V266 are designated as A6.28 and V6.30, respectively. The residues in the OTR sequence of the V2ROTR3iC chimera are named according to their positions in OTR.

Cell culture and transfection. COSM6 cells were cultured in DMEM containing 8% FCS, 2 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin. For membrane-binding assays, COSM6 cells were plated at 9 x 105 cells/100-mm dish. The next day, plasmid DNA was transfected in the cells with the Transfast reagent as described previously (45). Alternatively, plasmid DNA (6 µg/dish) was mixed with 600 µl DMEM containing 18 µl Fugene 6 and incubated for at least 15 min before addition to the dish. For PI turnover, intact cell binding, and cAMP accumulation experiments, COSM6 cells were plated in six-well plates at 1.2 x 105 cells/well. Plasmid DNA was transfected into the cells either with Transfast (45) or Fugene 6. Plasmid DNA (1 µg/well) was mixed with 100 µl DMEM containing 3 µl Fugene 6. Plasmid encoding Gq{alpha} was cotransfected with OTR-expressing plasmid for the PI turnover assay as described previously (31). Cell medium was replaced after 24 h.

Membrane preparation. After transfection (48 h), COSM6 cells were washed two times with PBS and harvested in chilled hypotonic buffer (20 mM HEPES, pH 7.4, 10 mM EDTA). The cells were homogenized at 4°C for 3 x 10 s with a Polytron (Brinkmann). Homogenates were spun at 500 g at 4°C for 10 min, and the resulting supernatants were centrifuged at 20,000 g at 4°C for 30 min. The resulting crude membrane pellets were suspended in 20 mM HEPES buffer (pH 7.4) and stored in aliquots at –80°C. Protein was determined using the Bradford reagent (Bio-Rad) with BSA as standard.

Ligand-binding assays. Stored membranes were thawed, centrifuged (20,000 g, 30 min), and resuspended at 0.1~0.2 mg protein/ml in binding buffer (50 mM Tris, pH 7.4, 0.1% BSA, 10 mM MgCl2). The isotope ([3H]OT or [3H]AVP) was diluted in binding buffer. The binding assay mixtures (200 µl) containing cell membranes (10~20 µg protein/well) and increasing concentrations of radiolabeled ligand (total binding) or the isotope plus 10–5 M unlabeled ligand (nonspecific binding) were incubated in a Multiscreen filter plate with shaking for 2 h at room temperature. The plate was then mounted on a Millipore vacuum manifold, and the binding reaction was stopped by rapid filtration under vacuum, followed by two washes with cold binding buffer without MgCl2. The filters containing bound ligand were punched out and counted in Scintisafe Econo 1 (Fisher, Fairlawn, NJ).

For intact cell binding, 2 days after transfection, COSM6 cells were washed one time with room temperature Hanks' balanced salt solution (HBSS) without Ca2+ and Mg2+. Each well received 0.6 ml Ca2+-free HBSS containing 5 mM MgCl2, 0.1% BSA, and 30 nM [3H]AVP in the presence (nonspecific) and absence (total binding) of 20 µM unlabeled AVP for 2.5 h at room temperature. The reaction was terminated by removing the medium, and the cells were immediately washed with cold HBSS containing 0.1% BSA. The cells were then lysed with 0.7 ml 1 M NaOH, and the lysate was neutralized with 0.7 ml 1 M HCl. Neutralized lysate (1 ml) was counted in ScintiSafe Econo 1. In all cases, specific binding was defined as the difference between total and nonspecific binding.

PI turnover. After transfection (24 h), COSM6 cells were washed two times with PBS and cultured in 1 ml DMEM, 0.5% FCS, and 0.4 µM myo-[3H]inositol at 37°C overnight. The labeled cells were washed two times with PBS and were incubated with HBSS (pH 7.4) containing 0.2% BSA and 10 mM LiCl at 37°C for 30 min. The cells were stimulated for another 30 min with different concentrations of OT or AVP as designated in the legends for Figs. 1–3. [3H]inositol 1,4,5-trisphosphate (IP3) was isolated by ion exchange chromatography as described previously (43). Data are expressed as agonist-dependent activity.


Figure 1
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  		Fig. 1. Multiple sequence alignment of human (h) oxytocin receptor (OTR) and human arginine vasopressin type 2 (V2R), type 1a (V1aR), and type 1b (V1bR) receptor together with bovine rhodopsin in the regions of the putative third cytoplasmic loop and a part of transmembrane (TM) 6, as predicted from evolutionary trace analysis (27, 43). Alignments within the putative intracellular domains are less well defined than in the TM regions. The boxed regions in V2R and OTR are the places where the sequences were exchanged in the construction of the V2R chimera containing the COOH-terminal region of the OTR third intracellular domain (V2ROTR3iC). The OTR COOH-terminal region of the third intracellular domain (OTR3iC) in this chimera is divided into four sections as indicated. For comparative purposes, the conserved P6.50 in the TM6 is indicated by a star; residues at 6.30 are marked with an arrowhead. Helix 6 of rhodopsin (protein database entry: 1F88) is designated in bold and enlarged letters. The putative cytoplasmic ends of Helices 6 in OTR, V2R, V1aR, and V1bR are designated according to homology with rhodopsin.

 

Figure 3
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  		Fig. 3. Importance of the RVSSVKL segment on extracellular signal-regulated kinase (ERK)1/2 phosphorylation in activated OTR. A: representative Western blot image of both phosphorylated and total ERK1/2 in cells expressing OTR, OTR-{Delta}3, and OTR-L6.25A. Cells transfected with pcDNA6 vector serve as negative control. B: ratio of bands quantitated from blots for phosphorylated and total ERK1/2 in cells transfected with pcDNA6 vector or plasmids encoding wild-type or mutated OTR in the absence (shaded bars) and presence (black bars) of 10–7 M OT. Data are expressed as means ± SE (n = 3) and were analyzed by ANOVA and Duncan's modified range test. OTR-{Delta}3 was compared separately with 0.4 µg OTR, conditions of comparable receptor expression. Significant differences between groups in a given category (P < 0.05) are designated with different lowercase letters.

 
cAMP assay. After transfection (48 h), COSM6 cells were washed two times with HBSS and preincubated with HBSS containing 20 mM HEPES and 1 mM IBMX for 15 min at 37°C. The cells were stimulated with increasing concentrations of AVP for 1 h at 37°C. The medium was removed, and the reaction was terminated by the addition of 0.5 ml of 0.1 N HCl. cAMP content in the cell lysate was measured by immunoassay, following the protocol recommended by the kit manufacturer (Assay Designs).

ERK1/2 phosphorylation and Western blot analysis. After transfection (24 h), COSM6 cells were placed in serum-free medium overnight and then in phenol red- and glucose-free DMEM containing 0.1% BSA for at least 1 h before treatment with 100 nM OT for 5 min. The samples were harvested, separated on 10% SDS-PAGE gels, and transferred to nitrocellulose membranes as described previously (45). The concentration of signal in bands was detected by enhanced chemiluminescence (Amersham Biosciences). Quantitation was accomplished by ImageQuant TL software analysis of data obtained from a Storm imager using ImageQuant software (Amersham Biosciences, Piscataway, NJ).

Receptor internalization assay. After transfection (48 h), COSM6 cells in six-well plates were washed two times with chilled PBS and kept on ice to prevent receptor internalization. Ca2+-free HBSS (1 ml) containing 5 mM MgCl2, 0.1% BSA, and 10 nM [3H]OT was added to each well of six-well plates. Nonspecific binding was determined in a separate well by inclusion of 1 µM unlabeled OT in the medium. Plates were incubated on ice for 3 h to reach equilibrium, followed by incubation at 37°C for different times. At the end of incubation, plates were washed two times with 1 ml cold Ca2+ and Mg2+-free HBSS containing 0.1% BSA and placed back on ice. Cells were treated with 0.5 ml cold acid wash solution (17) for 12 min. The wash solution was collected to determine the amount of membrane-bound receptors. The remaining cells were solubilized as described for the intact cell-binding assay, and the lysates were collected to determine internalized receptors. Specific binding was defined as the difference between total and nonspecific binding. Internalized receptors at each incubation time were expressed as a percentage of total receptors (specific counts in cell lysate/the total of membrane-bound and cell lysate-specific counts).

Immunocytochemistry and confocal microscopy. COSM6 cells were plated in 35-mm glass-bottom culture dishes at 1.0 x 105 cells/well. Transfection of OTR-L6.25A mutant was performed on the next day as described above. After transfection (48 h), cells were stained with Alexa 594-conjugated conconavalin A (ConA) and anti-OTR antibody as described previously (45). Fluorescent images within the same optical field for both ConA (543 nm) staining and OTR (488 nm) immunostaining were acquired in an epifluorescence mode at x60 magnification on a confocal microscope (Zeiss). The acquired images were exported to Adobe PhotoShop for further processing.

Data analysis. Binding data were analyzed with the Ligand program (P. J. Munson, National Institute of Child Health and Human Development, Bethesda, MD). Dose-response curves of PI turnover were analyzed with a four-parameter logistics curve-fitting program (M. L. Jaffe, Silver Spring, MD). Where indicated, data are presented as means ± SE and were analyzed by ANOVA and Duncan's modified multiple range test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Determinants in addition to K6.30 in the OTR3iC substitution enhance vasopressin-stimulated PI turnover in V2ROTR3iC. K6.30 is an important residue for OTR coupling. To determine if the presence of K at position 6.30 would be sufficient to increase ligand-stimulated PI turnover in V2R, the mutant receptor V2R-V6.30K was created. In addition, based on the observation that OTR-K6.28A did not change the function of wild-type OTR (43), V2R-A6.28K was also created as a putative control. Binding parameters of wild-type and mutant V2R receptors are summarized in Table 1. Expression of V2R-V6.30K was reduced by 40% compared with wild-type V2R. Substitution of K for either A6.28 or V6.30 did not affect the AVP-binding affinity of V2R. Furthermore, the ability of AVP to stimulate cAMP production at 1 and 10 nM was unchanged (data not shown).


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  		Table 1. Properties of V2R and V2R mutants

 
Although V2R expressed at high density can couple with Gq{alpha} (47), its ability to activate PLC was less than twofold under the conditions employed in this study (Table 1). V2R-A6.28K was no more active than wild-type V2R at stimulating PI turnover. Although V2R-V6.30K displayed a 1.5-fold increase in [3H]IP3 formation relative to V2R when exposed to 10–30 nM AVP (Table 1), this response did not approach the fourfold stimulation observed with V2ROTR3iC (data not shown). These data indicate that the single amino acid substitution at position 6.30 in V2R was unable to recapitulate the enhanced coupling to PLC introduced by the substitution of the entire OTR3i COOH-terminal region.

In an attempt to identify other determinants in the OTR3iC sequence, we applied a deletion strategy in the V2ROTR3iC chimera, where the inserted OTR sequence clearly conveyed coupling ability. Figure 1 shows the alignment of the putative 3i and part of TM6 regions of OTR, V1aR, V1bR, V2R, and rhodopsin as determined previously using evolutionary trace analysis (27) and the comparable sequence of the V2ROTR3iC chimera. The residues in section 4 (6.26–6.35) constitute the COOH-terminal region of OTR3iC. Residues in this region are highly conserved between OTR and V2R, with the exception of positions 6.28 and 6.30, which have been investigated singly in this and another study (43). Therefore, we did not make additional deletions in this region.

Section 1 contains 10 residues from 6.00 to 6.09 at the NH2-terminal end of the OTR3iC insertion. Deletion of this section from the V2ROTR3iC chimera did not change receptor properties with respect to expression on the cell membrane, binding to AVP, or, most importantly, AVP-stimulated IP3 formation (data not shown). The residues in section 2 (6.10–6.18) were deleted previously (43). Removing this region had no effect on expression, affinity, or vasopressin-induced PI turnover.

Section 3 is composed of the seven amino acid sequence RVSSVKL (6.19–6.25). This region is of potential interest because the VSSV/I sequence is conserved between Gq{alpha}-coupled OTR, V1aR, and V1bR but is absent in Gs{alpha}-coupled V2R (Fig. 1). When we deleted this region, the V2ROTR3iC-{Delta}3 receptor was not expressed well, and we could not analyze function. Preliminary alanine scanning of individual residues in section 3 of the V2ROTR3iC chimera indicated that substitution of A for S6.21, S6.22, or V6.23 reduced vasopressin-dependent PI turnover, substitution for L6.25 promoted coupling in the absence of vasopressin, and substitution for R6.19, V6.20, and K6.24 had no effect (data not shown). To obtain more insight into the roles of these residues in the native receptor, we examined the potential contribution of the RVSSVKL segment to receptor function in wild-type OTR.

Residues in the RVSSVKL segment impact OTR function. Deletion of the entire RVSSVKL segment (OTR-{Delta}3) resulted in 50% less expression of the membrane-bound receptor without any change on receptor affinity for [3H]OT (Table 2). This truncated OTR mutant was unable to stimulate PI turnover, even in the presence of 10 µM OT, when compared with OTR expressed at an equivalent concentration (Fig. 2A).


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  		Table 2. Properties of wild-type and OTR mutants

 

Figure 2
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  		Fig. 2. Alanine substitution for individual residues in the RVSSVKL region induces different phenotypes. A: effect of mutation of individual residues or the entire region ({Delta}3) on stimulation of phosphatidylinositol (PI) turnover by oxytocin (OT, 10 µM for OTR-{Delta}3 and OTR-R6.19A mutants and 100 nM for the rest). B: dose dependence of the ability of OTR and the S6.21A, S6.22A, V6.23A, and L6.25A mutants to stimulate PI turnover. C: constitutive activity of the OTR-L6.25A mutant, as demonstrated by an increase in basal [3H]inositol 1,4,5-trisphosphate (IP3) accumulation compared with that from cells transfected with pcDNA6 empty vector or vector expressing OTR. Data are expressed as means ± SE (n = 3–4 experiments) and were analyzed by ANOVA and Duncan's modified range test. All receptors were transfected at 1 µg, except OTR-{Delta}3 and OTR-R6.19A, which were compared separately with 0.4 µg OTR, conditions of comparable receptor expression. Significant differences between groups in a given category (P < 0.05) are designated with different lowercase letters.

 
To explore the importance of the individual residues in the RVSSVKL segment, they were mutated individually. Alanine substitution did not alter either the affinity of the mutant receptors for [3H]OT or receptor concentration in COSM6 cell membranes, except for the OTR-R6.19A mutant, which did not exhibit high-affinity binding (Table 2). A significant fraction of this mutant receptor was localized in the cell membrane, as determined by confocal microscopy and colocalization with ConA (data not shown). OTR-R6.19A was not able to activate PLC, even in the presence of 10 µM OT (Fig. 2A). Examination of receptors in which the individual residues in the sequence VSSVKL were mutated revealed that, despite unaltered OT affinity and membrane expression, the abilities of OTR-S6.21A, OTR-S6.22A, and OTR-V6.23A to activate PI turnover were reduced significantly (to 31, 36, and 40% of wild-type OTR, respectively, at 100 nM OT; Fig. 2A and Table 2). In contrast, the responses of OTR-V6.20A and OTR-K6.24A were similar to wild-type OTR (Fig. 2A and Table 2). The PI turnover responses for the S6.21A, S6.22A, and V6.23A OTR mutants were explored in greater detail. The diminished PI turnover in all three mutants is attributable to a change in maximal response and not EC50 (Fig. 2B and Table 2). In both of these studies, wild-type OTR-binding parameters were within the range reported by others (Kd = 0.32–25 nM; see Refs. 15, 18, 23, 25, and 34).

We observed a distinctly different phenotype for OTR-L6.25A. Alanine substitution for L6.25 did not affect the ability of OTR to increase PI turnover in response to OT stimulation. However, as shown in Fig. 2C, although basal PI turnover in cells transfected with wild-type OTR was comparable to that in cells transfected with pcDNA6 empty vector, the OTR-L6.25A mutant elicited a 60% higher basal activity in the absence of OT stimulation, representing constitutive activation. There was no difference observed in internalization kinetics between wild-type OTR and the OTR-L6.25A mutant (data not shown).

These data show that the RVSSVKL segment in the OTR3iC contains residues that facilitate the ability of OTR to activate PI turnover or repress constitutive activity.

Effect of mutations in section 3 on OT-stimulated ERK activity. Phosphorylation of ERK1/2 by activated OTR has been reported to utilize both Gq{alpha}/Gbeta{gamma} and Gi{alpha}/Gbeta{gamma} pathways (28, 39, 46). Therefore, it was of interest to determine if modification in the RVSSVKL sequence affected the ability of the mutated receptor to stimulate ERK phosphorylation. As expected, cells transfected with pcDNA6 empty vector did not respond to OT, whereas cells expressing OTR showed a twofold increase in ERK1/2 phosphorylation in response to OT (Fig. 3). Deletion of the RVSSVKL sequence from OTR completely eliminated ERK phosphorylation in response to stimulation with 100 nM OT. OTR-L6.25A did not show detectable constitutive activity in the ERK pathway (Fig. 3). Alanine substitution for S6.21, S6.22, and V6.23 led to impairment in OT-induced ERK phosphorylation, consistent with the observation in PI turnover (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Previously, the COOH-terminal OTR 3i domain and specifically K6.30 were found to convey enhanced ligand-stimulated PI turnover in the V2ROTR3iC chimera (43). Because the amino acid sequences in the region between the 3i domain and TM6 are essentially conserved between OTR and V2R, except for residues at 6.28 and 6.30, we hypothesized that substitution of a lysine at 6.30 might enable V2R to acquire enhanced ability to activate PLC. In the present study, V2R-V6.30K and wild-type V2R displayed similar affinity for [3H]AVP, suggesting that this modification did not induce a conformational change in the ligand-binding site or perturb general receptor conformation. Whereas V2R-V6.30K was more potent at stimulating PI turnover than wild-type V2R, the mutant receptor was significantly less effective at this coupling than V2ROTR3iC. This indicates that substitution of a single amino acid was unable to recapitulate in V2R the enhanced coupling phenotype introduced by the substitution of the entire OTR3i COOH-terminal region.

Because V2R-V6.30K did not acquire an enhanced ability to stimulate PI turnover, we reasoned that there must be other structural determinants in the OTR3iC sequence that are important for G protein activation. However, it would appear that these putative determinants are not sufficient by themselves to elicit G protein activation, since the K6.30V mutation completely eliminated AVP-stimulated PI turnover in the V2ROTR3iC chimera (43). In searching for such determinants in the OTR3iC region, we found that two regions (sections 1 and 2) could be omitted individually without affecting the ability of V2ROTR3iC to stimulate PI turnover. On the other hand, residues in the 6.19–6.25 region (RVSSVKL) possessed potentially interesting properties that were investigated in OTR.

Deletion of the entire RVSSVKL segment from OTR did not affect receptor affinity for [3H]OT but resulted in an OTR mutant that was incapable of activating PLC, consistent with the findings in the V2ROTR3iC chimera. Alanine substitution revealed that residues R6.19, S6.21, S6.22, V.6.23, and L6.25 contributed in different ways to OTR function. OTR-R6.19A was expressed in plasma membrane but did not exhibit high-affinity binding or respond to OT with PLC stimulation, suggesting that R6.19 may be involved in intramolecular interactions that are important for the conformational integrity of OTR. This is in striking contrast to the OTR construct lacking RVSSVKL (OTR-{Delta}3), which exhibited normal affinity for OT, possibly as a result of compensatory interactions by other residues in the 3i sequence or elsewhere. Alanine substitution for S6.21, S6.22, or V6.23 produced mutant receptors that displayed partially compromised abilities to increase PI turnover, with no change in receptor properties with respect to membrane expression, ligand affinity, and EC50. Because these residues are present in the inactive K6.30V mutant, these data indicate that S6.21, S6.22, and V6.23 play important, but not obligatory, roles in the coupling of OTR to G proteins.

Some conserved sequences, such as the KERK and the BBx(x)B motifs, have been implicated in GPCR coupling to G proteins but are receptor subtype specific (5, 14, 20, 26, 29). These motifs are not present in the 3i loop of OTR, whereas the VSSV/I sequence is present only in the V1a/V1b/OT subgroup of GPCRs. Multiple contact sites have been identified on the interfaces of both rhodopsin and the transducin G protein (16). The cytoplasmic ends of rhodopsin TM3 and TM6 form regions responsible for Gt{alpha} activation (8, 30, 38). An engineered cysteine substituted for S6.23 was found to cross-link to both the COOH and NH2 termini and a region within the {alpha}4-beta6 loop of the Gt{alpha} subunit (6, 22). Interestingly, the OTR residues S6.21, S6.22, and V6.23 implicated as affecting coupling are in this general area and may play similar roles.

Substitution of A- for L6.25 in OTR did not affect OT-stimulated PI turnover but exhibited a 60% enhancement of basal PI turnover over that of wild-type OTR. Constitutive activity previously reported for OTR-R3.50A and OTR-D3.49N mutants (7, 9) was roughly of the same order of magnitude. These data do not support a role for L6.25 in G protein activation but instead point to a potentially important role for this residue in keeping OTR in an inactive conformation, possibly as a consequence of intrareceptor hydrophobic interactions. In an OTR model based on the rhodopsin crystal structure (9), a significant reduction in the bend of TM6 was proposed to accompany the activation process. The straightening of the bend in TM6 would induce a significant movement at its cytoplasmic end. Interestingly, L6.25 is part of the sequence connecting the putative cytoplasmic end of OTR Helix 6 (TM6) with the region including the SSV sequence. The L6.25A substitution would decrease potential steric constraints at this position, perhaps contributing to increased receptor flexibility and constitutive activity or more directly to enhanced interaction with G proteins.

In addition to coupling to Gq{alpha}, OTR also activates Gi{alpha} and Gh{alpha} in some circumstances (12, 35). In turn, these G proteins may couple to distinct intracellular signaling pathways in different cell types, possibly accounting for the varying effects of OT on cell growth in different cancer cells (33). Phosphorylation of ERK1/2 is a convergent endpoint for signals from Gq{alpha}, Gi{alpha}, and Gbeta{gamma} pathways elicited by activated OTR (39, 46). The D3.49N substitution eliminated pertussis toxin-sensitive OTR activation of ERK1/2 phosphorylation in transfected HEK293 cells but exhibited constitutive PI turnover activity (9). These data support the hypothesis that the cytoplasmic end of the TM3 helix is important for the recognition by OTR of specific G{alpha} proteins. We have previously found that the OTR-K6.30V mutant failed to stimulate both PI turnover and ERK1/2 phosphorylation in response to OT (43). In the present study, we found a similar correlation when the entire RVSSVKL sequence was removed. However, although the OTR-L6.25A mutant receptor acquired constitutive activity with respect to IP3 production, we did not detect constitutive ERK1/2 phosphorylation. It is not clear whether this is actually the case or simply a result of our inability to detect a small change in ERK phosphorylation. Nonetheless, it is noteworthy that a similar dichotomy between ability to stimulate PI turnover and ERK1/2 phosphorylation was also observed in the OTR-D3.49N mutant receptor (9).

In summary, the RVSSVKL sequence in the COOH-terminal region of the OTR 3i domain has been identified in the present study as possessing features that influence OTR-G protein coupling. Residue R6.19 is important in maintaining the general properties of the OTR. OTR S6.21, S6.22, and V6.23 contribute subtle influences on coupling activity. L6.25 apparently plays a role in keeping the OTR in an inactive conformation, since substitution of an A at this position results in constitutive activity with respect to PI turnover. We speculate that single amino acid substitutions might reflect perturbations in interactions with other residues in the OTR receptor or with G proteins. These data point out the importance of residues in the OTR3iC in OTR function. Although the interhelical movements of TM3 and TM6 have been considered as conserved processes in the GPCR activation, the VSSV/I sequence is found only in the V1a/V1b/oxytocin subgroup of GPCRs, indicating it may be a unique feature in the activation of these receptors, particularly in regard to coupling to PLC.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported in part by National Institute of Child Health and Human Development Grant HD-09618 from the R. A. Welch Foundation.


   ACKNOWLEDGMENTS
 
We thank Jennifer Phillips for assistance in manuscript preparation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. M. Sanborn, Dept. of Biomedical Sciences, 102 Physiology Campus Delivery 1680, Colorado State Univ., Fort Collins, CO 80523 (e-mail: Barbara.Sanborn{at}colostate.edu)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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