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Endocrinology Vol. 139, No. 11 4563-4567
Copyright © 1998 by The Endocrine Society

ARTICLES

Epitope Tag Mapping of the Extracellular and Cytoplasmic Domains of the Rat Parathyroid Hormone (PTH)/PTH-Related Peptide Receptor1

Lin Y. Xie and Abdul B. Abou-Samra

Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Dr. Abdul B. Abou-Samra, Endocrine Unite, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The PTH/PTH-related peptide (PTHrP) receptor is predicted to span the plasma membrane seven times with an amino-terminal extracellular extension and a cytoplasmic carboxyl-terminal tail. To assess this prediction, we inserted 10- or 9-amino acid epitope tags from c-myc or hemophilus influenza hemaglutinin (HA), which are recognized by the monoclonal antibodies 9E10 and 12Ca5, respectively, in different extracellular and cytoplasmic regions of the receptor and examined the immunoreactivity of the epitopes in intact and permeabilized cells. The data show that the epitopes were well tolerated when introduced into the E2 region of the extracellular amino-terminus (E2-myc and E2-HA), in the first extracellular loop (EL1), in the second and third cytoplasmic loops (CL2c and CL3), or in the carboxyl-terminal tail (T-myc). Receptors tagged at these locations were well expressed, bound PTH with high affinity, and increased cAMP accumulation with a good efficiency. Receptors tagged in the second and third extracellular loops (EL2c and EL3c) or the first cytoplasmic loop (CL1c) bound the PTH radioligand with a low affinity, stimulated cAMP accumulation with a low efficiency, and had low expression levels. The receptors tagged on presumed extracellular regions, E2-myc, E2-HA, EL1, EL2c, and EL3c, were readily detected on the surface of intact cells with the monoclonal antibody against the epitope tag. In contrast, receptors tagged with the c-myc epitope in the cytoplasmic loops (CL1c, CL2c, and CL3) or in the carboxyl-terminal tail (T-myc) did not show any 9E10 binding in intact cells. These receptors, however, were well expressed on the cell surface, as detected by the binding of the monoclonal antibody, 12Ca5, to the HA tag that was introduced into the E2 region of these constructs. The c-myc epitopes, however, became accessible after permeabilization of the cell membrane. In conclusion, these data provide experimental evidence for the sidedness of the extracellular and cytoplasmic domains of the PTH/PTHrP receptor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
G PROTEIN-COUPLED receptors are predicted to span the plasma membrane seven times, with an extracellular amino-terminal extension and an intracellular carboxyl-terminal tail. The crystal structure of bacteriorhodopsin (1, 2) and bovine (3) and frog (4) opsins provided evidence that these molecules contains seven transmembrane helixes, three extracellular loops, and three intracellular loops.

The PTH/PTH-related peptide (PTHrP) receptor is a member of a new G protein-coupled receptor family that is distinct from all previously identified G protein-coupled receptors (5, 6). These receptors do not have any significant sequence homology with other G protein-coupled receptors or with rhodopsin, which have been shown to span the plasma membrane seven times (5, 6). However, these receptors contain several hydrophobic domains that are consistent with the seven transmembrane-spanning model of rhodopsin. This model, however, requires experimental verification.

Immunological methods using antibodies specific for different receptor domains could be useful to study the orientation of the receptor within the plasma membrane. For example, antibodies that recognize the amino-terminus of the ß-adrenergic receptor (7) and the amino-terminal extension and the third cytoplasmic loop of the LH/hCG receptor (8) were useful probes to study the sidedness (extracellular vs. intracellular) of these regions relative to that of the plasma membrane. This immunological approach, however, is limited by the availability of specific antibodies against the various receptor domains. Recently, epitope tagging of the amino- and carboxyl-terminal ends of several proteins has been used to assess expression and processing of the proteins within the cell (9). This approach is particularly useful to examine the sidedness of the presumed extracellular and cytoplasmic domains of G protein-coupled receptors. Epitope tags have been successfully inserted in each of the extracellular and cytoplasmic domains of bovine opsin as probes to determine the sidedness of these domains using immunodetection of intact and permeabilized cells (10). Thus, epitope tagging of various receptor domains may be used to study the cellular locations of these regions. In this study we show that silent 9- or 10-amino acid epitope tags, specifically recognized by the 9E10 and 12Ca5 monoclonal antibodies (8), placed in the middle of the coding sequence of the various regions of the PTH/PTHrP receptor by sequence conversion and/or insertion can be used to map the cellular location of the epitope relative to the plasma membrane.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
[Nle8,18,Tyr34]Bovine PTH-(1–34)NH2 (NlePTH) and human PTHrP-(1–36)NH2 (PTHrP) were purchased from Bachem (Irvine, CA), and all chemicals were of the highest grade available and were obtained from either Sigma Chemical Co., Inc. (St. Louis, MO), or Fisher Scientific International, Inc. (Pittsburgh, PA). Na125I (2125 Ci/mmol) and 125I-labeled sheep antimouse IgG were purchased from New England Nuclear (Boston, MA). Restriction enzymes were purchased from either U.S. Biochemical Corp. (Cleveland, OH), New England BioLabs (Beverly, MA), Promega Corp. (Madison, WI), or Life Technologies (Gaithersburg, MD). Diethylaminoethyl-dextran was obtained from Pharmacia Biotech (Piscataway, NJ). COS-7 cells were a gift from Dr. B. Seed, Laboratory of Molecular Biology of this institution. The monoclonal antibody hybridoma, 9E10, was obtained from American Type Tissue Cultures (Manassas, VA), and the 12Ca5 ascites fluid was purchased from Berkeley Antibodies (Berkeley, CA). Oligonucleotides were synthesized in this institution. Tissue culture media were prepared by the Massachusetts General Hospital medium facility. Flasks, plates, and other tissues cultures supplies were obtained from Corning (Oneonta, NY).

COS-7 cell cultures and transfection
COS-7 cells were cultured in DMEM containing 10% FBS and maintained in a humidified 95% air-5% CO2 incubator at 37 C. The cells were plated in 15-cm dishes until they become about 70% confluent, then were transfected with the diethylaminoethyl-dextran method using 5 µg plasmid DNA. One day after transfection, the cells were trypsinized and replated in 24-well plates (for PTH binding, PTH-induced cAMP accumulation, or double antibody binding) or in 6-well plates for immunofluorescence.

Construction of epitope-tagged receptors
The epitope tags were introduced in the sequence of the rat PTH/PTHrP receptor by site-directed mutagenesis using a modification of the method of Kunkle (11) and/or PCR. The veracity of the mutant complementary DNAs (cDNAs) was determined by restriction enzyme mapping and sequence analysis. A 9-amino acid epitope from hemophilus influenza (YPYDVPDYA) that is recognized by the monoclonal antibody 12Ca5 and/or a 10-amino acid epitope from c-myc (EQKLISEEDL or QQKLISEEDL) that is recognized by the monoclonal antibody 9E10 were introduced by sequence insertion, sequence replacement, and/or sequence conversion in the amino-terminal extracellular domain, the extracellular and cytoplasmic loops, and the carboxyl-terminal tail (Fig 1Go and Table 1Go). As EL2, EL3, CL1, and CL2 fails to express and/or to bind PTH, a second set of constructs was made, EL2c, EL3c, CL1c, and CL2c, to minimize sequence disruption and maximize expression and/or binding.



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  		Figure 1. Molecular design of epitope-tagged receptors. The figures schematizes the PTH/PTHrP receptor, with the location of the epitopes shown as fags. The corresponding name of the construct is also shown.

 

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  		Table 1. Molecular design of epitope-tagged receptors

 
RRA
Cells, grown in 24-well plates, were washed twice with 1 ml binding buffer (50 mM Tris-HCl, 100 mM NaCl, 5 mM KCl, 2 mM CaCl2, 5% heat-inactivated horse serum, and 0.5% FBS, pH 7.7) and incubated with 125I-labeled NlePTH in binding buffer at 15 C for 4 h in the presence or absence of 10-11–10-6 M nonradioactive NlePTH (12). In some experiments, increasing concentrations of the radioligand (100,000–10,000,000 cpm/well, which corresponds to 0.1–10 nM) was added in the presence and absence of 1 µM NlePTH. Cells were then washed three times with cold PBS and solubilized with 1 N NaOH, and radioactivity in the lysate was determined by {gamma}-counter (Model 6/400 Plus, Micromedic Systems, Inc., Horsham, PA). The binding data were analyzed with Scatchard plots.

Double antibody binding (13)
Cells grown in 24-well plates were washed 3 times with PBS (pH 7.4) containing 5% heat-inactivated FBS and incubated at room temperature for 2 h in 0.5 ml PBS in the presence of monoclonal antibody 9E10 or 12cA5 for 2 h at room temperature, rinsed three times with buffer, and incubated with 125I-labeled goat antimouse IgG (200,000 cpm/well; New England Nuclear). In some experiments, the cells were incubated with 2% paraformaldehyde in PBS and 5% dextrose, permeabilized with 0.1% Triton X-100 at room temperature for 4 min, rinsed (3 times) with PBS, incubated with the first antiserum at room temperature for 1 h, rinsed (3 times) with PBS, and incubated with the 125I-labeled second antibody. The incubation was terminated by washing the cells with PBS. The cells were then solubilized in 1 N NaOH, and radioactivity was counted.

Measurement of intracellular cAMP accumulation
Cells grown in 24-well plates were preincubated in serum-free DMEM medium containing 0.1% BSA, 10 mM HEPES (pH 7.42), and 1 mM 3-isobutyl-1-methyl-xanthine at room temperature for 10 min, then incubated at 37 C for an additional 15 min after adding agonists (100 nM PTH) or vehicles. The reaction was terminated by aspirating the medium, washing the cells with cold PBS, and freezing the plate on dry ice; the plates were stored at -80 C until cAMP RIA using previously described methods (14).

Statistical analysis
The mean ± SD of triplicate determinations were calculated. One-way ANOVA followed by Student’s t test was used to determine significance (P < 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To examine the sidedness of the different domains of the PTH/PTHrP receptor, we introduced the c-myc or HA epitopes in the amino-terminal extension (E2-myc and E2-HA), in the putative extracellular loops (EL1, EL2, EL2c, EL3, and EL3c) in the putative cytoplasmic loops (CL1, CL1c, CL2, CL2c, and CL3), and in the carboxyl-terminal tail (T-myc; Table 1Go and Fig. 1Go).

Introduction of the c-myc epitope in the E2 region of the putative amino-terminal extracellular domain of the PTH/PTHrP receptor did not impair receptor expression, ligand binding, or PTH-stimulated cAMP accumulation (Table 2Go). These data, which are similar to those previously reported for the HA-tagged PTH/PTHrP receptor (15), indicate that the amino-terminus of the PTH/PTHrP receptor is oriented extracellularly.


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  		Table 2. Functional characterization of the epitope-tagged receptors

 
To confirm the location of the putative extracellular loops, the c-myc epitope was introduced into the first, second, and third extracellular loops. The mutant receptors were first examined for ligand binding and PTH-stimulated cAMP accumulation. EL1 was well expressed, bound PTH with high affinity, and increased cAMP accumulation with high potency (Table 2Go). In addition, the epitope of EL1 was accessible for double antibody binding on the intact cells (Fig. 2Go). Interestingly, when the c-myc epitope was introduced in the E2 and EL1 regions in one construct, E2-EL1, the binding of 9E10 was increased compared with E2 or EL1 separately; this suggests that both epitopes are independently accessible (Fig. 1Go). In contrast, expression of EL2 and EL3 was dramatically reduced (Table 2Go and Fig. 2Go), and no ligand binding was detected (Table 2Go). Therefore, we designed two additional epitope tags, EL2c and EL3c, in which more sequence conversion and less insertion were undertaken (Table 1Go). EL2c and EL3c bound the PTH radioligand with an apparent affinity about 10-fold less than that of the wild-type receptor and increased cAMP accumulation with a sensitivity to PTH that was also decreased (Table 2Go). Additionally, the number of binding sites was reduced (Table 2Go). The immunoreactivity of the EL2c and EL3c epitopes was readily detected on intact cells; however, the binding was low compared with that of E2 and EL1 constructs (Fig. 2Go). These data indicate that the EL2c and EL3c regions are also extracellular because they are immunologically accessible on intact cells.



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  		Figure 2. Binding of the 9E10 monoclonal antibody to intact cells expressing the different epitope-tagged receptors. The cells, cultured in 24-well plates, were incubated with 9E10 ascites fluid (1:2,000) for 2 h and then with an 125I-labeled antimouse IgG (200,000 cpm/well) for an additional 2 h at room temperature. The data are normalized to the binding of the E2-myc. Total binding to E2-myc was 12,300 cpm/well, and nonspecific binding to COS-7 transfected with a plasmid vector DNA (pcDNA1) was 1,300 cpm.

 
To examine the cytoplasmic location of the putative first, second, and third cytoplasmic loops and the carboxyl-terminal tail, we introduced the c-myc epitope in these regions. The resulting constructs, CL2c, CL3, and T-myc, respectively, were well tolerated in term of expression levels, ligand-binding properties, and PTH-stimulated cAMP accumulation (Table 2Go). In contrast, CL1 and CL2 did not show any detectable expression, and the expression of CL1c was severely impaired. No detectable binding was detected for CL1 or CL2; however, weak low affinity and low capacity binding was detected for CL1c using saturation binding. Additionally, CL1c increased cAMP accumulation to 34% of the wild-type value, with an EC50 that was extremely high (230 nM). Nevertheless, the detectable ligand-binding and signaling properties of the CL1c mutant receptor suggest that this mutant assumes a functional structure. None of the c-myc epitopes of these mutants was accessible on intact cells (Fig. 2Go). As these mutants were constructed on the background of the E2-HA receptor, which contains an HA epitope in the E2 region of the amino-terminal extracellular extension, we examined the binding of 12Ca5 to intact cells expressing these mutant receptors (Fig. 3Go). COS-7 cells expressing CL2c, CL3, and T-myc bound the 12Ca5 monoclonal antibody with values at least 80% of the control (E2-HA). COS-7 cells expressing CL1c bound the 12Ca5 antibody with a value that was 22% of the control level; this value is consistent with the low receptor number calculated from Scatchard analysis (Table 1Go). The ability of 12Ca5 to bind to intact COS-7 cells expressing CL1c, CL2c, CL3, and T-myc indicates that these receptor mutants are located on the cell membrane, and their extracellular domains are accessible.



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  		Figure 3. Binding of the 12Ca5 monoclonal antibody to intact cells expressing the different epitope-tagged receptors. The cells, cultured in 24-well plates, were incubated with 12Ca5 ascites fluid (1:2,000) for 2 h and then with an 125I-labeled antimouse IgG (200,000 cpm/well) for an additional 2 h at room temperature. The data are normalized to the binding of the E2-HA. Total binding to E2-HA was 8,122 cpm/well, and nonspecific binding to COS-7 transfected with a plasmid vector DNA (pcDNA1) was 1,300 cpm.

 
To examine whether the c-myc epitope is functional, we assessed the binding of 9E10 to permeabilized COS-7 cells expressing CL1c, CL2c, CL3, and T-myc. Permeablized COS-7 cells expressing the wild-type receptor, R15B, were used to control for nonspecific binding. After permeabilization, the c-myc epitopes of CL1c, CL2c, CL3, and T-myc became readily accessible and bound the 9E10 antibody (Fig. 4Go). The values of 9E10 binding to permeabilized cells were parallel to those obtained with 12Ca5 on intact cells (compare Figs. 3Go and 4Go) and to the number of binding sites calculated by Scatchard analysis (Table 2Go). These data indicate that the c-myc epitopes introduced in the putative cytoplasmic regions of the PTH/PTHrP receptor become readily accessible if the cell membrane is permeabilized.



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  		Figure 4. Binding of the 9E10 monoclonal antibody to permeabilized COS-7 cells expressing the different epitope-tagged receptors. The cells, cultured in 24-well plates, were fixed with 2% paraformaldehyde in PBS and 5% dextrose, permeabilized with 0.1% Triton X-100 at room temperature for 4 min, and incubated with 9E10 ascites fluid (1:2,000) for 2 h and then with an 125I-labeled antimouse IgG (200,000 cpm/well) for additional 2 h at room temperature. The data are normalized to the binding of E2-myc.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A prediction of the sidedness of the different domains of the PTH/PTHrP receptor in relation to the plasma membrane was provided (5, 6) based on structural homology with other G protein-coupled receptors. Recently, ligand binding (15) and cross-linking (16) experiments suggested that certain regions of the PTH/PTHrP receptor lie on the extracellular side. Here we used a systematic approach, by inserting an epitope tag in the different receptor domains to directly examine their sidedness in relation to the plasma membrane. These experiments provided evidence that the amino-terminal extension and the carboxyl-terminal tail of the PTH/PTHrP receptor lie on the extracellular and cytoplasmic sides of the plasma membrane, respectively. Additionally, these data confirm the prediction (5, 6) that the PTH/PTHrP receptor structure contains three extracellular and three cytoplasmic loops.

Epitope tag mapping revealed that certain regions of the PTH/PTHrP receptor are relatively tolerant for sequence conversion. The most tolerant extracellular sequences are those in the E2 and EL1 regions. These regions could be deleted without impairment of the receptor ability to bind and to activate intracellular second messengers (15). In contrast, other extracellular regions, such as EL2 and EL3, did not tolerate mutations; these regions may be important for PTH binding and or for receptor conformation. In this regard, it has been previously shown that single point mutations within EL2 and EL3 cause dramatic loss of ligand binding affinity (15), and that EL3 is important for determining binding specificity for PTH/PTHrP, and PTH2R is important for PTHrP and PTH, respectively (17).

It is also interesting to note that sequences in the second and third cytoplasmic loops can be fully replaced with the c-myc or HA epitopes without impairing the receptor’s ability to stimulate adenylate cyclase. These data suggest that the molecular determinants for adenylate cyclase coupling reside outside the substituted regions of the second and third cytoplasmic loops. These data are consistent with the finding that certain mutations within the carboxyl-terminal end of second cytoplasmic loop cause loss of receptor coupling to Gq without affecting coupling to adenylate cyclase (18). It has been also shown that the amino-terminal end of the third cytoplasmic loop, RVLATKLR, plays an important role in coupling to adenylate cyclase and phospholipase C (19). This observation (19) is also consistent with our data that CL3-myc, in which the RVLATKLR sequence was not modified, bound PTH and increased cAMP accumulation.

The ability to obtain a fully functional receptor tagged in its carboxyl-terminal tail was predicted because we have shown previously that a PTH/PTHrP receptor truncated at residue 480 can bind and stimulate the intracellular effectors efficiently (20). In contrast, the first cytoplasmic loop did not tolerate sequence conversion or sequence insertion. The CL1 construct was not expressed at all, and the CL1c construct was weakly expressed and bound PTH with a low affinity. As the cytoplasmic loops are not in direct contact with the ligand, and the first cytoplasmic loop is highly conserved among all the receptors of the PTH/PTHrP receptor’s family, it is likely that the first cytoplasmic loop plays an important role in this family of receptors. In this regard, it has been shown that an alternatively spliced variant of the CRF receptor that contains an insertion within CL1 bound the CRF ligand with low affinity (21) and was weakly coupled to the G protein (22).

In addition to the signal peptide, the primary amino acid sequence of the PTH/PTHrP receptor contains nine other hydrophobic regions. The structure of this receptor, however, was predicted to be similar to that of another G protein-coupled receptor, i.e. containing seven (not nine) transmembrane-spanning domains (5, 6). One of the hydrophobic stretch regions, amino acid 105–126, is thought to be extracellular because it is located between the E2 region and the glycosylation sites both of which are shown to be extracellular (our data and Ref. 16 , respectively). The other hydrophobic region, amino acids 272–294, is located in the EL1 region that could be deleted without an effect on receptor function or expression (15). Additionally, our findings that an epitope in the EL-1 region is extracellular and that an epitope in the CL1 region is cytoplasmic suggest that the 272–294 region is probably extracellular.

In conclusion, our data provide experimental evidence about the topographic organization of the PTH/PTHrP receptor and support the seven transmembrane-spanning model of this receptor.


   Footnotes
 
1 This work was supported by the NIDDK, NIH (Grant DK-11794–26). Back

Received February 10, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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