Transmembrane Residues Together with the Amino Terminus Limit the Response of the Parathyroid Hormone (PTH) 2 Receptor to PTH-related Peptide*

Paul R. TurnerDagger , Suzanne Mefford, Tom Bambino, and Robert A. Nissenson§

From the Endocrine Unit, Veterans Affairs Medical Center and the Departments of Medicine and Physiology, University of California, San Francisco, California 94121

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The mechanisms of ligand binding and receptor activation for G-protein-coupled receptors in the secretin/parathyroid hormone (PTH) receptor subfamily are not understood. The PTH1 receptor (PTH1R) signals in response to both PTH and parathyroid hormone-related peptide (PTHrP), whereas the PTH2 receptor (PTH2R) responds only to PTH, not to PTHrP. To locate PTHrP discriminatory domains in the PTH2R, we generated PTH1R/PTH2R chimeras in which the extracellular amino-terminal domains were exchanged. Production of cAMP in response to 1 µM PTHrP or PTH was identical in cells expressing the PTH1R with the PTH2R amino terminus and in cells expressing the PTH2R with the PTH1R amino terminus. The ability of the chimeric receptor with the PTH2R amino terminus to respond fully to PTHrP showed that the body of the PTH2R must contain sites that limit the response to PTHrP. Mutations to PTH1R sequence were therefore made in each of the seven transmembrane domains of the PTH2R. Mutations in transmembrane domains 3 and 7 resulted in receptors able to respond to PTHrP. Thus, residues in more than one domain form a barrier or filter, allowing the receptor to discriminate between different ligands.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The mechanisms that allow G-protein-coupled receptors (GPCRs)1 to distinguish between different ligands are not known. The GPCR for parathyroid hormone (PTH) and for parathyroid hormone-related peptide (PTHrP) binds and signals via adenylate cyclase and phospholipase C in response to both PTH and the structurally very different ligand PTHrP (1). Both ligands activate this receptor (PTH1R) with equal potency. A recently identified member of this secretin/PTH/calcitonin subfamily (2) of GPCRs, the PTH2 receptor (PTH2R), signals only in response to PTH, not to PTHrP (3, 4). What are the structural features of the PTH2R required for this effective discrimination?

Features of the ligand required for this selective response have been identified. A single modification of Phe23 in hPTHrP-(1-34) to Trp, the residue in PTH (see Fig. 1), allows the ligand to bind to, but not activate, the PTH2R (5). A modification of a residue in the N-terminal region of the ligand (His5) to the residue found at this position in PTH (Ile) allows activation as measured by cAMP production (5, 6). These data are consistent with the idea that the C-terminal region of the peptide hormone is important for binding to the receptor and that the N terminus of the ligand is important for receptor activation (7). Unanswered by these studies are the structural features of the PTH2R that limit the response to PTHrP. The PTH1R and PTH2R share a high percent sequence homology in their seven transmembrane regions (~70%), with little homology present in the amino-terminal extracellular domain (3), making them well suited for studies to determine the location and nature of selectivity and activation mechanisms.

Previous work with chimeric receptors of between species homologues of the PTH1R (7) or with secretin and VIP receptors (8) has shown that the amino-terminal domain is a major determinant of binding specificity for related ligands. However, studies of chimeric class II receptors for dissimilar ligands such as PTH and secretin (9), calcitonin and PTH (10), or calcitonin and glucagon (11) receptors and also for related ligands such as peptide histidine isoleucineamide and VIP for VIP receptors (12) show that selectivity determinants are also located in the body of the receptor. Taken together, these results support a "two-site" model for receptor-ligand interaction in which ligands are able to bind to "site 1," located in the amino-terminal domain, with varying affinities. Those that bind are also able to interact with "site 2," located in the body of the receptor, which results in the stabilization of the active conformation of the receptor, G-protein activation, and intracellular signaling (13). Implicit in this model is the idea that the receptor contains a discrete activation region, perhaps located within a "pocket" formed by the alignment of the seven helical transmembrane domains. In this model, inappropriate ligands are unable to interact with this activation site 2 because access is prevented by a selectivity barrier or filter, imposed by residues in the body of the receptor. We have identified a putative component of such a selectivity filter or barrier near the extracellular side of the second transmembrane domains of the PTH and secretin receptors (9). This location was of interest since previous work has shown that other residues in TM2, located nearer the intracellular side of the transmembrane domain, are important for PTH1R activation (14-17). The objectives of this study were therefore to see if the PTH2R shared similar activation properties with the PTH1R and to determine which regions of the PTH2R limit the response to PTHrP.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Plasmid Construction-- The SacI-XbaI fragment from the pcDNA1 clone containing the opossum kidney PTH receptor sequence (kindly provided by Drs. H. Jüppner and G. V. Segre) (1) was subcloned into pBluescript II KS+ (Stratagene). The HindIII-NotI fragment of the clone containing the human PTH2R (kindly provided by Dr. T. B. Usdin) (3) was also cloned into pBluescript II KS+. Following mutagenesis, the 1.9-kilobase opossum kidney PTH1R fragment from a partial digest using BamHI and XbaI was subcloned into pcDNA1amp (Invitrogen) for transfection. The HindIII-NotI fragment containing the mutated PTH2R sequence was also cloned into pcDNA1amp.

For the N-terminal domain exchanges, an AccI restriction enzyme site was generated in the PTH1R (9); such a site exists already in the PTH2R. After AccI digestion, the N-terminal fragment was excised from the receptor/pBluescript vectors, with this exchange being made where the conserved transmembrane sequence first diverges between the two receptors. We therefore generated two chimeric receptors: one containing the body of the PTH1R and the N terminus of the PTH2R and one containing the body of the PTH2R and the N terminus of the PTH1R. All constructs were confirmed by sequencing through the mutated or spliced regions.

Site-directed Mutagenesis-- Oligonucleotide-mediated mutagenesis was performed using the protocol contained in a site-directed mutagenesis kit (CLONTECH) using AflIII-BglII switch selection primers. Mutagenic primers were designed both to introduce the mutation of choice and to either add or remove a restriction site to facilitate identification of the correct mutant clones.

Transient Transfection-- Mutated pcDNA1amp constructs (5 µg/T75 flask) were transiently transfected into HEK293 cells using the chloroquine/DEAE-dextran method (18). Parallel transfections were always carried out with a plasmid encoding the wild-type PTH2R and the wild-type PTH/PTHrP receptor and sometimes with the pcDNA1amp vector alone. Cells were subcultured into 6-well plates 24 h later and assayed for receptor function and expression 72 h post-transfection. Transfection efficiency was monitored by transfecting cells with green fluorescent protein (EGFP, CLONTECH), subcloned into pcDNA3amp (Invitrogen). Monitoring the percent fluorescent HEK293 cells visible 72 h post-transfection with green fluorescent protein showed that the transfection efficiency for a group of plasmids routinely varied from 10 to 25% from one transfection to the next.

Cyclic AMP Levels-- Transfected HEK293 cells in 6-well plates were incubated for 10 min at room temperature in 1 ml of Dulbecco's modified Eagle's medium containing 1 mM isobutylmethylxanthine, 1 mg/ml bovine serum albumin, and various concentrations of bPTH-(1-34) or hPTHrP-(1-34) (Bachem California). Cells were washed twice with ice-cold phosphate-buffered saline, and cellular cAMP was extracted with 1.5 ml of 95% ethanol. Cellular cAMP was quantified by radioimmunoassay (19).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Cells transfected separately with the constructs encoding the PTH1R and PTH2R were challenged with PTH and PTHrP. The structures of the two ligands used in this study, bPTH-(1-34) and hPTHrP-(1-34), are shown in Fig. 1a with identities indicated. The cAMP responses of cells transfected with constructs encoding the PTH1R or PTH2R to 1 µM PTH or PTHrP are shown in Fig. 1b. The PTH1R responses to PTH and PTHrP were of similar magnitude, whereas the PTH2R responded only to PTH and showed no detectable response above basal levels of cAMP production to 1 µM PTHrP. The effects of PTH on both receptors were antagonized using two forms of PTH-(7-34): [Tyr34]PTH-(7-34) and [Nle8,18]PTH-(7-34). A 1000-10,000-fold excess of antagonist was required to achieve significant inhibition of signaling by submaximal doses of PTH for both receptors (data not shown). These results are entirely consistent with previous results for this receptor (3-6).


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Fig. 1.   a, the ligands used in this study: bPTH-(1-34) and hPTHrP-(1-34). Identities are indicated by bars. b, the cAMP response of HEK293 cells transiently transfected with constructs encoding the wild-type PTH1R or PTH2R. Cells were exposed to 1 µM bPTH-(1-34) or hPTHrP-(1-34) 72 h post-transfection. The values plotted are the means ± S.E. of triplicate determinations and are expressed relative to the maximal response obtained with bPTH-(1-34) for each construct. Mean wild-type PTH1R basal levels were 7.8 ± 1.3 pmol/well; the mean maximal PTH1R response was 137.6 ± 9.2 pmol/well (n = six assays in triplicate). For the wild-type PTH2R, the mean basal cAMP level was 6.2 ± 0.9 pmol/well, and the mean maximal PTH response was 63 ± 12 pmol/well (n = nine assays in triplicate).

At present, it is thought that GPCR activation results from an agonist-induced altered conformation of the transmembrane domains of the receptor (13). The sequence identity in the body of the PTH2R to that of the PTH1R (Fig. 2) is high (70%), suggesting that the two receptors might share a similar activation mechanism. To test this, we subsequently mutated residues in the second transmembrane domain of the PTH2R (Fig. 2). Our purpose in making these mutations was to see to what extent functional similarities exist between the PTH1R and PTH2R. The mutations made were 1) histidine (position 220 in the PTH1R and position 180 in the PTH2R) to arginine and 2) arginine (position 230 in the PTH1R and position 190 in the PTH2R) to glutamate (Fig. 2). Mutation of these residues alters the signaling properties of both the PTH1R and secretin receptor (14, 15). In addition, the histidine-to-arginine mutation has been shown to result in a constitutively active human PTH1R (16, 17). The results obtained were similar in the two receptors: cells transfected with the H180R mutation in the PTH2R showed little responsiveness to PTH (Fig. 3a), but had grossly elevated basal cAMP levels in the absence of hormone, characteristic of a constitutively active receptor (16, 17). PTH and PTHrP addition had little or no impact on this receptor activity (Fig. 3a). The R190E mutation, as in the PTH1R (14), resulted in a poorly signaling receptor: the EC50 for PTH was right-shifted by ~10-15-fold, and the maximal cAMP response was ~30% that of the wild-type PTH2R (Fig. 3b). These results are consistent with the notion that the two receptors share a common activation mechanism.


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Fig. 2.   Diagram illustrating the primary sequence and proposed topological structure of the human PTH2R, showing the seven putative transmembrane-spanning regions (TM1-7). Boundaries for these domains are based on alignment with other members of the secretin/PTH receptor subfamily (26). Conserved residues with the opposum kidney PTH1R are shown in black circles. Also shown is the location of the AccI restriction site, which was used to exchange the N-terminal domains of the PTH1R and PTH2R. The nonconserved transmembrane domain residues mutated in this study are shown in gray circles. The remaining nonconserved residues are shown in white circles.


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Fig. 3.   a, basal and PTH- and PTHrP-stimulated (both at 1 µM) cAMP levels in cells transfected with pcDNA1amp alone (vector), the wild-type PTH2R, and the H180R and R190E mutant PTH2Rs. Values are the means ± S.E. of triplicate determinations. Similar results were obtained in four experiments. b, cAMP response curves for increasing doses of bPTH-(1-34) for the wild-type PTH2R and the R190E mutant PTH2R. Values are the means ± S.E. of triplicate determinations. Similar results were obtained in two additional experiments.

The lack of homology between the two N-terminal domains (Fig. 2) suggested that this could be the region entirely responsible for the discrimination between PTH and PTHrP. Having determined that the two receptors shared some functional similarities in terms of activation, we tested the hypothesis that the inability of the PTH2R to respond to PTHrP was due to a greatly reduced affinity of the N-terminal domain of this receptor for PTHrP. We utilized an AccI site, present in the PTH2R, but generated by mutagenesis in the PTH1R (9), to exchange the N terminus of the PTH1R with that of the PTH2R (Fig. 2). Our expectation was that the chimeric receptor with the body of the PTH1R and the N terminus of the PTH2R (1(2Nt)R) would not be able to respond to PTHrP. The reciprocal chimera (2(1Nt)R), containing the PTH1R N terminus, should show no discrimination and respond to both ligands.

To our surprise, however, both chimeras showed very robust responses to 1 µM PTHrP, identical in magnitude to the PTH responses (Fig. 4a). The potency of PTH and PTHrP as ligands was different, however, for the different receptors. Shown in Fig. 4b are the wild-type receptor cAMP dose-response curves for the two ligands. The PTH1R had similar EC50 values for both PTH and PTHrP (0.28 and 0.25 nM, respectively). The PTH2R responded only to PTH (EC50 = 0.9 nM), not to PTHrP. For the chimeras, PTH and PTHrP showed approximately similar potencies for the 2(1Nt)R chimera (EC50 = 1.1 and 1.3 nM, respectively). However (Fig. 4c), the 1(2Nt)R chimera had a 100-fold higher apparent affinity for PTH (EC50 = 0.05 nM) than for PTHrP (EC50 = 5 nM). This 100-fold lower potency of PTHrP still resulted in a maximal cAMP response at 1 µM PTHrP (Fig. 4c). Thus, discrimination between PTH and PTHrP did segregate with the N terminus to some extent, although responses to PTH and not PTHrP were visible only within a narrowly defined range of hormone concentration (Fig. 4c).


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Fig. 4.   a, the cAMP response of cells transiently transfected with constructs encoding the N-terminal chimeric receptors (1(2Nt)R and 2(1Nt)R). Cells were exposed to 1 µM bPTH-(1-34) or hPTHrP-(1-34). The values plotted are the means ± S.E. of triplicate determinations and are expressed relative to the maximal response obtained with bPTH-(1-34) for each construct. Similar results were obtained in seven independent experiments. The cAMP dose-response curves for PTH and PTHrP in cells transiently transfected with constructs encoding the wild-type (b) and N-terminal chimeric (c) forms of the PTH1R and PTH2R were also obtained. Values are the means ± S.E. of triplicate determinations. Similar results were obtained in three independent experiments.

These results with the amino terminus of the PTH2R showed that the body of the PTH2R must contain sites that restrict receptor activation by PTHrP. In an attempt to obtain structural information about the location of such residues in the body of the receptor, we chose to make mutant PTH2Rs in which nonconserved transmembrane residues were mutated to PTH1R sequence in each of the seven transmembrane domains. Due to the large number of such candidate residues (Fig. 2), we chose to mutate residues located in the upper, more "extracellular" half of each transmembrane domain (Fig. 2). Our reasoning for this, based on our previous work with the secretin receptor and PTH1R, was to test the idea that the activating ligand interacts with sites located in a pocket formed by the transmembrane regions and that there is a discrete number of residues that function as a selectivity filter to screen out inappropriate ligands. We hypothesized that these residues, if they are to function to prohibit entry to the activation regions, are therefore most likely to be located near the extracellular boundary. This strategy, which was rather loosely adhered to (we did not mutate every nonconserved residue in each transmembrane domain, and we did not mutate residues in the extracellular loops) resulted in the following mutations (Fig. 2): TM1 (F156L and A160T), TM2 (T192V and V196I), TM3 (V241T, M242V, and I244L), TM4 (A293V, A295V, V296T, and A297V), TM5 (G327V and L328V), TM6 (V369M, L370P, V371L, V380M, C380A, and L381T), and TM7 (C397Y, L399M, and F400L). These seven mutant PTH2Rs were screened for their ability to respond to 1 µM PTHrP (Fig. 5a) as well as to 1 µM PTH. While all the mutants were able to respond to PTH, only receptors encoding PTH1R sequence in TM3 or TM7 showed significant detectable responses to PTHrP (Fig. 5). The TM6 mutant showed an ~2-fold elevated basal cAMP level. This elevated basal cAMP level was also found in a mutant receptor in which 3 of the 6 residues mutated in the TM6 construct were mutated (V369M, L370P, and V371L), but not in the mutant receptor containing the remaining three mutations (V380M, C380A, and L381T) (data not shown). For those transmembrane domain mutant receptors showing no response to PTHrP, the maximal responses to PTH were not significantly different from those for the wild-type PTH2R (Fig. 5), and these receptors were not studied further.


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Fig. 5.   cAMP responses of HEK293 cells transfected with vector alone or with constructs encoding the wild-type PTH2R or mutant PTH2Rs containing from two to six mutations in each of the seven putative transmembrane domains. The values shown are the means ± S.E. of triplicate determinations in response to either no hormone (basal) or 1 µM PTH or PTHrP.

In an attempt to further define the mutations in TM3 and TM7 responsible for allowing the response to PTHrP, each of the 3 residues in TM3 and TM7 were mutated individually, and the resulting single-site mutants were screened for their ability to respond to PTHrP. In TM3, the I244L mutant receptor responded with as great a response to PTHrP as the TM3 triple-mutant receptor (Fig. 6a). The mutation V241T resulted in a receptor indistinguishable from the wild-type PTH2R (Fig. 6a). The M242V receptor gave only a poor response (2-fold stimulation) to PTH and no response to PTHrP (Fig. 6a). Possible reasons for the poor response to PTH could be reduced cell-surface expression or altered signaling properties of this receptor. However, the lack of any PTHrP responsiveness for M242V together with the finding that the I244L mutation was sufficient to account for the entire PTHrP response seen with the original TM3 mutant receptor suggest that M242V does not contribute to PTHrP responsiveness observed with the triple-mutant receptor.


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Fig. 6.   a, cAMP responses of HEK293 cells transiently transfected with constructs encoding the wild-type and three single-site TM3 mutant PTH2Rs to the following: no hormone (basal) or 5 µM PTH or PTHrP. The putative TM3 domain residues are shown to the right. The box contains residues thought to reside within the membrane; the top of the box represents the extracellular surface. Conserved residues are shown in black circles, mutated nonconserved residues are shown in gray circles, and other nonconserved residues are shown in white circles. The data shown represent the means ± S.E. of triplicate determinations from two experiments. b, cAMP responses of cells transiently transfected with constructs encoding the wild-type and single-site TM7 mutant PTH2Rs to 5 µM PTH or PTHrP. The data shown represent the means ± S.E. of triplicate determinations from two experiments.

For the TM7 mutations (Fig. 6b), the results were different from TM3 in that two sites seemed to contribute to the response to PTHrP: C397Y and F400L (Fig. 6b). These two mutations appear to function together since the maximal responses of receptors containing either point mutation alone were of smaller magnitude than that of the TM7 mutant receptor containing all three mutations. The third mutation, L399M, behaved as the wild-type PTH2R behaved, with no response to PTHrP evident (Fig. 6b).

Dose-response curves for PTHrP for the individual single-site TM3 mutant revealed an EC50 of 0.5 µM for I244L (Fig. 7) and ~0.9 µM for the combined mutations in the TM7 mutant receptor. A receptor containing both I244L and the three mutations in TM7 was generated to see if the presence of sites in two transmembrane domains would further improve the ability of PTHrP to activate. Only a small increase in efficacy for PTHrP was seen (Fig. 7). Furthermore, this response to PTHrP was still right-shifted relative to the response of the 1(2Nt)R chimera to PTHrP (Fig. 4c), suggesting that there are other residues in the body of the PTH2R that function to limit PTHrP responsiveness. Thus, sites in at least two transmembrane domains of the receptor, TM3 and TM7, appear to function to inhibit PTHrP from acting as an agonist.


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Fig. 7.   cAMP responses of HEK293 cells transiently transfected with PTH2R constructs to increasing doses of PTHrP. The constructs used encoded the wild-type (Wt) PTH receptor, the I244L mutant PTH2R (TM3), and the TM7 mutant receptor (C397Y, L399M, and F400L), in addition to a construct containing both I244L and the three mutations in TM7. Data shown represent the means ± S.E. of triplicate determinations from two experiments. Responses of the wild-type and mutant receptors to increasing concentrations of PTH were obtained simultaneously; the response curves for all the mutants were indistinguishable from that of the wild-type PTH2R (data not shown).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

This study shows that the inability of PTHrP to activate the PTH2R can be attributed 1) to components located in the N-terminal domain and 2) to residues located within the body of the receptor. The location of discriminatory components in the N terminus was not addressed in this study. The N-terminal extracellular domain is known to be important for binding affinity and may serve to fine-tune selectivity such as in cases of PTH/PTHrP interspecies differences (7). With closely related ligands like secretin and VIP, exchange of the N terminus of the secretin receptor for the VIP receptor results in a change from the responsiveness characteristic of a secretin receptor to that of a VIP receptor (8). Previous work has shown that the modification F23W in PTHrP-(1-34) improves binding to the PTH2R (5). The 1(2Nt)R chimera retains an apparent affinity difference between PTH and PTHrP (Fig. 2c), and one might therefore expect F23W to be as potent as PTH for this chimera. It is of interest to note that another putative ligand, active on the PTH2R, but not on the PTH1R, has been reported in hypothalamic extracts (20); the PTH2R may not be exposed to PTH under normal conditions, although presumably would be exposed to PTHrP.

The N terminus of the PTH2R does not, however, contain all of the determinants of selectivity. Studies of chimeric class II receptors for apparently unrelated ligands such as calcitonin and PTH (10), calcitonin and glucagon (11), and secretin and PTH (9) chimeric receptors have shown that determinants of selectivity are also located in the body of the receptor. Examples also exist for related ligands, such as peptide histidine isoleucineamide (a VIP-related peptide), which binds with high affinity to the rat VIP receptor and with low affinity to the human VIP receptor (12). This discrimination is determined by 3 amino acids located at the junction between exoloop 1 and TM3. Our results are similar for PTH/PTHrP discrimination by the PTH2R in that mutation of 1 residue in TM3 (I244L) and 2 in or adjacent to TM7 (C397Y and F400L) allowed PTHrP to activate the PTH2R. It is an exciting possibility that the residues we have identified are responsible for discrimination between His and Ile at position 5 in PTHrP and PTH (4, 5) and may represent sites of direct receptor-ligand interaction. If these residues constitute a "barrier" to PTHrP or a selectivity filter such as Asn192 for the secretin receptor (9), results from this study show that this barrier cannot be absolute. We postulate that such residues represent an energy barrier to possible ligands, which must be overcome if a ligand is to activate the receptor. The energy to overcome this activation barrier comes from the combined interaction of the ligand with site 1 in the N-terminal domain (and perhaps other sites in the extracellular loops) and from site 2 in the body of the receptor. For the PTH1R/PTH2R chimeras, the energy provided by the binding of PTHrP to the N terminus of the PTH1R must be greater than that provided by interaction with the N terminus of the PTH2R; thus, PTHrP is now able to activate the PTH2R with the PTH1R N terminus (Fig. 3b). The barrier to activation by PTHrP must be greater for the body of the PTH2R than for that of the PTH1R: the PTH2R N terminus provides enough energy from binding PTHrP to overcome this lower barrier in the 1(2Nt)R chimera. By introducing PTH1R sequence in TM3 and TM7, we postulate that we have reduced the size of this barrier enough to allow PTHrP to function. Presumably by introducing the corresponding PTH2R sequence into TM3 and TM7 in the PTH1R, especially into the chimera containing the PTH2R N terminus, we would be able to reduce the ability of PTHrP to activate.

The transmembrane domain residues we have identified as being important for allowing PTHrP to function are in TM3 and TM7. Residues in the corresponding transmembrane domains are involved in activation of other GPCRs such as rhodopsin and catecholamine receptors (21). Mechanisms of ligand binding and GPCR activation are varied, with some dependence seen on the size of the ligand (22). Small molecules such as catecholamines and photons, for example, are believed to interact with charged residues in a binding pocket formed by the seven transmembrane domains (21, 22). Ligand interaction in the best studied case, rhodopsin, is thought to result in a movement of the receptor transmembrane domains relative to each other, TM3 and TM6 in particular (23). This results in G-protein activation (13, 22). For the large peptide hormones in the secretin/PTH subfamily of GPCRs, the mechanisms of receptor activation are not well understood. For example, how protonation of His223 in the human PTH1R (16) or His220 in the opposum kidney PTH1R (17) results in a receptor locked into the active state is unclear. The role of the arginine in TM2 (Fig. 2) is also unclear (14, 15). In this study, we have shown that residues important for PTH1R activation are also important for PTH2R activation (H180R and R190E), and this supports the notion that these receptors share a common activation mechanism. These residues (His and Arg) are probably not sites of direct ligand binding, but may form part of the energy barrier that the activating ligand must overcome.

Is there a discrete activation domain in a pocket formed by an alignment of the transmembrane domains, or does the ligand interact with residues on the extracellular face of the receptor? This is an as yet unanswered question for receptors in this subfamily. However, the location of the receptor sites that interact directly with the ligand, although possibly amenable to study using complementation techniques, will probably require identification using more direct biochemical techniques, such as cross-linking, which has shown PTH binding to the juxtamembrane region of the N-terminal domain of the PTH1R (24).

The constitutive activity we report for H180R for the PTH2R was not found in COS-7 cells (17), a finding that may reflect higher levels of receptor expression or perhaps increased transfection efficiency in HEK293 cells. Basal cAMP levels of the TM6 mutants were also somewhat higher than for the wild-type PTH2R (Fig. 5). It is possible that expression of these TM6 mutant receptors was higher and that the higher expression resulted in this increase in basal "constitutive" activity (17).

In summary, we have characterized PTH1R/PTH2R chimeras that respond to PTHrP. Our data suggest a two-site model in which a high affinity interaction of the agonist with the receptor's N-terminal domain and interaction with a second site in the body of the receptor are required for receptor activation. We have identified discrete residues in TM3 and TM7 that form part of this second site and that function to allow the PTH2R to discriminate between PTH and PTHrP. In support of this, Gardella and co-workers (25) recently reported that I244L confers on the PTH2R the ability to respond to modified forms of PTHrP. In addition, they identified a PTH2R-to-PTH1R mutation in exoloop 2 (Y318I), not targeted in the present study, that is permissive for PTHrP responsiveness. As in the present study, they generated N-terminal chimeric receptors that responded to PTHrP, although the N-terminal chimera with the N terminus of the human PTH1R (25) only responded weakly to the modified PTHrP. This difference could be ascribed to differences in sequence of the N-terminal domains (opossum versus human), the different site used by this group for exchange of the N terminus, or the different ligands used in the two studies. These results provide further evidence for a selectivity filter or barrier as a mechanism of general importance for receptors in the secretin/PTH subfamily. The identification of additional residues that limit response to a ligand and the ability of such residues to be transplanted as a functional unit or to be abrogated as well as identification of the receptor activation domains will undoubtedly be of great importance in the development of agonists and antagonists for the treatment of human disease.

    ACKNOWLEDGEMENT

We thank Dr. T. B. Usdin for supplying the cDNA for the PTH2R.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant DK 35323 (to R. A. N).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.

Dagger To whom correspondence should be addressed: Endocrine Unit, VAMC (111N), 4150 Clement St., San Francisco, CA 94121. Tel.: 415-221-4810 (ext. 3331); Fax: 415-750-6929; E-mail: pturner{at}itsa.ucsf.edu.

§ Research Career Scientist of the Department of Veterans Affairs.

1 The abbreviations used are: GPCRs, G-protein-coupled receptors; PTH, parathyroid hormone; bPTH, bovine PTH; PTHrP, PTH-related peptide; hPTHrP, human PTHrP; PTH1R, PTH1 receptor; PTH2R, PTH2 receptor; VIP, vasoactive intestinal peptide; TM, transmembrane domain.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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