Multiple Regions of Ligand Discrimination Revealed by Analysis of Chimeric Parathyroid Hormone 2 (PTH2) and PTH/PTH-Related Peptide (PTHrP) Receptors

J. A. Clark, T. I. Bonner, A. S. Kim and T. B. Usdin

Section on Genetics National Institute of Mental Health Bethesda, Maryland 20892-4090


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PTH and PTH-related peptide (PTHrP) bind to the PTH/PTHrP receptor and stimulate cAMP accumulation with similar efficacy. Only PTH activates the PTH2 receptor. To examine the structural basis for this selectivity, we analyzed receptor chimeras in which the amino terminus and third extracellular domains of the two receptors were interchanged. All chimeric receptors bound radiolabeled PTH with high affinity. Transfer of the PTH2 receptor amino terminus to the PTH/PTHrP receptor eliminated high-affinity PTHrP binding and significantly decreased activation by PTHrP. A PTH/PTHrP receptor N terminus modified by deletion of the nonhomologous E2 domain transferred weak PTHrP interaction to the PTH2 receptor. Introduction of the PTH2 receptor third extracellular loop into the PTH/PTHrP receptor increased the EC50 for PTH and PTHrP, while preserving high-affinity PTH binding and eliminating high-affinity PTHrP binding. Similarly, transfer of the PTH/PTHrP receptor third extracellular loop preserved high-affinity PTH binding by the PTH2 receptor but decreased its activation. Return of Gln440 and Arg394, corresponding residues in the PTH/PTHrP and PTH2 receptor third extracellular loops, to the parent residue restored function of these receptors. Simultaneous interchange of wild-type amino termini and third extracellular loops eliminated agonist activation but not binding for both receptors. Function was restored by elimination of the E2 domain in the receptor with a PTH/PTHrP receptor N terminus and return of Gln440/Arg394 to the parent sequence in both receptors. These data suggest that the amino terminus and third extracellular loop of the PTH2 and PTH/PTHrP receptors interact similarly with PTH, and that both domains contribute to differential interaction with PTHrP.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The PTH/PTH-related peptide (PTHrP) (1) and PTH2 receptors (2) are members of a relatively small group of G protein-coupled receptors, which includes the receptors for secretin, vasoactive intestinal polypeptide, glucagon, CRF, calcitonin, and several related peptides (3). These receptors share a sequence homology of 30–70% and a number of highly conserved motifs, but structurally resemble the majority of seven-transmembrane domain receptors only in their overall predicted topology and in signaling through G proteins. The high degree of homology between these receptors has facilitated a number of recent studies of chimeric receptors aimed at identifying regions involved in ligand recognition (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18). These studies, together with deletion and site-directed mutagenesis studies, have allowed some generalizations about the functional architecture of the receptors (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18). They all have a relatively large, extracellular, amino terminus of 100–200 residues which contributes significantly to ligand-binding affinity and discrimination. For the PTH/PTHrP receptor, in particular, the N-terminal region appears to be responsible for differences between the interaction of the receptor from different species with PTH analogs (19) and to interact with the carboxyl-terminal region of PTH(1–34) (20). Contact between PTH and a region of the N terminus predicted to be close to the extracellular end of the first transmembrane domain is also supported by chemical cross-linking data (21). Additional regions contributing to ligand interaction, including the third extracellular loop, have been identified by evaluation of chimeras and mutants in which surface expression is maintained but ligand affinity is lost (4, 5, 10).

PTH binds to and activates the PTH/PTHrP receptor (22) and the PTH2 receptor (2, 10, 23). However, although the PTH/PTHrP and PTH2 receptors both recognize PTH, they differ greatly in their recognition of PTH-related protein (PTHrP). Depending on the species, mature PTH and PTHrP are peptides of 84 and 139–173 residues, respectively. They share eight of their 13 N-terminal residues but have no further sequence identity (22, 24). The N-terminal 34 residues of either peptide are sufficient to bind and activate the PTH/PTHrP receptor at nanomolar concentrations (22). While PTH(1–34) also binds and activates the PTH2 receptor at nanomolar concentration, activation by PTHrP is barely detectable at micromolar concentrations (2, 10, 23). Binding of radiolabeled PTHrP to the PTH2 receptor is not detectable, whereas PTHrP displaces the binding of radiolabeled PTH to the PTH2 receptor with an IC50 of approximately 2 µM, demonstrating very low affinity of PTHrP for the PTH2 receptor (10). Functional activation of the PTH/PTHrP receptor by two very different endogenous peptide ligands is both biologically and biochemically unusual, and the relationship between the binding sites for the two peptides is unknown. The identification of the PTH2 receptor, which recognizes only one of these peptides, provides an opportunity to examine the structural basis for this phenomenon. Two recent studies have explored the ligand sequences underlying differential recognition of PTH and PTHrP by the PTH2 receptor and have identified residues in the peptides that facilitate or prevent binding and activation of the PTH2 receptor (10, 23). The receptor structures that contribute to the differences in ligand interaction between the PTH/PTHrP and PTH2 receptors are unexplored.

We have used chimeras between the PTH/PTHrP and PTH2 receptors to gain further understanding of their interactions with their peptide ligands. Because of the similarity between these two receptors (51% sequence identity, 70% similarity) and because they both recognize PTH(1–34), we thought it likely that many receptor chimeras would be functional and informative. Considering previous data demonstrating the importance of the N-terminal and third extracellular loops for ligand recognition, we performed a set of extracellular domain exchanges. Surface expression and receptor function were verified by examining the chimeric receptors’ interaction with PTH(1–34), after which detailed comparison of the interaction with PTHrP(1–34) and PTH(1–34) was performed in an attempt to identify domains within the PTH2 receptor that prevented the interaction with PTHrP and domains within the PTH/PTHrP receptor that facilitated it.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Human (h) PTH/PTHrP and PTH2 receptors incorporating a C-terminal hemagglutinin epitope tag were used in this study. Both wild-type receptors (PrP and P2) bound 125I-[Nle8,21,Tyr34]rPTH(1–34) (125I-NlePTH) with nanomolar affinity (Table 1Go) and the PTH/PTHrP receptor (PrP) bound 125I-[Tyr36]PTHrP(1–36) (125I-PTHrP) with high affinity (Table 1Go). Both hPTH(1–34) and PTHrP(1–34) stimulated PTH/PTHrP receptor- mediated cAMP synthesis with subnanomolar EC50 values (Table 1Go), while hPTH(1–34) stimulated PTH2 receptor-mediated cAMP synthesis with a nanomolar EC50 (Table 1Go). These binding and activation data are in agreement with those previously described for wild-type receptors (1, 2).


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Table 1. Effects of Interchanging the Amino Termini of the PTH/PTHrP and PTH2 Receptors and Deletion of the PTH/PTHrP Receptor E2 Domain on Functional Properties of the Receptors

 
To identify regions of the PTH/PTHrP receptor responsible for PTHrP binding and receptor activation, or regions of the PTH2 receptor that prevent PTHrP interaction, we generated a series of PTH/PTHrP and PTH2 receptor chimeras. Chimeric receptors (Fig. 1BGo) were designed to test the contribution of the PTH/PTHrP amino terminus and third extracellular loop to ligand recognition. When transiently expressed in COS cells, all chimeras bound 125I-NlePTH (Tables 1Go, 2Go, and 3Go; Fig. 2Go, A and C, Fig. 3Go, A and C, and Fig. 4Go, A and C) demonstrating that they were expressed on the plasma membrane. In addition, high-affinity binding of radioiodinated PTH suggests that the topology of the chimeric receptors has not been significantly disrupted. Saturation binding analyses were performed for each of the receptors studied to assess functional receptor expression.



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Figure 1. Diagram of the Human PTH/PTHrP Receptor and PTH/PTHrP-PTH2 Receptor Chimeras

A, Schematic of the hPTH/PTHrP receptor primary sequence. Filled circles represent residues that are identical in both the PTH/PTHrP and PTH2 receptors. Shaded circles are conservative amino acid differences in the PTH2 receptor, and open circles represent residues that are different in the two receptors. The X denotes conserved Cys residues. The bracketed region N delineates the boundaries of the amino-terminal region of the PTH/PTHrP receptor (amino acid residues 1–214), which includes the first transmembrane domain. Bracketed region 3L denotes the third extracellular loop boundaries (amino acid residues 429–441) of the PTH/PTHrP receptor used in making chimeric receptors. Corresponding residues for the amino-terminal region and the third extracellular loop in the PTH2 receptor are 1–171 and 384–395, respectively. The heavy brackets denote the region {Delta}N which is the E2 domain (amino acid residues 62–106). The asterisk in the third extracellular loop denotes Gln440 in the PTH/PTHrP receptor and Arg394 in the PTH2 receptor. The arrows point to the amino acid residues corresponding to the restriction enzyme sites used in constructing the chimeric receptors. B, Schematics of the hPTH/PTHrP-PTH2 receptor chimeras used in this study. Solid and shaded lines represent hPTH/PTHrP and PTH2 receptor sequences, respectively. PrP, amino acid residues 1–592 hPTH/PTHrP receptor (hPTH/PTHrPR); PrP{Delta}N, hPTH/PTHrP 1–61, 107–592; Prp-NP2, hPTH2 receptor (hPTH2R) 1–171, hPTH/PTHrPR 215–592; PrP-3LP2, hPTH/PTHrPR 1–428, hPTH2R 384–394, hPTH/PTHrPR 441–592; PrP-3L*P2, hPTH/PTHrPR 1–428, hPTH2R 384–393, hPTH/PTHrPR 440–592; PrP{Delta}N-3LP2, hPTH/PTHrPR 1–61, 107–428, hPTH2R 384–394, hPTH/PTHrPR 441–592; PrP-N3LP2, hPTH2R 1–171, hPTH/PTHrPR 215–428, hPTH2R 384–394, hPTH/PTHrPR 441–592; PrP-N3L*P2, hPTH2R 1–171, hPTH/PTHrPR 215–428, hPTH2R 384–393, hPTH/PTHrPR 440–592; P2, hPTH2R 1–539; P2-NPrP, hPTH/PTHrPR 1–214, hPTH2R 172–539; P2-{Delta}NPrP, hPTH/PTHrPR 1–61, 107–214, hPTH2R 172–539; P2-3LPrP, hPTH2R 1–383, hPTH/PTHrPR 429–440, hPTH2R 395–539; P2-3L*PrP, hPTH2R 1–383, hPTH/PTHrPR 429–439, hPTH2R 394–539; P2-N3LPrP, hPTH/PTHrPR 1–214, hPTH2R 172–383, hPTH/PTHrPR 429–440, hPTH2R 395–539; P2-N3L*PrP, hPTH/PTHrPR 1–214, hPTH2R 172–383, hPTH/PTHrPR 429–439, hPTH2R 394–539; P2-{Delta}N3LPrP, hPTH/PTHrPR 1–61, 107–214, hPTH2R 172–383, hPTH/PTHrPR 429–440, hPTH2R 395–539; P2-{Delta}N3L*PrP, hPTH/PTHrPR 1–61, 107–214, hPTH2R 172–383, hPTH/PTHrPR 429–439, hPTH2R 394–539.

 

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Table 2. Functional Properties of PTH/PTHrP and PTH2 Receptors with Their Third Extracellular Loops Switched

 

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Table 3. Characterization of PTH/PTHrP and PTH2 Receptors with Interchanged Amino Termini and Third Extracellular Loops

 


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Figure 2. Ligand-Binding Properties and cAMP Responses of PTH/PTHrP-PTH2 Receptor Chimeras Involving the Amino Termini and E2 Domain

A and C, Closed symbols represent ligand-binding data from competition of 125I-[Nle8,21,Tyr34]rPTH(1–34) with [Nle8,21,Tyr34]rPTH(1–34), and open symbols represent data from competition of 125I-[Tyr36]PTHrP with PTHrP(1–34). Data are plotted as percent of control where control is ligand bound in the absence of unlabeled ligand. Values are the mean ± SEM of triplicate determinations from representative experiments performed three to four times for each receptor. B and D, Responses of wild-type and chimeric receptors to hPTH(1–34) (closed symbols) or PTHrP(1–34) (open symbols). Data from each assay were converted from picomoles per well to stimulation as described in Table 1Go. Values are the mean of duplicate determinations, which differed by no more than 10%, from representative experiments performed three to seven times for each receptor. Wild-type and chimeric receptors were expressed in COS-7 cells as described in Materials and Methods.

 


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Figure 3. Ligand-Binding Properties and cAMP Responses of PTH/PTHrP-PTH2 Receptor Chimeras Involving the Third Extracellular Loop

A and C, Closed symbols represent ligand-binding data from competition of 125I-[Nle8,21,Tyr34]rPTH(1–34) with [Nle8,21,Tyr34]rPTH(1–34), and open symbols represent data from competition of 125I-[Tyr36]PTHrP with PTHrP(1–34). Data are plotted as percent of control where control is ligand bound in the absence of unlabeled ligand. Values are the mean ± SEM of triplicate determinations from representative experiments performed three to five times for each receptor. B and D, Responses of wild-type and chimeric receptors to hPTH(1–34) (closed symbols) or PTHrP(1–34) (open symbols). Data from each assay were converted from picomoles per well to stimulation as described in Table 1Go. Values are the mean of duplicate determinations, which differed by no more than 10%, from representative experiments performed three to seven times for each receptor. Wild-type and chimeric receptors were expressed in COS-7 cells as described in Materials and Methods.

 


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Figure 4. Ligand-Binding Properties and cAMP Responses of PTH/PTHrP-PTH2 Receptor Chimeras Involving the Amino Termini, E2 Domain, and the Third Extracellular Loop

A and C, Closed symbols represent ligand-binding data from competition of 125I-[Nle8,21,Tyr34]rPTH(1–34) with [Nle8,21,Tyr34]rPTH(1–34), and open symbols represent data from competition of 125I-[Tyr36]PTHrP with PTHrP(1–34). Data are plotted as percent of control where control is ligand bound in the absence of unlabeled ligand. Values are the mean ± SEM of triplicate determinations from representative experiments performed three to four times for each receptor. B and D, Responses of wild-type and chimeric receptors to hPTH(1–34) (closed symbols) or PTHrP(1–34) (open symbols). Data from each assay were converted from picomoles per well to stimulation as described in Table 1Go. Values are the mean of duplicate determinations, which differed by no more than 10%, from representative experiments performed three to seven times for each receptor. Wild-type and chimeric receptors were expressed in COS-7 cells as described in Materials and Methods.

 
N-Terminal Substitutions
A PTH/PTHrP receptor, in which amino acids 62–106 encoded by exon E2 in the PTH/PTHrP receptor gene (25) have been removed (PrP{Delta}N, Fig. 1Go), was made to facilitate comparisons with the PTH2 receptor, which lacks a homologous sequence. PTH(1–34) and PTHrP(1–34) bound and activated PrP{Delta}N with affinities (Table 1Go and Fig. 2AGo) and EC50 values (Table 1Go and Fig. 2BGo) indistinguishable from the values of wild-type receptors, confirming previously reported results (4, 19).

Receptor chimeras in which the N-terminal domains of the PTH/PTHrP and PTH2 receptors were interchanged were designed to test whether the amino terminus is responsible for the differential interaction of PTHrP with these receptors. Neither the apparent affinity (KD) of 125I-NlePTH nor the EC50 for stimulation of cAMP accumulation by PTH for the PTH/PTHrP receptor containing a PTH2 receptor N-terminus (PrP-NP2) were significantly different from those of the parent receptor (Table 1Go and Fig. 2Go, A and B). However, the EC50 for PTHrP stimulation of cAMP accumulation increased 10-fold (Table 1Go and Fig. 2BGo) and 125I-PTHrP binding could no longer be detected. The 10-fold increase in EC50 suggests that PTHrP affinity has decreased below the point at which 125I-PTHrP binding can be detected. Three to seven independent transfections were performed for each chimeric receptor in this study, and although the absolute amount of cAMP accumulated varied somewhat between experiments, the increase over the basal level and the EC50 for a given construct and forskolin were remarkably constant. Maximal stimulation of cAMP accumulation by PTHrP was not changed by substitution of the PTH2 receptor N terminus. Receptor constructs in which maximal stimulation of cAMP accumulation does change are described below. These data suggest that maximal cAMP accumulation under our assay conditions is a useful operational measure of receptor function. This measure includes contributions intrinsic to the receptor molecule as well as its interaction with G proteins. The fixed KD and Bmax for 125I-NlePTH, EC50 for PTH(1–34), and cAMP accumulation stimulated by PTH(1–34) and PTHrP(1–34) suggest that lost 125I-PTHrP binding and increased EC50 for PTHrP are due to a decrease in affinity for PTHrP brought about by substitution of the PTH2 receptor N-terminal for the wild-type PTH/PTHrP receptor amino terminus.

Replacement of the PTH2 receptor N terminus by the N-terminal sequence of the PTH/PTHrP receptor (P2-NPrP) had no significant effect on the EC50 for PTH activation of the receptor as compared with the wild-type receptor (Table 1Go and Fig. 2CGo). However, binding of 125I-NlePTH was not sufficiently above background for calculation of KD and Bmax values. This substitution did not confer detectable 125I-PTHrP binding or ability of PTHrP to stimulate cAMP accumulation. The construct contains, within its N terminus, the E2 domain (amino acid residues 62–106), which does not have a homologous sequence within the PTH2 receptor. Substitution of the PTH2 receptor N-terminal by a PTH/PTHrP receptor N-terminal sequence lacking the E2 domain (P2-{Delta}NPrP) yielded a receptor with a KD and Bmax for PTH that are not significantly different from the parent PTH2 receptor (Table 1Go) and introduced weak activation by PTHrP (Table 1Go and Fig. 2DGo). Binding of 125I-PTHrP could not be detected. Thus, while the PTH2 receptor N terminus prevents PTHrP interaction, and the PTH/PTHrP receptor N terminus partially transfers it, these data suggest that additional regions of the receptor are involved in the differential interaction of PTHrP with the PTH/PTHrP receptor.

Third Extracellular Loop Interchanges
To determine which other regions of the receptors interact differentially with PTHrP, we designed chimeric receptors in which the third extracellular loops of the PTH/PTHrP and PTH2 receptors were interchanged, since previous studies have implicated this domain in PTH(1–34) interaction (5). Exchange of the third extracellular loops (PrP-3LP2 and P2-3LPrP, Fig. 1Go) did not change the apparent affinity for 125I-NlePTH(1–34) from that of the parent receptors for either construct (Table 2Go). Substitution of the PTH2 receptor third extracellular loop into the PTH/PTHrP receptor (PrP-3LP2) decreased the apparent affinity for PTHrP 5-fold (Table 2Go and Fig. 3AGo). The EC50 values for both PTH and PTHrP were increased by this substitution, and the maximum response to both peptides was decreased as compared with the parent PTH/PTHrP receptor, although this decrease did not reach statistical significance (Table 2Go and Fig. 3BGo). The corresponding substitution of the PTH/PTHrP receptor third extracellular loop into the PTH2 receptor (P2-3LPrP) increased the EC50 for PTH(1–34) 10-fold and did not introduce detectable activation by PTHrP (Table 2Go and Fig. 3DGo). Thus while the reduction in PTHrP affinity created by substituting the PTH2 receptor third extracellular loop into the PTH/PTHrP receptor suggests involvement in ligand interaction by this domain, the results are more complicated, because each of these third loop interchanges produced receptors that functioned less well than the parent receptors.

Trp437 and Gln440 in the third extracellular loop of the PTH/PTHrP receptor have been shown by mutation to be important for PTH(1–34) binding and are thought to contribute to interaction with the amino terminus of the peptide (5). The Trp residue is conserved between the PTH/PTHrP and PTH2 receptors but the Gln residue is not. Gln440 of the PTH/PTHrP receptor corresponds to Arg394 in the PTH2 receptor third extracellular loop. To determine whether the changes in binding and activation were due to changing this single residue, we created third extracellular loop interchanged receptors in which Arg394-to-Gln (PrP-3L*P2) and Gln440-to-Arg (P2-3L*PrP) mutations had been made, returning these residues to those of the parent receptors. These mutations had no significant effect on the apparent affinities of the chimeric receptors for 125I-NlePTH or 125I-PTHrP. Return of this residue to the one of the parent receptor decreased the EC50 values for cAMP stimulation 6- to 10-fold, moving them toward but not reaching the EC50 values of the wild-type receptors (Table 2Go and Fig. 3Go, B and D). Increase in the maximal response of these chimeric receptors was relatively small as compared with the 6- to 10-fold decrease in the EC50 values. These effects suggested to us that third extracellular loop residues might interact with other domains of the receptors. To further examine the interaction of residues in the third extracellular loop with those in the amino terminus, we examined a PTH/PTHrP receptor in which the E2 domain was removed and the third extracellular loop substituted with that from the PTH2 receptor (PrP{Delta}N-3LP2, Fig. 1Go). Binding of 125I-NlePTH did not differ between this receptor and the wild-type PTH/PTHrP receptor. Elimination of these amino-terminal residues significantly increased the EC50 values for both PTH(1–34) and PTHrP(1–34) (Table 2Go and Fig. 3DGo) and decreased 125I-PTHrP binding below detection. These data suggest that the E2 domain is not without effect on receptor function or conformation.

Exchange of Both N-Terminal Domains and Third Extracellular Loops
Chimeric receptors were generated to examine the effects of interchanging both the N terminus and third extracellular loops of the PTH/PTHrP and PTH2 receptors. The affinity for 125I-NlePTH of a PTH/PTHrP receptor in which both of these domains were substituted with the corresponding sequences of the PTH2 receptor (PrP-N3LP2) did not differ from that observed for the wild-type PTH/PTHrP receptor (Table 3Go and Fig. 4AGo). While 125I-NlePTH binding appeared unaffected by these changes, the ability of PTH(1–34) to stimulate receptor-mediated cAMP synthesis was lost (Table 3Go). Mutation of Arg394 to Gln in the third extracellular loop recovered the ability of PTH and PTHrP to stimulate the receptor, although the EC50 values remained high (Table 3Go and Fig. 4BGo). No specific binding of 125I-PTHrP to either of the double chimeras was detected.

PTH2 receptors in which the N-terminal domain and the third extracellular loop were replaced with sequences of the PTH/PTHrP receptor (P2-N3LPrP and P2-N3L*PrP) had reduced affinity for 125I-NlePTH. Removing amino acid residues 62–106 from the PTH/PTHrP receptor-donated amino terminus results in the return of high-affinity binding of 125I-NlePTH by these chimeras (P2-{Delta}N3LPrP and P2-{Delta}N3L*PrP, Table 3Go and Fig. 4CGo). While neither of the PTH2 receptor-based chimeras with wild-type PTH/PTHrP receptor N-terminal and third extracellular loops (P2-N3LPrP and P2-N3L*PrP) are activated by either PTH(1–34) or PTHrP(1–34), deletion of amino acid residues 62–106 (P2-{Delta}N3LPrP and P2-{Delta}N3L*PrP) introduces weak activation by both PTH(1–34) and PTHrP(1–34) (Table 3Go and Fig. 4DGo). It is also worth noting that mutation of Gln440 in the PTH/PTHrP receptor-donated extracellular loop to Arg in P2-{Delta}N3L*PrP results in a decrease in the EC50 for PTH(1–34), as compared with the same receptor without the mutation (P2-{Delta}N3LPrP) (Table 3Go and Fig. 4DGo), although this decrease did not reach statistical significance. These data suggest that PTH(1–34) and PTHrP(1–34) interact with both the N-terminal and third extracellular loop domains of these receptors and that both regions contribute to the differential interaction with PTHrP.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this study we looked for regions of the PTH2 receptor that discriminate between PTH and PTHrP and regions within the PTH/PTHrP receptor that recognize PTHrP. This is the first study to focus on receptor domains important for PTHrP interaction, whereas PTH/PTHrP receptor domains important for binding PTH have been studied extensively (4, 5, 10, 17, 18, 21). We characterized a series of PTH/PTHrP-PTH2 receptor chimeras designed to examine involvement of the PTH/PTHrP receptor amino terminus and third extracellular loop in PTHrP interaction. Deletion analyses of the PTH/PTHrP receptor (4), study of interspecies PTH/PTHrP receptor chimeras (19, 26), and study of chimeras between the PTH/PTHrP receptor and other members of the secretin/calcitonin/PTH receptor family (5, 20) have shown that the amino terminus and third extracellular loop are critical determinants of PTH interaction with the PTH/PTHrP receptor. While data presented in the present manuscript show that these regions are also critical determinants for PTHrP interaction with the PTH/PTHrP receptor, it is evident that additional, as yet unidentified regions, of the receptor are involved as well.

125I-NlePTH binding demonstrates that all of the receptors studied are expressed on the cell surface and that the general architecture of the chimeric receptors has not been dramatically disrupted. This is consistent with data from previous studies of chimeras between more dissimilar receptors in this family, which has suggested that domains with homologous sequences have homologous function (5, 13, 15, 16, 20, 27, 28). In addition, because 125I-NlePTH binding is unaltered by interchanging the amino termini and third extracellular loops of the PTH2 and PTH/PTHrP receptors, this study shows that these regions must be involved in PTH binding by the PTH2 receptor as well as the PTH/PTHrP receptor. While these regions have been studied extensively as regards PTH binding to the PTH/PTHrP receptor (4, 5, 10, 17, 18, 21, 29), study of PTH binding to the PTH2 receptor has not been described.

An epitope tag (the hemagglutinin epitope) was incorporated into the C terminus of the receptors studied to aid in cellular localization of nonfunctional receptors. In some recent studies receptor expression has been quantitated by antibody binding to a hemagglutinin epitope replacing the E2 domain within the amino terminus of the PTH/PTHrP receptor (4, 19). We chose not to incorporate an epitope into this region because we were studying its function. In fact, we observed that the E2 domain does influence receptor function when evaluated in combination with the PTH2 receptor. This was unexpected since we confirmed the previous observation that this region was neutral as regards PTH interactions with the PTH/PTHrP receptor (4, 19) and extended it by demonstrating that the E2 domain is also neutral as regards PTHrP binding and activation of the wild-type PTH/PTHrP receptor. When it became apparent that all of the chimeric receptors bound 125I-NlePTH, we used saturation binding analyses to assess functional receptor expression.

Evaluation of chimeric receptors in which the amino termini of the PTH/PTHrP and PTH2 receptors have been interchanged showed that the PTH/PTHrP amino terminus contributes significantly to PTHrP recognition by the wild-type receptor. Lee and colleagues (4, 5) demonstrated that PTH binding and activation of the PTH/PTHrP receptor is disrupted by mutation or deletion of the amino terminus. Jüppner et al. (19) showed that the PTH/PTHrP amino terminus determines the binding affinity of amino-terminally truncated PTH analogs. More recently, Bergwitz et al. (20) showed that PTH recognition is transferred to the calcitonin receptor by replacing the calcitonin receptor amino terminus with that of the PTH/PTHrP receptor. With these findings in mind we reasoned that PTHrP recognition sites may also reside, at least in part, in the receptor’s amino terminus. A chimera with the full-length PTH/PTHrP receptor amino terminus on the PTH2 receptor bound PTH and was fully activated by PTH, but there was no detectable interaction of PTHrP with this receptor. Deletion of the E2 domain from the PTH/PTHrP receptor amino terminus resulted in a chimera that was activated by high concentrations of PTHrP, indicating a low-affinity interaction of the peptide with the receptor. Replacement of the PTH/PTHrP amino terminus with that of the PTH2 receptor did not affect PTH binding or activation of the receptor; however, PTHrP binding was not detectable, and the EC50 for PTHrP receptor activation was significantly increased. These data show that some recognition of PTHrP is transferred with the amino terminus of the PTH/PTHrP receptor and that the PTH2 receptor amino terminus does not support high-affinity PTHrP binding. Thus, our findings support the suggestion that the amino termini of these two receptors interact differently with PTHrP or direct a differential interaction with it.

Study of chimeric receptors in which the E2 domain (residues 62–106 of the PTH/PTHrP receptor amino terminus) has been deleted reveal that these residues are involved in receptor function. These findings were unexpected as we showed that the E2 domain is neutral in PTHrP binding, and activation of the wild-type PTH/PTHrP receptor and previous work showed this region to be neutral in PTH interaction with the receptor (4, 19). The effect of deletion of the E2 domain on receptor function was context dependent. A chimeric receptor with a PTH/PTHrP receptor backbone in which the E2 domain has been deleted (PrP{Delta}N-3LP2) exhibited an increase in EC50 values for both PTH and PTHrP and a decrease in 125I-PTHrP binding below detectable levels, while 125I-NlePTH binding remained unchanged. These data show that deletion of the E2 domain is more detrimental to PTHrP binding to the receptor than PTH binding, but that activation of the receptor by both peptides is weakened to the same extent. In the context of receptors with a PTH2 receptor backbone (P2-NPrP and P2-{Delta}NPrP), deletion of the E2 domain revealed an incompatibility of these residues with PTHrP interaction. P2-NPrP bound 125I-NlePTH weakly and was activated by PTH, but binding of 125I-PTHrP and activation of adenylyl cyclase by PTHrP were undetectable. Deletion of the E2 domain, creating P2-{Delta}NPrP, resulted in 125I-NlePTH binding that was not different from wild-type receptor binding. There was an increase in the EC50 for activation of the receptor by PTH, but now PTHrP activated this receptor at high concentrations. This incompatibility was even more evident in double chimeras in which both the amino terminus and the third extracellular loop of the PTH/PTHrP receptor were introduced into the PTH2 receptor, since these receptors were unable to activate adenylyl cyclase despite binding PTH. However, deletion of the E2 domain in these double chimeras (P2-{Delta}N3LPrP and P2-{Delta}N3L*PrP) resulted in activation of these receptors by both PTH and PTHrP. The nature of the observed incompatibilities is not defined, but it is clear that the deletion of residues 62–106 in the PTH/PTHrP amino terminus of the chimeras examined had marked effects on PTH and PTHrP activation of, and 125I-PTHrP binding to, these receptors.

Because replacement of the PTH/PTHrP receptor amino terminus with that of the PTH2 receptor did not eliminate PTHrP activation, it is evident that additional regions of the receptor are involved in PTHrP interaction. In an effort to identify additional sites in the PTH/PTHrP receptor that recognize PTHrP and in the PTH2 receptor that discriminate between PTH and PTHrP, we looked at chimeric receptors in which the third extracellular loops of the PTH/PTHrP and PTH2 receptors had been switched. We observed that peptide-mediated receptor activation and PTHrP binding were particularly affected by interchange of these domains. Previous work showed that deletion or mutation of the third extracellular loop of the PTH/PTHrP receptor disrupts PTH binding (4, 5). We observed that replacement of the third extracellular loop of the PTH/PTHrP receptor with that of the PTH2 receptor affected PTHrP binding but had no effect on PTH binding. Similarly, binding of PTH to a PTH2 receptor with the third extracellular loop from the PTH/PTHrP receptor resembles that for the wild-type receptor. These data suggest that the PTH2 and PTH/PTHrP receptor third extracellular loops have similar interactions with PTH, while the PTH/PTHrP receptor third extracellular loop preferentially interacts with PTHrP. Functional activation of these chimeras was affected as reflected by an increase in EC50 values for PTH. However, mutation of Arg394 in a PTH/PTHrP receptor with a PTH2 third extracellular loop results in lower EC50 values for both peptides, and a PTH2 receptor with a PTH/PTHrP third extracellular loop and a Gln440-to-Arg mutation is activated by both peptides with EC50 values indistinguishable from wild-type values. PTHrP binding to PTH/PTHrP receptors with PTH2 third extracellular loops with or without an Arg394-to-Gln mutation was reduced in affinity as compared with the wild-type receptor, indicating that other residues in the third extracellular loop of the PTH2 receptor contribute to an incompatibility with PTHrP binding. These data suggest that the third extracellular loops of these receptors are more crucial for peptide-mediated activation of the receptor than peptide binding and that Gln440 and Arg394 play a critical role in the transduction process. Previous studies suggested that binding of peptides to members of this receptor family occurs in such a way that the carboxyl terminus of the peptide interacts with the amino terminus of the receptor, while the amino terminus of the peptide interacts with other regions in the carboxy-terminal portion of the receptor (19, 20). We speculate that the carboxy-terminal portion of PTH is able to bind to chimeric receptors in which the third extracellular loops have been swapped but that interchange of the third extracellular loops has altered the structure of these receptors such that the receptors are unable to transduce a peptide-mediated signal through G proteins as effectively as wild-type receptors. Gln440 in the PTH/PTHrP receptor and Arg394 in the PTH2 receptor are particularly important in maintaining a receptor structure that is compatible with peptide-mediated signal transduction.

Simultaneous interchange of both the amino termini and the third extracellular loops of the PTH2 and PTH/PTHrP receptors in three of the receptors studied (PrP-N3LP2, P2-N3LPrP, and P2-N3L*PrP) dissociated agonist binding and activation of the receptors. These receptor chimeras exhibited high-affinity binding of 125I-NlePTH but were not activated by PTH or PTHrP. Dissociation of receptor binding and receptor activation was also observed in a previous study in which mutation of Arg233 in the second transmembrane domain and Gln451 in the seventh transmembrane domain of the PTH/PTHrP receptor resulted in disruption of receptor signaling but had little effect on agonist binding (10). In the current study, additional sequence changes, mutation of Arg394 to Gln in a PTH/PTHrP receptor with a PTH2 receptor amino terminus and third extracellular loop, or deletion of the E2 domain in PTH2 receptors with PTH/PTHrP receptor amino termini and third extracellular loops with or without a Gln440 to Arg mutation, restored peptide activation of these receptors. Addition of the third extracellular loop and the E2-deleted amino terminus of the PTH/PTHrP receptor to the PTH2 receptor resulted in more effective activation of this chimera by PTHrP than a PTH2 receptor with the E2-deleted PTH/PTHrP receptor amino terminus, suggesting that interaction of the amino terminus and third extracellular loop in the PTH/PTHrP receptor is important for PTHrP recognition.

We were successful in making a PTH2 receptor that responds to PTHrP. However, we were unsuccessful in transferring high-affinity PTHrP binding to that receptor. It is possible that failure to transfer high-affinity PTHrP binding is due to domain incompatibilities within the various receptor chimeras. This explanation does not seem likely though; nearly all the receptor chimeras retained relatively high-affinity 125I-NlePTH binding, despite changes in the apparent binding affinity of PTHrP and the ability of peptides to activate the receptors. It is more likely that we have not identified all the PTH/PTHrP receptor domains responsible for high-affinity interaction with PTHrP. Lee et al. (5) showed that there are regions of the PTH/PTHrP receptor, such as the first extracellular loop, that are not involved in PTH recognition. The primary sequence of the first extracellular loop of the PTH/PTHrP receptor differs from that of the PTH2 receptor, making this a candidate region for recognition of PTHrP. Several groups studying the PTH/secretin/calcitonin and rhodopsin families of G protein-coupled receptors have shown that residues in the transmembrane domains are involved in agonist binding and activation of these receptors (4, 10, 30, 31, 32, 33, 34, 35, 36, 37). Residues that are not conserved between the PTH2 and PTH/PTHrP receptors are scattered throughout the receptor transmembrane domains, and these may be involved in PTHrP recognition. The results presented here provide a foundation for future work toward understanding specific determinants responsible for PTH/PTHrP and PTH2 receptor recognition of agonists and receptor activation by them.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Peptides and Reagents
PTHrP(1–34), [Tyr36]PTHrP(1–36), [Nle8,21, Tyr34] rat PTH(1–34), and human PTH(1–34) were purchased from Bachem (Torrance, CA). Forskolin was from Sigma Chemical Co. (St. Louis, MO), Na125Iodide was from ICN Biomedicals (Costa Mesa, CA), and [125I]succinyl cAMP was from Dupont NEN (Boston, MA). Restriction endonucleases and Vent DNA polymerase were purchased from New England Biolabs (Beverly, MA), and Taq DNA polymerase was from Promega (Madison, WI).

Plasmid Constructions
PTH receptor chimeras were generated using PCR with oligonucleotide primers directed to specific sites in the human PTH/PTHrP (the generous gift of Dr. H. Jüppner) and human PTH2 receptor (2) sequences (Fig. 1Go, A and B). Oligonucleotide primers incorporated BsmBI sites at the point of chimera fusion and specific restriction sites for subcloning into the appropriate plasmid vectors. BsmBI is a Type II-S restriction endonuclease that cleaves at a distance from the recognition site for the enzyme. Digestion of fragments amplified with BsmBI primers results in removal of its recognition site and cleavage at the desired site of chimera fusion so that the desired fragments are produced with a single amplification (38).

PrP was made by amplifying the hPTH/PTHrP receptor with a primer encoding a 5'-BamHI site, followed by a consensus Kozak sequence (CCACC) and then bases 1–21 of the coding sequence for the receptor, paired with a 3'- primer directed against bases 1762–1779 followed by a SalI site. The digested BamHI/SalI fragment was subcloned into pcDNAHA.

P2 was amplified with a primer encoding a 5'-HindIII site, followed by a consensus Kozak sequence (CCACC) and then bases 1–21 of the hPTH2 receptor coding sequence paired with a 3'-primer directed against bases 1632–1649 followed by a SalI site. PCR fragments were digested with HindIII and SalI and ligated into pcDNAHA.

pcDNAHA was constructed by ligating a primer encoding the hemagglutinin epitope into SalI/XbaI-digested pcDNAI/amp (Invitrogen; Carlsbad, CA).

PrP{Delta}N, in which bases 184–218 of the coding sequence have been deleted, was constructed by amplification of the hPTH/PTHrP receptor with a primer directed against the initiation methionine (described above) paired with a 3'-primer directed against bases 160–183 followed by a BsmBI site, and a primer encoding a 5'-BsmBI site followed by bases 319–339 paired with a 3'-primer encoding bases 1162–1197, which includes an EagI site. PCR fragments were digested with HindIII/BsmBI or BsmBI/EagI and ligated into HindIII/EagI-digested PrP.

P2-NPrP was constructed by amplification of the hPTH/PTHrP receptor with a primer encoding a 5' HindIII site, followed by a consensus Kozak and bases 1–21 paired with a 3'-primer directed against bases 622–642 followed by a BsmBI site and amplification of the hPTH2 receptor with a primer encoding a 5'-BsmBI site followed by bases 514–534 paired with a 3'-primer directed against bases 736–768 including a BamHI site. PCR fragments were digested with HindIII/BsmBI or BsmBI/BamHI and ligated into HindIII/BamHI-digested P2.

PrP-NP2 was made by amplifying the hPTH2 receptor with a primer directed against the initiation codon (described above) paired with a primer directed against bases 493–513 followed by a BsmBI site and amplification of the hPTH/PTHrP receptor with a primer encoding a 5'-BsmBI site followed by bases 643–663 paired with the downstream primer used in making PrP (described above). PCR fragments were digested with HindIII/BsmBI or BsmBI/SalI and ligated into HindIII/SalI-digested pcDNAHA.

PrP-3LP2 was constructed by amplification of the hPTH/PTHrP receptor with a 5'-primer encoding bases 1177–1203 including an EagI site paired with a 3'-primer encoding bases 1270–1284 of the hPTH/PTHrP receptor, followed by bases 1149–1172 of the hPTH2 receptor and a BsmBI site and amplification with a primer encoding a 5'-BsmBI site, followed by bases 1167–1181 of the hPTH2 receptor, and bases 1321–1338 of the hPTH/PTHrP receptor paired with the downstream primer used to make PrP (described above). PCR fragments were digested with EagI/BsmBI or BsmBI/SalI and ligated into EagI/SalI-digested PrP.

PrP-3L*P2 was constructed by amplification of PrP with the same primers used to generate the EagI/BsmBI fragment as described above for PrP-3LP2 and with a primer encoding a 5'-BsmBI site, followed by bases 1167–1181 of the hPTH2 receptor with a CGC encoding Arg in place of the CAG encoding Gln, and bases 1321–1338 of the hPTH/PTHrP receptor paired with a 3'-primer directed against the hemagglutinin epitope with an XbaI site. PCR fragments were digested with EagI/BsmBI or BsmBI/XbaI and ligated into EagI/XbaI- prepared PrP.

P2-3LPrP was constructed by amplification of the hPTH2 receptor with a 5'-primer directed against bases 754–780 including a BamHI site paired with a 3'-primer encoding a BsmBI site, followed by bases 1134–1148 of the hPTH2 receptor, and bases 1285–1311 of the hPTH/PTHrP receptor and a 5'-primer encoding a BsmBI site, followed by bases 1306–1320 of the hPTH/PTHrP receptor, and bases 1182–1199 of the hPTH2 receptor paired with the downstream primer used in constructing P2 (described above). PCR fragments were digested with BamHI/BsmBI or BsmBI/SalI and ligated into BamHI/SalI- prepared P2.

P2-3L*PrP was constructed the same as for P2-3LPrP accept for a CAG (Gln) to CGC (Arg) change in the primer encoding a BsmBI site, bases 1306–1320 of the hPTH/PTHrP receptor, and bases 1182–1199 of the hPTH2 receptor.

P2-{Delta}NPrP was made by ligating a HindIII/BspEI fragment from PrP{Delta}N into HindIII/BspEI-digested P2-NPrP.

PrP{Delta}N-3LP2 was made by ligation of a HindIII/BspEI fragment of PrP{Delta}N into HindIII/BspEI-digested PrP-3LP2.

P2-N3L*PrP was made by ligation of a BamHI/SalI fragment from P2-3L*PrP into BamHI/SalI-digested P2-N3LPrP.

P2-{Delta}N3LPrP was made by ligation of a HindIII/BspEI fragment from PrP{Delta}N into HindIII/BspEI-digested P2-N3LPrP.

P2-{Delta}N3L*PrP was made by ligation of a PshAI/SalI fragment from P2-3L*PrP into PshAI/SalI-digested P2-{Delta}N3LPrP.

PrP-N3L*P2 was made by ligation of a EagI/XhoI fragment from PrP-3L*P2 into EagI/XhoI-digested PrP-N3LP2.

DNA sequence analysis of each construct confirmed the absence of PCR- introduced mutations.

Cell Culture and Transient Expression in COS-7 Cells
COS-7 cells were cultured in DMEM (GIBCO/BRL; Gaithersberg, MD) supplemented with 10% FCS (Sigma), 100 U/ml penicillin G, 100 µg/ml streptomycin sulfate (GIBCO/BRL) in a humidified atmosphere supplemented with 5% CO2. Cells were plated at a density of 2.5 x 105 cells per well in 24-well plates and transfected after 16–24 h by addition of 0.5 µg/well of PTH receptor plasmid DNA in diethylaminoethyl-dextran/chloroquine (39) for 3–4 h followed by a 2-min treatment with 10% dimethylsulfoxide in PBS (40). The dimethylsulfoxide solution was replaced with complete medium and cells were incubated overnight. Within 24 h of transfection, media were changed and cells were incubated for a total of 72 h post transfection before use in adenylyl cyclase stimulation or ligand-binding assays. In each experiment three to six wells of cells were transfected with 0.5 µg/well of a plasmid encoding ß-galactosidase. Transfection efficiency ranged from 25–50% based on histochemical determinations of ß-galactosidase expression on the day of assay.

Determination of Cellular cAMP Response
Transfected COS cells were washed once with an assay buffer composed of modified Krebs-Ringer-HEPES medium (41), 1% BSA, 100 µM Ro 20–1724 (Research Biochemicals International; Natick, MA), and 1 µg/ml bacitracin. After a 10-min incubation in assay buffer at room temperature, 150 µl of assay buffer without and with a concentration range of hPTH(1–34), PTHrP(1–34), or forskolin was added to each well. Cells were incubated with peptide for 10 min at room temperature on a rotary shaker table. The assay was terminated by addition of an equal volume of 0.1 N hydrochloric acid and 0.1 mM calcium chloride (42), and plates were set on ice for 30 min and then stored at -20 C. Initial determinations of cAMP concentrations were made using cAMP RIA methods described by Brooker et al. (43). More recent cAMP determinations used a solid phase modification (44) of the cAMP RIA. Immulon II removawells (Dynatech, Chantilly, VA) were coated overnight with 100 µl protein G (1 mg/ml in 0.1 M NaHC03, pH 9.0) at 4 C, rinsed with PBS-gelatin-Tween (PBS containing 0.1% gelatin, 0.2% Tween-20) three times quickly and then once for 30 min, and then incubated overnight with 100 µl of a sheep antibody to cAMP diluted in 50 mM sodium acetate, pH 4.75 (Atto Instruments, Rockville, MD; dilution of stock to 2.5 x 10-6, determined empirically). After rinsing with PBS-gelatin-Tween, the RIA was set up by adding acetylated cAMP standards or acetylated aliquots from stimulated cells and 5,000–7,000 cpm [125I]succinyl cAMP to the plates in a final volume of 150 µl. Plates were incubated overnight at 4 C, rinsed four times with sodium acetate buffer, and blotted dry, after which individual wells were broken off and bound radioactivity was determined in a {gamma}-counter. EC50 values were determined by nonlinear least squares fit of the data to the equation Ao + (Am*L)/(EC50 + L) where Ao is the basal level of cAMP, Am the maximal accumulation under these conditions, L is the added ligand concentration, and EC50 the apparent half-maximal concentration for stimulation of cAMP accumulation.

Radioligand Preparation
125I-Nle8,21,Tyr34]rPTH(1–34)NH2 and 125I-[Tyr36]PTHrP(1–36) were prepared by combining 10 µg peptide, 2 mCi carrier free Na125I, and 10 µg chloramine-T for 30 sec in a final volume of 40 µl containing 0.1 M sodium phosphate, pH 7.4. The reaction was terminated by addition of 20 µl of 10 mM cysteine and free iodine separated from the peptides by absorption to a C-18 Sep-Pak cartridge, which was rinsed with 0.1% tri-fluoroacetic acid (TFA) and eluted with 3 ml 40% isopropanol in 0.1% TFA. HPLC purification was performed by diluting the Sep-Pak-purified material 3-fold with 0.1% TFA and then loading it onto a Vydac C4 analytical HPLC column that was equilibrated in 0.1% TFA containing 20% acetonitrile and then eluted with a gradient of acetonitrile. The two major radioactivity-containing peaks were identified as the mono- and diiodinated peptides based on the ratio of radioactivity to optical absorbance (monitored at 220 nm). Binding kinetics for both the mono- and diiodinated species of 125I-[Nle8,21,Tyr34]rPTH(1–34) were virtually identical (data not shown); therefore, diiodinated 125I-[Nle8,21,Tyr34]rPTH(1–34) was used throughout this study. Monoiodinated 125I-[Tyr36]PTHrP(1–36) was used for all PTHrP-binding assays.

Radioligand Binding
After one wash with binding buffer (100 mM sodium chloride, 50 mM Tris HCl, pH 7.5, 5 mM potassium chloride, 2 mM calcium chloride, 5% heat-inactivated horse serum, 0.5% FCS), 150 µl of binding buffer without or with 2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) was added to each well of transfected COS cells and plates were set on ice. Fifty microliters of binding buffer with AEBSF without or with various concentrations of [Nle8,21, Tyr34]rPTH(1–34)NH2, or PTHrP(1–34) was added to the cells on ice. After addition of 200,000 cpm/well of 125I-[Nle8,21, Tyr34]rPTH(1–34)NH2, or 125I-[Tyr36]PTHrP(1–36), the cells were placed at 15 C for 2–4 h. Binding was terminated on ice by washing twice with ice-cold binding buffer, and cell-associated 125I was extracted with 1.0 N sodium hydroxide. Samples were transferred to tubes and bound radioactivity quantified in a {gamma}-counter. Nonspecific binding was 1–4% of the total added counts. Total binding in the absence of unlabeled ligand was less than 25% of the added counts. Data are plotted as percent of control where control is counts per min bound in the absence of unlabeled ligand. KD, Bmax, and nonspecific binding values were derived from ligand-binding data using the MacIntosh version of the program LIGAND (45).

Statistical Analysis
The different measures for each set of homologous chimeras (e.g. KD for 125I-NlePTH binding to PTH2 receptor-based chimeras) were initially compared using ANOVA (P < 0.05). Post hoc analysis of data containing significant differences was performed using Tukey’s t test (P < 0.05) to identify which chimeras differed.


    ACKNOWLEDGMENTS
 
The authors would like to thank J. Northup and S. Hoare for helpful discussions and critical review of the manuscript; M. Brownstein for his generous support and critical review of the manuscript; L. Baptiste for technical assistance; M. K. Floeter for assistance with the statistical analyses; and H. Jüppner for the hPTH/PTHrP receptor plasmid and initial batches of radiolabeled peptides.


    FOOTNOTES
 
Address requests for reprints to: J. A. Clark, Section of Genetics, National Institute of Mental Health, Building 36, Room 3B-12, 36 Convent Drive, MSC 4090, Bethesda, Maryland 20892-4090. E-mail: janet{at}codon.nih.gov

Received for publication August 7, 1997. Revision received November 4, 1997. Accepted for publication November 7, 1997.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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