Section on Genetics National Institute of Mental Health Bethesda, Maryland 20892-4090
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ABSTRACT |
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INTRODUCTION |
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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 139173 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(134) 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(134), 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(134), after which detailed comparison of the interaction with PTHrP(134) and PTH(134) 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.
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RESULTS |
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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 1 and
Fig. 2
, A and B). However, the EC50 for PTHrP stimulation
of cAMP accumulation increased 10-fold (Table 1
and Fig. 2B
) 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(134), and cAMP accumulation stimulated by PTH(134) and
PTHrP(134) 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 1 and Fig. 2C
).
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 62106), 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-
NPrP) yielded a receptor with a
KD and Bmax for PTH that are not significantly
different from the parent PTH2 receptor (Table 1
) and introduced weak
activation by PTHrP (Table 1
and Fig. 2D
). 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(134) interaction (5). Exchange of the third extracellular loops
(PrP-3LP2 and P2-3LPrP,
Fig. 1) did not change the apparent affinity for
125I-NlePTH(134) from that of the parent receptors for
either construct (Table 2
). Substitution
of the PTH2 receptor third extracellular loop into the PTH/PTHrP
receptor (PrP-3LP2) decreased the apparent
affinity for PTHrP 5-fold (Table 2
and Fig. 3A
). 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 2
and Fig. 3B
). The corresponding
substitution of the PTH/PTHrP receptor third extracellular loop into
the PTH2 receptor (P2-3LPrP) increased the
EC50 for PTH(134) 10-fold and did not introduce
detectable activation by PTHrP (Table 2
and Fig. 3D
). 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(134)
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 2 and Fig. 3
, 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
N-3LP2, Fig. 1
). 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(134) and PTHrP(134) (Table 2
and Fig. 3D
) 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 3 and Fig. 4A
). While 125I-NlePTH
binding appeared unaffected by these changes, the ability of PTH(134)
to stimulate receptor-mediated cAMP synthesis was lost (Table 3
).
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 3
and Fig. 4B
). 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 62106 from the
PTH/PTHrP receptor-donated amino terminus results in the return of
high-affinity binding of 125I-NlePTH by these chimeras
(P2-N3LPrP and
P2-
N3L*PrP, Table 3
and Fig. 4C
). 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(134)
or PTHrP(134), deletion of amino acid residues 62106
(P2-
N3LPrP and
P2-
N3L*PrP) introduces weak activation by
both PTH(134) and PTHrP(134) (Table 3
and Fig. 4D
). It is also
worth noting that mutation of Gln440 in the PTH/PTHrP receptor-donated
extracellular loop to Arg in P2-
N3L*PrP
results in a decrease in the EC50 for PTH(134), as
compared with the same receptor without the mutation
(P2-
N3LPrP) (Table 3
and Fig. 4D
), although
this decrease did not reach statistical significance. These data
suggest that PTH(134) and PTHrP(134) 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.
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DISCUSSION |
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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 receptors 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 62106 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
(PrPN-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-
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-
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-
N3LPrP
and P2-
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 62106 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.
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MATERIALS AND METHODS |
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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. 1, 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 121 of the coding sequence for the receptor, paired with a 3'- primer directed against bases 17621779 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 121 of the hPTH2 receptor coding sequence paired with a 3'-primer directed against bases 16321649 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).
PrPN, in which bases 184218 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
160183 followed by a BsmBI site, and a primer encoding a
5'-BsmBI site followed by bases 319339 paired with a
3'-primer encoding bases 11621197, 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 121 paired with a 3'-primer directed against bases 622642 followed by a BsmBI site and amplification of the hPTH2 receptor with a primer encoding a 5'-BsmBI site followed by bases 514534 paired with a 3'-primer directed against bases 736768 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 493513 followed by a BsmBI site and amplification of the hPTH/PTHrP receptor with a primer encoding a 5'-BsmBI site followed by bases 643663 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 11771203 including an EagI site paired with a 3'-primer encoding bases 12701284 of the hPTH/PTHrP receptor, followed by bases 11491172 of the hPTH2 receptor and a BsmBI site and amplification with a primer encoding a 5'-BsmBI site, followed by bases 11671181 of the hPTH2 receptor, and bases 13211338 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 11671181 of the hPTH2 receptor with a CGC encoding Arg in place of the CAG encoding Gln, and bases 13211338 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 754780 including a BamHI site paired with a 3'-primer encoding a BsmBI site, followed by bases 11341148 of the hPTH2 receptor, and bases 12851311 of the hPTH/PTHrP receptor and a 5'-primer encoding a BsmBI site, followed by bases 13061320 of the hPTH/PTHrP receptor, and bases 11821199 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 13061320 of the hPTH/PTHrP receptor, and bases 11821199 of the hPTH2 receptor.
P2-NPrP was made by ligating a
HindIII/BspEI fragment from PrP
N
into HindIII/BspEI-digested
P2-NPrP.
PrPN-3LP2 was made by ligation of a
HindIII/BspEI fragment of PrP
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-N3LPrP was made by ligation of a
HindIII/BspEI fragment from PrP
N
into HindIII/BspEI-digested
P2-N3LPrP.
P2-N3L*PrP was made by ligation of a
PshAI/SalI fragment from
P2-3L*PrP into
PshAI/SalI-digested
P2-
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 1624 h by addition of 0.5 µg/well of PTH receptor
plasmid DNA in diethylaminoethyl-dextran/chloroquine (39) for 34 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 2550% 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 201724 (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(134), PTHrP(134), 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 5,0007,000 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 -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(134)NH2
and 125I-[Tyr36]PTHrP(136) 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(134)
were virtually identical (data not shown); therefore, diiodinated
125I-[Nle8,21,Tyr34]rPTH(134)
was used throughout this study. Monoiodinated
125I-[Tyr36]PTHrP(136) 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(134)NH2, or PTHrP(134) was
added to the cells on ice. After addition of 200,000 cpm/well of
125I-[Nle8,21,
Tyr34]rPTH(134)NH2, or
125I-[Tyr36]PTHrP(136), the cells were
placed at 15 C for 24 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 -counter. Nonspecific binding was 14% 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 Tukeys
t test (P < 0.05) to identify which
chimeras differed.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication August 7, 1997. Revision received November 4, 1997. Accepted for publication November 7, 1997.
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REFERENCES |
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