Endocrine Research Unit Veterans Affairs Medical Center and Departments of Medicine and Physiology University of California San Francisco, California 94121
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ABSTRACT |
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The N-terminal region of PTH1r was divided into three segments that were replaced either singly or in various combinations with the homologous region of the secretin receptor (SECr). Substitution of the carboxy-terminal half (residues 105186) of the N-terminal region of PTH1r for a SECr homologous segment did not reduced affinity for PTH but abolished signaling in response to PTH. This data indicate that receptor activation is dissociable from high affinity hormone binding in the PTH1r, and that the N-terminal region might play a critical role in the activation process. Further segment replacements in the N-termini focus on residues 105186 and particularly residues 146186 of PTH1r as providing critical segments for receptor activation. The data obtained suggest the existence of two distinct PTH binding sites in the PTH1rs N-terminal region: one site in the amino-terminal half (residues 162) (site 1) that participates in high-affinity PTH binding; and a second site of lower affinity constituted by amino acid residues scattered throughout the carboxy-terminal half (residues 105186) (site 2). In the absence of PTH binding to site 1, higher concentrations of hormone are required to promote receptor activation. In addition, elimination of the interaction of PTH with site 2 results in a loss of signal transduction without loss of high-affinity PTH binding.
Divers substitutions of the extracellular loops of the PTH1r highlight the differential role of the first- and third extracellular loop in the process of PTH1r activation after hormone binding.
A chimera containing the entire extracellular domains of the PTH1r and the transmembrane + cytoplasmic domains of SECr had very low PTH binding affinity and did not signal in response to PTH. Further substitution of helix 5 of PTH1r in this chimera increased affinity for PTH that is close to the PTH affinity for the wild-type PTH1r but surprisingly, did not mediate signaling response. Additional substitutions of PTH1rs helices in various combinations emphasize the fundamental role of helix 3 and helix 6 on the activation process of the PTH1r.
Overall, our studies demonstrated that several PTH1r domains contribute differentially to PTH binding affinity and signal transduction mechanism and highlight the role of the N-terminal domain and helix 3 and helix 6 on receptor activation.
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INTRODUCTION |
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Our previous studies involving the two chimeric receptors NPTH/SECr
(the N terminus of the PTH1r attached to the remainder of the SECr) and
NSEC/PTHr (the reciprocal chimeric receptor) support the view that the
N-terminal extracellular domain as well as the carboxy-core of the
PTH1r are both critical for PTH(NOREF>134) binding and signal transduction
(17). To further define the structural basis of the interaction between
PTH(NOREF>134) and the PTH1rs N terminus and carboxy core domains, we
performed a detailed pharmacological analysis of 27 SEC/PTH1r chimeras.
This approach was feasible because of the high sequence identity (60%)
within the transmembrane (TM) segments of the secretin and PTH1
receptors (Fig. 1), and because of the mutual absence of
cross-recognition of these hormones with each others receptor. The
results indicate that 1) two distinct PTH(NOREF>134) binding sites are
present within the receptors N terminus: the first site (site 1)
resides on the amino-terminal half of the PTH1rs N terminus and is
implicated in high affinity binding of PTH, while a second site (site
2) of lower affinity is located on the carboxy-terminal half and is
necessary to promote receptor signaling. Moreover, the data suggest
that site 1 acts in concert with site 2 for optimal function of the
receptor (hormone binding and cAMP response); 2) that the first- and
third extracellular loops of the PTH1r are critical for high-affinity
PTH(NOREF>134) binding and signal transduction; 3) helix 5 in combination
with the extracellular domains (N-termini and loops) regulates PTH1r
affinity for PTH; 4) effective signaling in response to PTH requires
the presence of the PTH1rs TM3 and TM6.
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RESULTS |
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Construction and Expression of Chimeras Receptors in HEK-293
Cells
We constructed four sets of chimeric receptors. Figures 2 and 3
provide a schematic diagram of the various chimeric receptors studied.
Studies with the first set of chimeric receptors were carried out to
map the regions of the N-terminal domain of the PTH1r that cooperate
functionally with the receptors carboxy core. To accomplish this, we
divided the PTH1rs amino-terminal extracellular domain into three
domains according to the position of the strictly conserved cysteine
residues within the N-terminal region of class II GPCRs
(i.e. Cys 105, 114, 128, and 145 for the PTH1r; Fig. 1
):
domain A (residues 1106), domain B (residues 106145), and domain C
(residues 145186) (Fig. 4
). Each
domain was introduced, either singly or in combination, in place of the
corresponding domain of the chimera NSEC/PTHr. The second and third set
were designed to analyze the effect on receptor function (hormone
binding and cAMP response) of progressive replacement of the sequence
of the secretin receptor within the chimera NPTH/SECr with that of the
PTH1r. In set 2, PTH1r sequence was progressively introduced from the
cytoplasmic end of TM1 through the third extracellular loop (3e). In
set 3, PTH1r sequence was progressively introduced in the opposite
direction, from the carboxy-terminal tail through the first
extracellular loop (1e). The fourth set was designed to distinguish the
relative contribution to ligand binding and signaling of the entire
extracellular domain (N-terminal region + extracellular loops)
vs. the TM segments. The binding affinity for PTH(NOREF>134) and
cAMP response of the chimeric receptors was analyzed in stably
transfected HEK-293 cells. Table 1
summarizes the PTH(NOREF>134) binding and signaling properties of the PTH1
and hybrid receptors. The expression levels of the chimeric receptors
were generally similar to that of the PTH1r (Table 1
). None of the
chimeric receptors was able to bind or respond to secretin (data not
shown).
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Role of the N-Terminal Extracellular Domain of the PTH1r in Signal
Transduction
Compared with NSEC/PTHr, chimera A showed a strong increase in
binding affinity for PTH(NOREF>134), with a potency close to that observed
for the wild-type PTH1r (Ki = 9.2 vs.
5.6 nM). Despite this gain in hormone binding
affinity, chimera A was totally unresponsive to PTH(NOREF>134) in terms of
stimulation of intracellular cAMP production. This was not due to
decreased levels of expression of chimera A relative to the wild-type
PTH1r (Table 1). Thus, the signaling defect of chimera A may be due to
improper positioning of PTH within the PTH1r. This suggests that
structural determinants in the domain BC of the PTH1r are required to
properly position the hormone in the receptor.
To determine whether domain BC as an effect on the signaling properties
of PTH1r, the pharmacological properties of chimera receptors B, C, and
BC were analyzed. Although chimera C did not display a gain of affinity
for the binding of PTH(NOREF>134) (IC50>1000
nM), it did display a marked increase in cAMP responses to
high concentrations (10-5 M) of
PTH(NOREF>134). In a receptor-agonist system with equal magnitude for the
biological response, relative potencies (EC50)
should reflect relative binding affinities (Ki)
(20). For chimera C the maximal extent of PTH stimulation of cAMP
formation was the same as in cells expressing PTH1r. Thus, the high
EC50 ( 3, 500 nM) value for
PTH(NOREF>134) observed for chimera C suggests that PTH1r domain C
could be part of an interaction of low affinity
(IC50
3, 500 nM) with the hormone,
sufficient to promote a full signaling response. Domain C appears to be
involved in the activation of the PTH1r. Data obtained with chimera B
are not informative because of the absence of hormone binding and the
low cAMP response toward PTH.
Simultaneous substitution of domains B and C of the PTH1r (chimera BC) strongly increased the potency of PTH(NOREF>134) for increasing cellular cAMP levels (EC50=22.7 nM), as well as conferring a maximal cAMP response similar to that of the wild-type PTH1r. As expected from the functional (cAMP) response, the binding affinity was also increased (Ki= 30.7 nM). Therefore, domain BC of the PTH1r appears to be part of a PTH(NOREF>134) binding site that is involved in signal transduction. Thus, the conformation conferred by domain BC appears to be involved in transducing the signal from the N-terminal extracellular domain to the carboxy core domain of the receptor.
When we compared the properties of wild-type PTH1r and chimera BC, we observed that the increase in the Ki value (6-fold) observed for the chimera BC as compared with the PTH1r did not reflect the increase in the EC50 (200-fold) for PTH-stimulated cAMP production. This discrepancy could reflect 1) a decrease in cell surface receptor expression (i.e. the roughly similar value for Ki and EC50 suggest the absence of spare receptors in cells expressing the chimera BC) and/or 2) a contribution of the binding site in domain A to signal transduction in the wild-type PTH1r. To test whether the interaction of PTH(NOREF>134) with domain A could act in concert with additional hormone interaction sites in domains B or C, we constructed and expressed chimeras AB and AC. The apparent binding affinity of PTH(NOREF>134) to chimera AB (K i= 12.5 nM) and to chimera AC (Ki = 15.6 nM) was markedly increased as compared with chimeras B and C, respectively, and was similar to the affinity of PTH(NOREF>134) for chimera A (Ki = 9.2 nM). Although introduction of domain A increased the binding affinity of chimeras B and C, it did not enhance their signaling properties.
Altogether, these data not only confirm the importance of the PTH1rs N-terminal domain for hormone binding, but also indicate its critical contribution to signal transduction. Our data identify two domains (domains A and BC) of the N-terminal extracellular domain of PTH1r that differentially affect PTH binding and receptor activation. It appears that the N-terminal domain participates in at least two interactions with the hormone: one interaction with domain A (site 1) that is not required for full efficacy of the cAMP response; and an additional interaction with domain BC (site 2) that is necessary to promote the signal transduction process. Contact of the hormone with sites 1 and 2 appears to be required for optimal functionality of the receptor.
Differential Role of the Extracellular Loop 1 and Extracellular
Loop 3 of PTH1r in Hormone Binding and Receptor Activation
Chimeric receptors (CRs) that contain the N-terminal sequence and
the first one, three, or four TM segments including the connecting
loops of the PTH1r (chimeric receptors CR1, CR2, and CR3) showed no
specific 125I-PTHrP(NOREF>134) binding and no cAMP
responses to PTH(NOREF>134), even though these chimeric receptors displayed
cell surface expression that was 70100% that of the wild-type PTH1r
(Table 1). Further substitution of the sequence extending from the N
terminus through TM6 of the PTH1r resulted in a chimera (CR4) that
displayed high-affinity PTH binding (Ki = 12
nM vs. 5.6 nM for the
wild-type PTH1r) (Fig. 5A
). CR4 also
initiated PTH-stimulated cAMP production (maximal response =75% of the
wild-type receptor), but the potency of PTH was markedly reduced
(EC50 = 42 nM
vs. 0.1 nM for the wild-type receptor)
(Fig. 5B
). Additional replacement of the third extracellular loop
(chimera CR5) resulted in a remarkable 400-fold increase in the potency
of PTH(NOREF>134) in stimulating cAMP production
(EC50 = 0.1 nM), with only
a 2.5-fold increase in apparent binding affinity for PTH(NOREF>134) (Fig. 5
, A and B, and Table 1
). These results indicate that the third
extracellular loop of the PTH1r plays an important role in the process
of receptor activation after agonist binding. The fact that CR5 is
virtually identical to the wild-type PTH1r with respect to ligand
binding affinity and signaling indicates that PTH1r-specific residues
in the region extending from TM7 to the C terminus do not play a
critical role in promoting high-affinity PTH binding or receptor
signaling to adenylyl cyclase.
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Substitution of the extracellular loop 1 in CR10 (resulting in CR11)
and of the extracellular loop 3 in CR4 (resulting in CR5) show
disparate effects on PTH binding and cAMP inducibility. Indeed, as
compared with the first extracellular loop substitution (CR10
vs. CR11: Ki decreases 6-fold and
EC50 decreases 40-fold), the third extracellular
loop substitution (CR4 vs. CR5: Ki
decreases 2.5-fold and EC50 decreases 420-fold) shows a 10-fold better
PTH potency for cAMP induction (Fig. 5B). On the other hand, the first
exoloop substitution shows a 2.4-fold better affinity for PTH binding
(Fig. 5A
). These disparate effects underscore the differential roles of
exoloop 1 and exoloop 3 of PTH1r on PTH binding and receptor
activation.
Helices 3, 5, and 6 of PTH1r Are Important for High-Affinity
Hormone Binding and Receptor Activation
Substitution of TMs 1, 2, 4, and 7 resulted in a receptor (chimera
CR12) with properties similar to those of the wild-type PTH1r (Table 1
and Fig. 6
). Introduction of the
secretin receptors second extracellular loop into chimera CR12 had no
influence on the binding and signaling properties of the resulting
receptor (data not shown). These results support the nonessential role
of the divergent residues in TMs 1, 2, 4, and 7 as well as the second
extracellular loop (2e) for PTH1r function, and suggest that, in the
presence of the N-terminal domain, the first and the third
extracellular loops, as well as TMs 3, 5, and 6 of the PTH1r define the
principal determinants of high affinity PTH(NOREF>134) binding and
receptor activation.
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None of the chimeras in which only a single TM was substituted
(CR14: introduction of TM3 into CR13; CR15: introduction of TM5 into
CR13; and CR16: introduction of TM6 into CR13) were able to rescue the
pharmacologically inactive CR13 receptor (Fig. 6, A and B). However,
CR15 displayed the most important gain in PTH(NOREF>134) binding affinity.
Indeed this chimera showed a Ki value close to
that observed for the wild-type PTH1r (Ki = 9.5
vs. 5.6 nM) and an equivalent
Bmax value. Chimera CR14 displayed reduced
binding affinity (Ki = 50
nM), and chimera CR16 showed little if any
specific PTH binding. Functionally, chimeras CR15 and CR16 were totally
unresponsive to PTH(NOREF>134) stimulation; this was unexpected for chimera
CR15 in view of its ability to bind PTH(NOREF>134) with high affinity. In
contrast, chimera CR14 was able to respond modestly to PTH(NOREF>134)
(maximal cAMP response = 41% that of the wild-type PTH1r;
EC50 = 220 nM) (Table 1
and
Fig. 6B). These results indicate that PTH was recognized when TM3
or TM5 of PTH1r was incorporated into CR13. It is important to note
that these data do not show that either TM 3 or 5 makes a direct
contact with PTH, but suggest that the conformation conferred by TM5
and TM3 plays a role in PTH binding and signal transduction,
respectively.
Helices 3 and 6 of PTHR1 Receptor Are Determinant for Signal
Transduction
To further explore the helix specificity by which introduction of
TMs 3, 5, and 6 can restore the function lost in CR13, three additional
chimeric receptors (CR17: introduction of TMs 5 and 6; CR18:
introduction of TMs 3 and 5; CR19: introduction of TMs 3 and 6) were
constructed and characterized.
Compared with CR13, CRs 17, 18, and 19 displayed a remarkable
increase in PTH binding and cAMP induction. However, they show distinct
differences in cAMP induction profiles (Fig. 6D). CR19 with the lower
PTH affinity binding (Ki = 45 nM vs. 8.9
nM and 11.9 nM for CR17 and
18, respectively) has the strongest effect in the cAMP response toward
PTH (EC50=10 nM
vs. 140 nM and 94
nM for CR17 and CR18, respectively). These data
support an important role for TM3 and TM6 in the process of signal
transduction in the PTH1r.
These data indicate that, in the presence of the PTH1rs extracellular domains, specific molecular interactions between TMs 3, 5, and 6 play a decisive role to govern the overall function of the PTH1r. Structural elements in TM5 appear to contribute to high affinity PTH binding, and determinants in TM3 and TM6 contribute to a lesser extent to PTH binding but are essential for effective signal transduction.
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DISCUSSION |
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The binding to chimeras A (Ki = 9.2
nM) and BC (Ki = 30.7
nM) demonstrated that the unfavorable interaction of
PTH(NOREF>134) with the chimera NSEC/PTHr (Ki>1000
nM) can be overcome by replacing domain A or BC of the
secretin receptor with the corresponding domain of the PTH1r (residues
1104 for domain A; and residues 105186 for domain BC). This
suggests the existence of two distinct PTH(NOREF>134) binding sites in the
PTH1rs N-terminal domain: one in domain A (site 1) displays high
affinity for PTH, and a second in domain BC (site 2) displays slightly
lower affinity ( 3.5-fold).
Despite displaying high-affinity for PTH(NOREF>134), chimera A was not able to induce cAMP accumulation, indicating a complete loss of the transduction process associated with PTH(NOREF>134) stimulation. This absence of functional activity was not attributable to a decrease in cell surface receptor expression. Rather it was caused by conformational incompatibilities, possibly involving the N terminus and the carboxy-core domain of the receptor, that interfere with a proper positioning of PTH. The remarkable gain of PTH-stimulated cAMP accumulation with chimera BC, as compared with NSEC/PTHr, indicates that hormone interaction with site 2 (PTH1rs domain BC) was essential to promote receptor activation. However, chimera BC did not fully reproduce the binding and signaling properties of the wild-type PTH1r (Ki = 30.7 vs. 5.6 nM; EC50 = 22.7 vs. 0.1 nM), suggesting that hormone interaction with site 1 (domain A) may optimize the function of the receptor by lowering the Ki and EC50 values. We speculate that the interaction of PTH(NOREF>134) with site 1 (domain A) is not requisite for the recognition of PTH by domain BC, but could facilitate the association of PTH with site 2 (domain BC), thus allowing initiation of the signal transduction process.
Our study revealed that full stimulation of the cAMP response was achieved only when the PTH1rs domain C was present in the chimeras (chimeras C, AC, and BC), suggesting that the conformation conferred by domain C was required for maximal PTH-stimulated cAMP production. That chimera C, in the absence of high-affinity PTH(NOREF>134) binding, was able to stimulate a full cAMP response at a high hormone concentration (10-5 M) suggests that domain C of the PTH1r could be part of a low-affinity binding site for PTH(NOREF>134) that is sufficient to promote receptor activation. These results are in agreement with a recent photoaffinity cross-linking study demonstrating close proximity between PTH(NOREF>134) and amino acids 173189 in domain C of the human PTH1r (14).
Therefore, our results indicate that the receptors N-terminal domain not only has a fundamental role in PTH(NOREF>134) binding, but also makes a critical contribution to the process of signal transduction. Since the N-terminal domain of a GPCR cannot interact with G proteins on the cytoplasmic face of the plasma membrane, it is not clear how this region of the receptor can control the signal transduction process. The current model for activation of GPCRs is that receptor occupancy by an agonist results in the stabilization or induction of the active receptor conformation that is competent to activate the cognate G protein (23). According to this model, we speculate that domain BC of the PTH1 receptor (site 2) could be of a structural domain of the receptor that stabilizes or induces the active receptor conformation after PTH binding. In our recent study using PTH1/PTH2 chimeric receptors, we proposed a possible two-site model for PTH(NOREF>134)-PTH1r interaction (2), in which the combined hormone interaction with the receptors N-terminal domain and the receptors carboxy core are required for receptor activation. The results of the present study allow us to extend this model: as chimera A displays PTH binding affinity 3.5-fold higher than the chimera BC, we propose that the initial contact between PTH and the receptor could occur with receptor elements in domain A as well as elements in the carboxy core. Indeed, cross-linking studies have identified an interaction between residue 23 in PTH and the extreme amino terminus of the PTH1R (14). This binding event facilitates an energetically less favorable interaction between PTH and elements in domain BC and in the carboxy core. The latter interaction initiates the conformational switch that results in signal transduction.
Although our data clearly suggest that the receptor N-terminal domain
plays a role in receptor activation, previous reports (16, 24) might
deemphasize the importance of the N-terminal domain in the activation
process. Indeed, the substitution of the N termini of PTH1- and of GHRH
receptors by the corresponding N termini of other class II receptors
[as the calcitonin receptor and the VIP receptor] have resulted in
chimeras N-Cal/PTHr and N-VIP/GHRHr that fully stimulate adenylyl
cyclase in response to high concentrations ( 1 µM) of
PTH and GHRH, respectively. These data contrast with the very low cAMP
response to PTH of our chimera N-SEC/PTHr. Alignment of the N-terminal
regions between the secretin-, PTH-, and calcitonin receptors indicates
a significant higher amino acid sequence identity between corresponding
domain C of PTH- and calcitonin receptor (36%) vs.
corresponding domain C of PTH- and SEC receptor (16%). Because our
data suggest that domain C of PTHR is sufficient to promote receptor
activation, a possible explanation for the differences between the
signaling response of chimeras N-Cal/PTHr and N-SEC/PTHr, might be that
some of the determinants required for PTH binding are present in the
N-terminal domain (corresponding to domain C) of the calcitonin
receptor and sufficient to promote full activation.
The Carboxy Core Domain
The very low PTH binding affinity and lack of functional response
to PTH displayed by the chimera NPTH/SECr
(IC50>1000 nM) demonstrate that
domains within the PTH1rs carboxy core must complement the
receptors N-terminal region with respect to hormone binding. To
identify these domains, we generated chimeric receptors by
progressively replacing SECr domains in the chimera NPTH/SECr with the
corresponding domains of PTH1r (sets 1 and 2). Analysis of the
PTH(NOREF>134) binding and signaling properties of these chimeras led to
the following conclusions:
1. The divergent residues (i.e. amino acids that differ
between PTH1r and SECr) present in TM1, TM2, TM4, TM7, the second
extracellular loop, and the carboxy- terminal tail are not important
for PTH1r function (hormone binding and cAMP response). Of course, this
does not preclude a functional role for amino acids in these TM domains
that are conserved between PTH1r and SECr. Indeed, previous studies
(25, 26, 27) have demonstrated that conserved residues in TM2
(e.g. His 222, Ser 226, Arg 230, and Ser 233) and TM7
(e.g. Gln 445) of the PTH1r are determinants of receptor
function (Fig. 1).
2. Analysis of chimeras CRs 4, 5, 10, and 11 indicates that the PTH1rs first- and third extracellular loop participates differentially in receptor activation as well as in high affinity PTH binding.
3. Critical role of TMs 3, 5, and 6 for the overall activation process
of the PTH1r. Analysis of the third set of chimeric receptors, designed
to distinguish the role of the extracellular domains from the TM 3, 5,
and 6 segments on receptor function, suggests that the extracellular
domain and these three TMs act cooperatively to confer high-affinity
binding of PTH. In support of this, introduction of TMs 3, 5, and 6
into the inactive receptor CR13, which contains all of the
extracellular domains of the PTH1r receptor, rescue PTH1rs functional
characteristics (see data for CR12). The present study cannot determine
whether bound PTH is in direct contact with these TM domains of the
receptor, or if the gain of function of CR12 is secondary to allosteric
effect between TMs 3, 5, 6, and the extracellular domains. However,
recent cross-linking studies have demonstrated direct contact of PTH
with amino acid residue Met425 on TM6 of the human PTH1r (corresponding
to Met419 in the opossum PTH1r, Fig. 1) (28).
Although chimera CR15 (introduction of TM5 into CR13) was able to bind PTH with relatively high affinity, it was unable to initiate activation of adenylyl cyclase. Thus, TM components in TMs 3 and 6 in addition to TM5 are required for effective signal transduction in the agonist-occupied receptor. Indeed, the improved ability for PTH to stimulate the adenylate cyclase in chimera CR19 (introduction of TMs 3+6 into CR13; EC50 = 10 nM) compared with chimera CR17 (introduction of TMs 5+6 into CR13; EC50 = 140 nM) and chimera CR18 (introduction of TMs 3+5 into CR13; EC50 = 94 nM) underline the importance of TMs 3 and 6 in the activation process of PTH1r. The importance of TM3 and TM6 in signal transduction has also been reported for a variety of class I GPCRs (29). Studies on rhodopsin have indicated that receptor activation involves the rotation of TM6 and its movement away from TM3 at the cytoplasmic side of the plasma membrane (30), and evidence for a similar conformational shift in the PTH1R has recently been reported (31).
In conclusion, by using a gain-of-function chimeragenesis strategy, we
have delineated PTH1rs region that differentially influence PTH
binding and receptor activation. PTH binding site is formed by residues
scattered throughout the receptors N terminus and first and third
extracellular loops. Although the present results do not establish
whether PTH is in contact with TMs 3, 5, and 6, our data support the
view that helix interactions between these TM domains are critical for
maintaining the PTH1r conformation required for high-affinity PTH
binding. We present a composite diagram in Fig. 7 that indicates the regions of the PTH1r
involved in maintaining high-affinity PTH binding (black
box), and those required to promote receptor signaling
(striped box). One may speculate on a possible mechanism of
activation of the PTH1r in accordance with a two-site model: the N
terminus [residues 1104; deletion of exon 2, which is
residues 62103, has no influence on the function of PTH1r (15)];
exoloops 1 and 3 and TM5 constitute site 1 of the receptor accounting
for an interaction of high affinity with PTH(NOREF>134). As discussed
earlier, it is unclear whether TM5 is a direct site of contact with PTH
or whether it influences binding affinity indirectly. High-affinity
binding to site 1 facilitates the interaction of PTH with a second site
of lower affinity, located in the N-terminal extracellular domain
(residues 104186) and in TMs 3 and 6, to promote signal transduction.
As the receptors first- and third extracellular loops as well as TMs
3 and 6 appear to play an important role in signal transduction, we
suspect that an interaction of PTH with the domains 1e+TM3 and TM6+3e
alters the arrangement of TM3 and TM6, resulting in the activation of
the receptor. These results, together with evolving structural models,
will provide new insights into the molecular basis of hormone-induced
signal transduction for class II GPCRs.
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MATERIALS AND METHODS |
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Construction of Chimeric Receptors and Nomenclature
The opossum PTH1 and rat secretin receptor cDNAs were used for
the construction of the chimeric receptors (32, 33). The construction
of the chimeric receptors NPTH/SECr and NSEC/PTHr was previously
described (17). The cDNAs for the N-terminal extracellular region of
chimeras A [PTH1r(1104)SECr(66142)], B
[SECr(166)PTH1r(106145)SECr(108142)], C
[SECr(1107)PTH1r(146186)], AB
[PTH1r(1145)SECr(108142)], AC
[PTH1r(1104)SECr(66107)PTH1r(146186)], and BC
[SECr(166)PTH1r(106186)] (set 1 in Fig. 2) were engineered by a
recombinant PCR technique using the Pfu DNA polymerase
(Stratagene, La Jolla, CA), as previously described (34).
The resulting amplified products were purified and digested with
HindIII/AccI, and subcloned into the
corresponding sites of the recombinant vector pBS/PTHr, a derivative of
pBluescript that contains the cDNA of the opossum PTH1r. Chimeric
receptors CR1 to CR11 (set 2 and 3 in Fig. 3
) were constructed using
preexisting restriction sites in the receptors cDNAs (Fig. 3A
) and by
splicing restriction endonuclease fragments derived from the wild-type-
or hybrid receptors cDNAs with synthetic oligonucleotide adaptors
ranging in size from 24 to 55 bases, as previously described (35). The
cDNA fragments were ligated into the vector pBluescript
(Stratagene, La Jolla, CA) that had been linearized by
digestion with HindIII and NotI. Chimeric
receptor CR13 was constructed according to a scheme previously
described (6). Chimeric receptors CR12 and CR14 to CR19 (set 4) were
engineered by a recombinant PCR technique previously described (34),
using the Pfu DNA polymerase (Stratagene) and
the CR13 cDNA as a DNA template. Proper construction of the hybrid
cDNAs was confirmed by DNA sequence analysis.
HindIII/NotI fragments of the chimeric cDNAs were
then introduced into the respective sites in pCEP4
(Invitrogen, San Diego, CA), a mammalian expression
vector.
Cell Culture and Transfection
Stable cell lines that selectively express the PTH1r, the
secretin receptor, or the chimeric receptors were established in the
human embryonic kidney cell line 293-EBNA (HEK-293). These cells were
grown in DMEM supplemented with 10% FCS and 1%
penicillin/streptomycin at 37 C in 5% CO2
atmosphere. At 18 h before transfection, the cells were split into
75-cm2 flasks at 30% confluency. Cells were
transfected using the calcium phosphate precipitation method with
24 h incubation in the presence of 10 µg of plasmid DNA per
flask. Two days after transfection, selection of stably transfected
cells was initiated by the addition of hygromycin (200 µg/ml).
Selection was generally complete after 34 weeks of hygromycin
treatment. Stock cell lines were cultured in the continuous presence of
hygromycin, except when subcultured for experiments in which case
hygromycin was omitted.
Competitive Radioligand Binding
The transfected cells were split into six-well culture plates at
a density of 106 cells per well. Twenty four to
48 h later, they were incubated with DHB (DMEM containing 20
mM HEPES and 0.1% BSA) for 2 h at 4 C and then
incubated in 1 ml DHB with 100,000 cpm
125I-hPTHrP(NOREF>134)amide or
125I-secretin ( 0.1 nM) and
various concentrations of bPTH(NOREF>134) or porcine secretin at 4 C for
2 h. After extensive washes with ice-cold PBS, cells were lysed
with 1 ml 0.8 N NaOH, and cell-bound radioactivity was
determined by a
-counter.
Adenylyl Cyclase Activity Determination
Intracellular cAMP accumulation was assessed in transfected
cells split in 12-well plates and incubated for 10 min at room
temperature in 0.5 ml DHB containing 1 mM IBMX and various
concentrations of bPTH(NOREF>134) or porcine secretin. Cells were washed
twice with ice-cold PBS, and cAMP was extracted with 1 ml of 100%
ethanol and measured by RIA (36).
Determination of Cell Surface Receptor Expression
Transfected cells cultured in 12-well plates were incubated at
room temperature for 2 h with 1 ml DMEM containing 5%
heat-denatured FCS in the presence of 1.5 µg/ml mouse monoclonal
antibody OK-1. Cells were washed three times with PBS and incubated for
2 h at room temperature with 200,000 cpm/ml of
125I-labeled goat anti-mouse Ig (Amersham Pharmacia Biotech). After three washes with ice-cold PBS, cells
were lysed with 1 ml 0.8 N NaOH, and cell-bound
radioactivity was determined. No specific binding was detected with
mock-transfected HEK-293 cells. Specific antibody binding for the wild
type PTH1r was 7.5%/well of added
125I-labeled goat antimouse Ig. Specific binding
was normalized to the cell number.
Data Analysis
Dose-response curves were analyzed by computer-assisted
nonlinear regression GraphPad software (GraphPad Software, Inc., San Diego, CA), using the logistic equation: effect =
basal +
Emax.[A]/([A]+EC50)
where Emax is the maximal cAMP accumulation,
[A] is the concentration of agonist, and EC50
is the agonist concentration responsible for 50% of the
Emax. Binding isotherms were fit to a one-site
competitive binding curve; the equilibrium dissociation constants
(Kd) and receptor numbers were obtained with
GraphPad software. The inhibition constant Ki was
calculated according to Cheng and Prusoffs equation (37). The data
were calculated with the assumption of one-to-one stoichiometry of
agonist to receptor and a homogeneous distribution of receptors in the
cells. All experiments were replicated at least three times in
independent experiments, and the results are expressed as mean ±
SEM.
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FOOTNOTES |
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This work was supported by funds from the Medical Research Service of the Department of Veterans Affairs, and from Lilly Research Laboratories. Dr. Nissenson is a Research Career Scientist of the Department of Veterans Affairs.
Received for publication February 5, 2001. Revision received March 19, 2001. Accepted for publication March 21, 2001.
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