(Received for publication, October 27, 1994; and in revised form, February 6, 1995)
From the
The functional role of the rat parathyroid
hormone(PTH)/PTH-related peptide (PTHrP) receptor's
carboxyl-terminal region was characterized by comparing the binding and
signaling properties of receptors that have 78 and 111 amino acid
deletions (R513 and R480, respectively), with those of the 591-amino
acid wild-type (WT) receptor. R480 and R513 have 4- and 1.5-fold lower
apparent K values for rat
PTH-(1-34) (rPTH), compared with the WT receptor (WT, 1.81
± 0.19 nM; R513, 1.24 ± 0.12 nM; R480,
0.48 ± 0.05 nM, mean ± S.E.). PTH (100
nM)-stimulated cAMP accumulation and polyphosphoinositide
hydrolysis both correlated positively with receptor expression.
However, whereas PTH-stimulated polyphosphoinositide hydrolysis was
indistinguishable among WT and either truncated mutant at comparable
levels of expressed receptors, maximal PTH-stimulated cAMP accumulation
was 4-6- and 2-3-fold higher in cells expressing R480 and
R513, respectively. Furthermore, pretreatment of COS-7 cells with 100
ng/ml of pertussis toxin (PTX) enhanced PTH-stimulated cAMP
accumulation in cells expressing the WT receptor, but failed to do so
in cells expressing either R480 or R513. Thus, sequences in the
PTH/PTHrP receptor's carboxyl-terminal tail lower the affinity of
the WT receptor for agonist; directly interact with, or indirectly
facilitate the interaction of the receptor with a PTX-sensitive G
protein that inhibits adenylyl cyclase; and decrease the efficacy with
which the receptor interacts with G
.
The parathyroid hormone (PTH)()/PTH-related peptide
(PTHrP) receptor (1, 2) belongs to an unique family
within the seven membrane-spanning guanine-nucleotide regulatory
protein (G protein)-coupled receptor superfamily. This family includes
mammalian receptors for calcitonin (3) , secretin(4) ,
glucagon(5) , glucagon-like peptide-1(6) , growth
hormone-releasing hormone(7) , vasoactive intestinal
peptide(8) , vasoactive intestinal peptide-2(9) ,
pituitary adenylyl cyclase-activating peptide(10) , gastric
inhibitory peptide(11) , and corticotrophin-releasing
factor(12) . Additionally, an insect diuretic hormone receptor (13) and a partial genomic sequence from Caenorhabditis
elegans(14) are homologous with the PTH/PTHrP receptor,
indicating that this newly discovered family is widely conserved
through evolution.
In primary and transformed cells derived from
bone and kidney, the agonist-occupied PTH/PTHrP receptor activates
multiple intracellular effectors, including adenylyl cyclase and
phospholipase C(15, 16) . Activation of phospholipase
C results in rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate,
which generates two second messengers, inositol 1,4,5-triphosphate
(IP) and
diacylglycerol(17, 18, 19) . IP
increases intracellular free calcium
([Ca
]
) by stimulating
its release from the endoplasmic
reticulum(20, 21, 22) , whereas
diacylglycerol activates protein kinase
C(23, 24, 25) . There also is evidence that
PTH stimulates arachidonic acid metabolism(26) , changes
membrane potentials(27) , and decreases intracellular
pH(28) .
The goal of the present study was to analyze the
role of the carboxyl-terminal tail of the PTH/PTHrP receptor in
activating adenylyl cyclase and phospholipase C, by comparing the
properties of R480 and R513, truncated rat PTH/PTHrP receptor mutants
with carboxyl-terminal deletions of 111 and 78 amino acids, with those
of the wild-type (WT) rat receptor, which is 591 amino acids in length.
R480 has been shown to contain the minimal length necessary for full
ligand binding, whereas R513 is the rat receptor equivalent of OK-H, a
truncated version of the opossum PTH/PTHrP receptor (OK-O) studied
previously (29) . When these three PTH/PTHrP receptors were
expressed at closely similar cell surface densities, PTH stimulation of
these truncated receptors strikingly increased cAMP accumulation
without changing their capacities to stimulate polyphosphoinositide
(PI) hydrolysis, compared with the WT receptor. The PTH/PTHrP
receptor's carboxyl-terminal intracellular region contains
domains that lower the binding affinity of the WT receptor for agonist,
directly interact with or indirectly facilitates the interaction of the
receptor with a pertussis toxin (PTX)-sensitive G protein that inhibits
adenylyl cyclase, and decreases the efficacy with which the receptor
couples to G
Figure 1: Schematic depiction of the 591-amino acid WT rat PTH/PTHrP receptor and R513 and R480, mutant receptors with 78- and 111-amino acid truncations of their carboxyl-terminal regions. Extracellular (EC) and intracellular (IC) regions are indicated
Figure 2:
Time course of PTH-stimulated cAMP
accumulation and PI hydrolysis in COS-7 cells expressing the WT rat
PTH/PTHrP receptor. COS-7 cells (10 cells/150-mm dish) were
transfected with 5 µg of DNA, replated into 24-well plates, and
incubated with PTH (100 nM) or vehicle alone, under conditions
described under ``Experimental Procedures.'' Intracellular
cAMP accumulation (A) and (B) IP
(
),
IP
(
), and IP
(
) were measured at
37 °C from 1 to 60 min. Experiments were conducted in triplicate,
and results were calculated after subtraction of basal values, which
were obtained in the absence of hormone. Data (mean ± S.E.) are
from one of two experiments, both of which had closely similar results.
When not shown, S.E. values are so small that they fall within the
symbols.
We first characterized the full-length WT receptor's
capacities to bind ligands and antireceptor antibody and to stimulate
adenylyl cyclase and phospholipase C. In COS-7 cells transiently
expressing the WT receptor (2-3 10
receptors/transfected cell), the apparent K
for
I-PTHrP binding was 2.1 ± 0.3
nM, whereas EC
values for cAMP accumulation and
PI hydrolysis were 0.3 ± 0.1 nM and 4.0 ± 0.2
nM, respectively (Fig. 3; since the PI hydrolysis
response was not quite maximal at 10
M PTH,
this EC
is an approximation). Significant stimulation of
cAMP accumulation was detected in cells treated with as low as
10
M PTH, a concentration that did not
detectably displace the radioactive ligand. PTH-stimulated PI
hydrolysis, however, was substantially less sensitive than
PTH-stimulated cAMP accumulation; it was first detected at ligand
concentrations between 10
and 10
M. When the two second messenger responses are
normalized by setting maximal responses to 100%, lower levels of
receptor occupancy elicited relatively greater increases in cAMP
accumulation, compared with the level of receptor occupancy needed for
similar increases in PI hydrolysis.
Figure 3:
Dose-response relationship of PTH binding
and PTH-stimulated cAMP accumulation and PTH-stimulated PI hydrolysis
in COS-7 cells expressing the WT rat PTH/PTHrP receptor. COS-7 cells
(10/150-mm dish) were transfected with 5 µg of DNA,
replated, and incubated with PTH (10
to
10
M) or vehicle alone, under conditions
described under ``Experimental Procedures.'' Radioreceptor
assays were performed with
I-PTHrP, at 16 °C for 4 h.
Specific binding (
) was calculated by subtracting binding in the
presence of PTH (1 µM) from total binding and expressed as
a percentage of specific binding in the absence of unlabeled rPTH;
cyclic AMP accumulation (
) and total IPs (
) generated were
measured after 15 and 30 min, respectively, and are expressed as a
percentage of maximally stimulated values. Data (mean ± S.E.)
are from seven radioreceptor assays, and three bioassays each for cAMP
and IP metabolites, all of which were performed in triplicate. When not
shown, S.E. values are so small that they fall within the
symbols.
Cell surface expression markedly differed among the mutant and WT receptors, when the same amount (5 µg) of plasmid DNA was used for transfection. Since we sought to study the properties of these receptors at similar expression levels, we first determined the levels of cell surface expression for each receptor, after transfecting COS-7 cells with varying amounts of plasmid DNA (0.05-5 µg of WT and 0.1-10 µg of R513 and R480). The number of PTH binding sites per cell, as calculated from Scatchard analysis, was dependent on the amount of introduced plasmid DNA, but markedly differed among these receptor constructs (Fig. 4A). Levels of receptor expression comparable with those obtained with the WT receptor could only be achieved by transfecting the COS-7 cells with higher amounts of either truncated receptor construct.
Figure 4:
The
relationship between the amount of plasmid DNA used for transfecting
COS-7 cells and the number of expressed receptors. COS-7 cells were
transfected with varying amounts of plasmid DNA (0.05-10 µg)
containing WT () or truncated mutant receptors R513 (
) and
R480 (
). A, the number of receptors per transfected
cell, as calculated by Scatchard analysis. B, the specific
I-labeled goat anti-rabbit IgG bound was determined,
after the cells were incubated sequentially with sheep anti-receptor
antibody, G48, and rabbit anti-sheep IgG (see ``Experimental
Procedures''). Data (mean ± S.E.) are from one of four
representative experiments, all of which were performed in triplicate.
When not shown, S.E. values are so small that they fall within the
symbols.
To further characterize the relationship between cell surface expression of the three receptors and the amount of the transfected plasmid DNA, we assessed whether changes in cell surface expression were due to variation in the number of cells expressing receptors or to variations in the number of receptors expressed per cell. When COS-7 cells were transfected with 0.1 or 5 µg of plasmid DNA containing the WT receptor construct, there was no significant difference in the percentage of COS-7 cells that stained positively by immunofluorescence (Table 1). In all experiments, the transfection efficiency for the WT receptor construct in COS-7 cells was consistently 17-20%, regardless of the amount of DNA used, and was indistinguishable from the transfection efficiency for either mutant receptor construct (Table 1). Thus, the amount of cDNA introduced determined PTH/PTHrP receptor's cell surface expression per cell, but did not influence the number of transfectable cells in this transient expression system. To match the expression level of each receptor, we varied the amount of DNA used for transfection, while maintaining a ratio of plasmid DNA for R591/R513/R418 of 1/25/50, respectively. The results from G48 antireceptor antibody binding confirmed observations based on Scatchard analysis (Fig. 4B).
Radioreceptor assays were
conducted in triplicate for each construct, over a range of 5
10
to 10
receptors/transfected cell. Scatchard
analysis showed that the apparent K
values of
rPTH-(1-34) for WT, R513, and R480 receptors were 1.81 ±
0.19 nM (n = 17), 1.24 ± 0.12 nM (n = 11), and 0.48 ± 0.05 nM (n = 13), respectively, and were consistent within
each group over this level of receptor expression. All were
statistically different from each other: WT versus R513 (p < 0.05), WT versus R480 (p < 0.001), R513 versus R480 (p < 0.01).
Next, we correlated the level of receptors expressed with the activated receptor's capacity to stimulate cAMP accumulation and PI hydrolysis. Ligand binding, cAMP accumulation, and PI hydrolysis were always compared in cells from the same transfection. PTH (100 nM)-stimulated PI hydrolysis in COS-7 cells expressing WT, R513 (Fig. 5A), and R480 receptors (Fig. 6A) was linearly dependent upon the level of receptor expression and was indistinguishable among the WT and the two truncated receptors ( Fig. 5and Fig. 6). In contrast, although PTH-stimulated cAMP accumulation also correlated with the level of receptor expression, it was 2-4- and 4-6-fold higher in the cells expressing R513 (Fig. 5B) and R480 (Fig. 6B), respectively, compared with the WT receptor, at all levels of receptor expression.
Figure 5:
The
signal transduction properties of the WT rat PTH/PTHrP receptor
compared with R513, as a function of the number of receptors expressed.
COS-7 cells were transfected with varying amounts of plasmid DNA
containing either WT () or mutant R513 (
) receptors,
radioreceptor assays were then performed, and the number of receptors
expressed per transfected cell was determined by Scatchard analysis.
The capacity of COS-7 cells expressing these receptors to generate IP
metabolites (A) and to increase intracellular cAMP (B) in response to treatment with PTH (100 nM) then
was determined, as described under ``Experimental
Procedures.'' Data (mean ± S.E.) are from one of three
representative experiments, all of which were performed in triplicate.
When not shown, S.E. values are so small that they fall within the
symbols.
Figure 6:
The
signal transduction properties of the WT rat PTH/PTHrP receptor
compared with R480, as a function of the number of receptors expressed.
COS-7 cells were transfected with varying amounts of plasmid DNA
containing either WT () or mutant R480 (
) receptors,
radioreceptor assays were then performed, and the number of receptors
expressed per transfected cell was determined by Scatchard analysis.
The capacity of COS-7 cells expressing these receptors to generate IP
metabolites (A) and to increase intracellular cAMP (B) in response to treatment with PTH (100 nM) then
was determined, as described under ``Experimental
Procedures.'' Data (mean ± S.E.) are from one of three
representative experiments, all of which were performed in triplicate.
When not shown, S.E. values are so small that they fall within the
symbols.
We then assessed the
dose-response relationships for PTH-stimulated cAMP accumulation in
COS-7 cells expressing comparable levels of WT, R513, and R480
receptors. To achieve these levels, we transfected cells with 0.1, 2.5,
and 5.0 µg of plasmid DNA containing WT, R513, and R480 receptor
constructs, respectively. These transfections resulted in expression of
620,000, 505,000, and 520,000 per cell for WT, R513, and R480
receptors, respectively, as determined by Scatchard analysis. Maximal
cAMP stimulation was 2- and 6-fold higher in COS-7 cells expressing
R513 and R480, respectively, than in those expressing the WT receptor (Fig. 7); however, the EC values of PTH-stimulated
cAMP accumulation were similar for the three constructs.
Figure 7:
PTH-stimulated intracellular cAMP response
of COS-7 cells expressing comparable levels of WT and mutant receptors.
PTH-stimulated intracellular cAMP accumulation was determined, over a
hormonal concentration range of 10 to
10
M, in COS-7 cells transfected with
varying amounts of plasmid DNA (see ``Experimental
Procedures'' for details). As determined by Scatchard analysis,
similar levels of receptors per transfected cell were expressed (WT,
620,000; R513, 505,000; R480, 520,000), when COS-7 cells were
transfected with 0.1, 2.5, and 5 µg of plasmid DNA, respectively.
Data (mean ± S.E.) are from one of three representative
experiments, all of which were performed in triplicate. When not shown,
S.M. values are so small that they fall within the
symbols.
We and
others have reported previously that pretreatment of a rat
osteoblast-like osteosarcoma cell line, ROS
17/2.8(38, 39) , and human osteoblasts in primary
culture (40) with PTX augmented PTH-stimulated cAMP
accumulation, without any significant alteration of the basal cAMP
levels. These results suggested coupling of the native PTH receptor to
a PTX-sensitive inhibitory G protein, putatively a member of the
G family. Since truncated receptors had higher maximal
PTH-stimulated cAMP accumulation than WT receptors, we tested the
hypothesis that a putative PTX-sensitive G
protein might
couple to the WT receptor, but not to the truncated receptors. COS-7
cells expressing similar levels of WT and truncated (R513 or R480)
PTH/PTHrP receptors (5-6
10
binding
sites/cell) were treated with 100 ng/ml PTX for 14 h before stimulating
the cells with 10
to 10
M PTH. PTX augmented PTH-stimulated cAMP production by 2-fold at all
concentrations of PTH in cells expressing WT receptors, but it failed
to alter PTH-stimulated cAMP accumulation in cells expressing either
mutant receptor (Fig. 8).
Figure 8:
The
effect of PTX pretreatment on PTH-stimulated cAMP accumulation in COS-7
cells expressing WT (left), R513 (middle), and R480 (right) receptors. COS-7 cells were transfected with varying
amounts of plasmid DNA containing either WT or the two mutant
receptors, radioreceptor assays were then performed, and the number of
receptors expressed per transfected cell was determined by Scatchard
analysis. Cells expressing similar numbers of WT, R513, and R480
receptors (5-6 10
binding sites/cell) were
treated with PTX (
, 100 ng/ml) or vehicle alone (
) for the
last 14 h in serum-free DMEM medium containing 0.1% BSA, prior to
stimulation with PTH (10
to 10
M). Data (mean ± S.E.) are from one of two
representative experiments, both of which were performed in triplicate.
When not shown, S.E. values are so small that they fall within the
symbols.
Our most striking observation is that rat PTH/PTHrP receptors with truncated carboxyl-terminal, intracellular regions signal adenylyl cyclase with markedly higher efficacy, compared with the WT receptor, but have indistinguishable capacities to hydrolyze PI.
We initially noted lower ligand binding in cells transfected with R480 and R513, compared with the WT receptor, when the same amount of DNA was used for transfection. Because the properties of WT and truncated receptors might be influenced by the levels at which they were expressed, we first sought conditions that matched levels of functional receptors. These preliminary studies demonstrated that the number of receptors expressed per transfected cell was markedly influenced by both the amount of plasmid DNA and by the specific construct used for transfection, but that the percentage of transfected cells was independent of either parameter; that is, each transfected COS-7 cell expressed WT receptors more efficiently over a broad range of added DNA, compared with their expression of either R480 and R513. Careful control of the conditions for COS-7 cell's incubation and transfection and of the amount of plasmid DNA used for transfection enabled us to compare the properties of the truncated and WT receptors at closely similar levels of receptors per cell and over a relatively wide range of expressed receptors.
The magnitude of both maximal
PTH-stimulated cAMP accumulation and PI hydrolysis depended on the
level of cell surface expression in COS-7 cells. The rates of PI
hydrolysis were indistinguishable between either truncated mutant and
WT receptor when cells expressing matched levels of the three receptors
were treated with 100 nM PTH, over a wide range of receptor
levels. In contrast, R480 and R513 increased cAMP accumulation by
5-6- and 2-3-fold, respectively, compared with the WT
receptor expressed at comparable levels, when treated with the same
dose of hormone. These data confirm and extend those of an earlier
observation from our laboratory, which demonstrated that PTH-stimulated
increases in intracellular [Ca] and
increases in the rates of PI hydrolysis correlated directly with the
number of PTH/PTHrP receptors stably expressed in LLC-PK1 cells, a
porcine kidney cell line without endogenous PTH/PTHrP
receptors(22, 41) .
In a previous study(29) , we had noted that a carboxyl-terminally truncated opossum kidney PTH/PTHrP receptor (OK-H) that contains 508 of the 585 amino acids of the full-length receptor (OK-O), and is the opossum homolog of R513, seemed to have unimpaired capacity to activate adenylyl cyclase, but reduced capacity to stimulate the rate of PI hydrolysis. In recent experiments in which receptor expression was carefully controlled, however, we found, instead, that OK-H had an unimpaired capacity to stimulate PI hydrolysis and enhanced capacity to stimulate cAMP accumulation (data not shown). We had misinterpreted our early experiments because we had failed to rigorously control for the number of receptors expressed per cell.
Our observation that the intact, cloned PTH/PTHrP receptor interacts with a PTX-sensitive G protein linked to inhibition of adenylyl cyclase extends earlier observations we and others have made in studies of endogenous receptors on ROS 17/2.8 cells (38, 39) and primary cultures of human osteoblasts(40) , where PTX treatment enhanced intracellular cAMP accumulation, without altering basal levels. Since the PTX effect was seen only with the WT receptor, the receptor's interaction with this inhibitory G protein must rely, directly or indirectly, on the sequence between residues 513 and 591. Future efforts are needed to further identify the domain(s) involved in mediating this function.
Other aspects of our observations also will require additional experiments before they are fully understood. For example, the higher efficacy (maximal cAMP responsiveness) with which R480 activates adenylyl cyclase, compared with the WT receptor, can be attributed in part to the absence of a site that enables the PTH/PTHrP receptor to couple to a PTX-sensitive G protein which inhibits adenylyl cyclase and which is present between residues 513 and 591 of the receptor's sequence. Other factors, however, must also be involved in modulating this response, because PTH treatment of R513 elicits a maximal cAMP response that is lower than the response of R480, although both mutant receptors are insensitive to PTX, but higher than the response of the WT receptor, which is sensitive to PTX.
Second, the higher binding affinities with which R480 and R513 bind
agonist, compared with the WT receptor, would predict lower EC values of both adenylyl cyclase and phospholipase C responses, if
the increased binding energy were associated with a conformation that
activated G proteins. This was not observed with either effector
response; neither the EC
nor the maximal phospholipase C
response was modified, nor was any change noted in the EC
of the adenylyl cyclase response, although the strikingly higher
maximal cAMP accumulation might have made it difficult to appreciate
any change. Taken together, these data suggest that the affinity of
ligand binding to the these receptors can be dissociated from
activation of the two effector pathways. This appears to clearly be the
the case with activation of phospholipase C. With respect to activation
of adenylyl cyclase, these truncated PTH/PTHrP receptors appear to have
higher capacities to increase intracellular cAMP accumulation, compared
with the WT receptor. It is difficult to relate this property directly
to the changes in ligand binding affinity, however, because no change
in the EC
values was noted. Additionally, the different
affinities with which these two truncated receptors bind agonist, when
expressed in COS-7 cells, suggest that multiple intracellular receptor
sequences influence this property. Interestingly, Parker and Ross (42) showed that truncated avian
-adrenergic receptors
activated adenylyl cyclase with higher efficacy than the full-length
receptor. Although these truncated receptors had modestly increased
affinity for ligands, the influence of PTX on the adenylyl cyclase
response was not assessed.
Studies of PTH/PTHrP receptors
transiently expressed in COS-7 cells, however, have limitations as well
as certain advantages. They allow relatively rapid comparisons of
different receptors at various levels of expression, which can be
rigorously controlled at levels above 10 receptors/transfected cell, and they provide a system that allows
the properties of these receptors to be assessed independently without
complicating variables, such as clonality that influence stably
expressed receptors. On the other hand, receptor expression is
difficult to control at more modest levels. Furthermore, the signaling
properties of the PTH/PTHrP receptor necessarily depend on the
particular complement of G proteins and other cytoplasmic factors
present in COS-7 cells. Studies in which PTH/PTHrP receptors are
characterized in the context of a cellular milieu other than COS-7
cells, therefore, are likely to provide additional information.
These results highlight the functional importance of the
carboxyl-terminal region of the PTH/PTHrP receptor, as manifested
mainly by the striking discordance between the increased PTH-stimulated
cAMP accumulation and the unchanged response in PTH-stimulated PI
hydrolysis, when the properties of mutant PTH/PTHrP receptors with
deletions of this region are compared with those of the WT receptor.
Sequences in the PTH/PTHrP receptor's carboxyl-terminal
intracellular region have multiple influences on receptor function:
they lower the apparent affinity of the WT receptor for agonist;
contain domains distal to amino acid 513 that directly interact with,
or indirectly facilitate its interaction with, a PTX-sensitive G
protein, presumably a member of the G family that inhibits
adenylyl cyclase; and they decrease the efficacy (maximal
agonist-stimulated cAMP accumulation) with which the receptor interacts
with G
.