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Division of Molecular Medicine, Department of Medicine, Cornell University Medical College and The New York Hospital, New York, New York 10021
Address all correspondence and requests for reprints to: Marvin C. Gershengorn, Cornell University Medical College, 1300 York Avenue, New York, New York 10021. E-mail: mcgersh{at}mail.med.cornell.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In general, GPCRs require agonist binding for activation. Constitutive
(or agonist-independent) signaling activity in mutant receptors has
been well documented, but only a few GPCRs have been identified with
agonist-independent activity in the wild-type (or native) form. For
example, native dopamine D1B and PG EP3a receptors, which are members
of the rhodopsin/ß2-adrenergic receptor subfamily, have
been found to possess constitutive activity (8, 9). A number of members
of the rhodopsin/ß2-adrenergic receptor subfamily, for
example, receptors for thyroid-stimulating hormone (10) and LH (11),
have been found to be mutated and exhibit agonist-independent activity
and cause disease in humans. To our knowledge, only the PTH/PTHrP
receptor in the hCTR subfamily has been so implicated (12).
Experimentally, several single-amino acid mutations have produced
agonist independent activity. 
1B, ß2, and
2 adrenergic receptors, for example, mutated at single
sites in the third cytoplasmic loop show constitutive activity (13, 14). In some cases, a large deletion mutation in the carboxyl terminus
tail or in the intracellular loops of GPCRs has led to constitutive
activity. For example, in the TRH receptor, a truncation deletion of
the carboxyl terminus renders the receptor constitutively active (15, 16), and a smaller deletion in the second extracellular loop of the
thrombin receptor causes constitutive activity also (17). The finding
that certain receptors exhibit constitutive activity has led to a
modification of traditional receptor theory (18). It is now thought
that receptors can exist in at least two conformations, an inactive
conformation (R) and an active conformation (R1), and that an
equilibrium exists between these two states that markedly favors R over
R1 in the majority of receptors in the absence of agonist. In some
native receptors and in the mutants described above, it has been
proposed that there is a shift in equilibrium in the absence of agonist
that allows a sufficient number of receptors to be in the active R1
state to initiate signaling.
In this report, we present data of agonist-independent activity of hCTR-1 and hCTR-2 by showing that these two receptor isoforms cause elevation of cAMP and of cAMP-responsive gene transcription in two cell types in culture. Furthermore, we show that mutations in a conserved His residue in the putative intracellular loop one, homologous to a mutation that caused the PTH/PTHrP receptor to be constitutively active in a patient with Jansen-type Metaphyseal Chondrodysplasia (12), does not affect signaling properties of hCTR-2.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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COS-1 cells were grown in 100-mm dishes in DMEM with 5% Nu-Serum in a humidified incubator in an atmosphere of 5% CO2 at 37 C. Diethylaminoethyl-dextran transfections were carried out, as previously described, using receptor-cDNA containing plasmid concentrations between 2 and 5000 ng/ml (6). Liposome-mediated gene transfers were performed by incubating cells in serum-free DMEM for 5 h at 37 C in a plasmid/liposome mixture using 20 mg/ml of a liposome transfection reagent containing a 1:1 ratio of dioleoylphosphatidylethanolamine and N-[1-(2, 3-dioleoyloxy)propyl]-N, N,N- trimethylammonium methylsulfate, prepared by Dr. Anthony Scotto (Cornell University Medical College). Where appropriate, the luciferase construct was transfected using a plasmid concentration of 5 µg/ml. In this procedure, approximately 40,000 cells/well were seeded into 6 multiwell plates and, 24 h later, washed several times with serum-free DMEM before transfection. Immediately after a 5-h incubation, an equal volume of DMEM containing 10% Nu-serum was added to each well for overnight incubation. All binding, stimulation, and luciferase studies were performed 48 h after diethylaminoethyl-dextran transfection and 24 h after liposome transfection. Untransfected Mardin-Darby canine kidney (MDCK) cells (provided by Dr. Enrique Rodriguez-Boulan, Cornell University Medical College) were stably transfected by electroporation with receptor plasmid and the selectable marker in pcDNA1/Neo (Invitrogen, San Diego, CA) by standard techniques (16). Geneticin (G418)-resistant clones were isolated and assessed for receptor expression.
Ligand binding
After transfection, receptor binding was assessed in intact
cells using saturation binding assays as described (21). In brief,
cells (100,000 cells per 12 multiwell) were washed with HBSS containing
25 mM HEPES, pH 7.4 (HBSS). Between 5 and 300
pM 125I-salmon CT (125I-sCT), in
the absence or presence of 1 µM unlabelled sCT (to
determine nonspecific binding), was added to replicate wells, and
binding occurred at 4 C overnight. The binding buffer contained BSA (1
mg/ml), bacitracin (1 mg/ml), and phenylmethylsulfonylfluoride (1
mM). Assays were terminated by aspirating the binding
buffer, washing with chilled HBSS three times, and solubilizing the
cells with 0.4N NaOH. An aliquot of this solution was then counted in a
counter and the data expressed as cpm bound. In some experiments,
binding was measured with a single concentration of
125I-sCT, and nonspecific binding was determined in the
presence of 1 µM unlabelled sCT.
cAMP formation
Eighteen to 24 h after transfection, cells were washed with
HBSS and incubated with HBSS supplemented with 0.5 mM
isobutylmethylxanthine for 30 min at 37 C as described (21). The buffer
was aspirated, cells were washed three times, and then were lysed with
1 ml stop solution (methanol-concentrated HCl-EDTA). Chloroform was
added, and aqueous and lipid phases were separated. An aliquot of the
aqueous phase was dried, redissolved in acetylation buffer, and assayed
by RIA.
IP formation
Eighteen hours after transfection, cells were harvested
and seeded in wells of a 12-multiwell dish in DMEM containing 1
µCi/ml myo-[3H]inositol as described (22). Twenty-four
hours later, mediums were removed, and HBSS containing 10
mM LiCl was added for 30 or 60 min. The incubation was
terminated, cells were lysed as above, and IPs were measured using ion
exchange chromatography as described previously (22). In some
experiments, IPs and cAMP were measured in the same cell lysates.
Luciferase activity
Cells transfected with pCRE-fos-LUC, without or
with the indicated amounts of pFLAG-hCTR-2 or phCTR-2, were incubated
in DMEM in the absence or presence of sCT or a competitive antagonist
of CT action, N-acetyl-sCT-(8–32)amide (23),
for the times indicated as described (19). Cells were washed with PBS
and lysed with 0.5 ml/10 cm dish of lysis buffer (25 mM
GlyGly, 15 mM MgSO4, 4 mM EGTA, 1
mM DTT, and 1% Triton X-100, pH 7.8). Cell extracts were
diluted 1:10 or 1:20 in reaction buffer (25 mM GlyGly, 15
mM MgSO4, 4 mM EGTA, 1
mM DTT, 15 mM KH2PO4, 2
mM ATP, pH 7.8). Luciferase activity assay was started by
combining equal volumes of diluted cell extracts with 0.4
mM luciferin in reaction buffer, and the signal was read
with a luminometer for 10 sec (19) (Monolight 2010, Analytical
Luminescence Laboratory, San Diego, CA).
Data analysis
Binding and dose-response curves were analyzed and statistical
differences determined using GraphPad Prism (GraphPad Software, Inc.,
San Diego, CA). Statistical analyses of other data were by t
test.
Materials
COS -1 cells were obtained from American Type Culture Collection
(Rockville, MD). DMEM, HBSS, and Geneticin, were obtained from
GIBCO/BRL (Grand Island, NY). Mouse anti-FLAG M2 monoclonal antibodies
were from IBI-Kodak (New Haven, CT). Nu-Serum was from Collaborative
Research (Bedford, MA). sCT and N-
acetyl-sCT-(8–32)amide were gifts from Dr. R. Gamse (Sandoz
Corporation, Basel, Switzerland). 125I-cAMP was from
Biomedical Technologies, Inc. (Stoughton, MA). Antibodies directed
against cAMP were obtained from Calbiochem (La Jolla, CA) and goat
antirabbit IgG antibodies were from Arnel Products, Inc. (New York,
NY). All restriction enzymes and sequencing kits were from New England
Biolabs (Beverly, MA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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);
mock-transfected cells, which are cells that were transfected with
plasmid without DNA encoding hCTRs, were not different from
untransfected cells (data not shown). Similar increases in basal cAMP
formation were observed in cells transfected with pFLAG-hCTR-1 or
pFLAG-hCTR-2 (data not shown). In contrast, no increases in basal IP
production were observed in cells expressing either hCTR-1 or hCTR-2.
These findings are consistent with the idea that hCTRs preferentially
couple to cAMP formation (6).
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. There
was a direct relationship between the amount of 125I-sCT
specifically bound and the level of basal cAMP formation vs.
the amount of plasmid used for transfection up to 700 ng/ml. A
statistically significant increase in basal cAMP accumulation was found
with 70 ng/ml of pFLAG-hCTR-2 (P < 0.01); there were
no measurable increases in basal cAMP formation with lower amounts of
plasmid, even though there was expression of hCTR-2. This may reflect
our finding that with the lower amounts of pFLAG-hCTR-2, fewer than 7%
of the cells expressed hCTR-2 (data not shown). There was no difference
in the affinity of specific 125I-sCT binding assessed in
competition binding experiments in cells transfected with all doses of
pFLAG-hCTR-2 (data not shown); the apparent equilibrium inhibitory
constants (Ki) were similar to our previously published
values for hCTR-2 (15 ± 3.4 nM) (6). These data show,
moreover, that FLAG epitope-tagged receptors exhibit a binding affinity
similar to that of wild-type hCTR-2. More importantly, the same
relationship between receptor number and basal formation of cAMP was
observed in cells transfected with phCTR-2 (data not shown). The level
of cAMP production in the absence of agonist was not the maximal level
that could be attained via these receptors, because sCT stimulated a
severalfold increase in cAMP formation over basal; the level of
stimulation varied with the concentration of plasmid transfected (data
not shown).
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(unfilled
bars) illustrates that, like the correlation between increasing
amounts of pFLAG-hCTR-2 plasmid used for transfection and basal cAMP
production, there was a direct relationship between plasmid
concentration and basal luciferase activity. In fact, significant
increases in the level of basal luciferase activity occurred with the
same amount (70 ng/ml) of pFLAG-hCTR-2 as for cAMP production. A
similar relationship was observed in cells transfected with phCTR-2
(data not shown). Thus, the increase in basal cAMP production caused by
hCTR-2 leads to an increase in gene transcription and, therefore, a
cellular response downstream from second messenger generation. As with
cAMP production, the increase in basal cAMP-dependent gene
transcription via the expressed hCTR-2 was not maximal, because
addition of sCT further increased the level of induction (3- to
4-fold).
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-acetyl-sCT-(8–32)amide.
N-acetyl-sCT-(8–32)amide is a competitive
antagonist of CT binding to hCTRs with an apparent equilibrium
inhibition constant of approximately 1 nM (23). In these
experiments, N-acetyl-sCT-(8–32)amide was
present in the medium from the time of transfection, and when
indicated, sCT was added for the last 4 h. There was no effect of
N-acetyl-sCT-(8–32)amide on luciferase
activity (Fig. 3
, hatched bars). Thus, there was no evidence
for a CT-like factor in the medium that activated hCTRs, confirming our
conclusion that these receptors exhibit agonist-independent activity.
Moreover, because there was no inhibition of the unstimulated
cAMP-dependent transcriptional activity in the presence of
N-acetyl-sCT-(8–32)amide, it seems that
N-acetyl-sCT-(8–32)amide is a neutral
antagonist without any negative antagonist (or inverse agonist)
activity. This conclusion was corroborated by a lack of effect of
N-acetyl-sCT-(8–32)amide on the basal rate
of cAMP formation in COS-1 cells expressing hCTRs (data not shown).
To address the possibility that the observed constitutive activity of
hCTRs was peculiar to COS-1 cells, we studied this phenomenon in stably
transfected clonal cell lines. We chose MDCK cells because kidney
tubule cells normally express CT receptors (24). We used two cell lines
that express 18,500 ± 2,700 hCTR-2s/cell or 12,700 ± 2,100
hCTR-1s/cell (data not shown). We chose these lines because the levels
of hCTR expression were similar to those found in cells endogenously
expressing hCTRs, for example, T47D breast cancer cells (25), and much
lower than are found in transiently transfected COS-1 cells (6). MDCK
cells stably transfected with pFLAG-hCTR-1 or pFLAG-hCTR-2 exhibited
greater rates of cAMP production than untransfected MDCK cells or
mock-transfected MDCK cells, which are cells stably transfected with
plasmid without DNA encoding hCTRs (data not shown) and 27-fold and
32-fold increases in luciferase activity, respectively, compared with
untransfected MDCK cells (Fig. 4). Thus, the increases
in agonist-independent activity exhibited by hCTR-1 and
hCTR-2 are found in COS-1 and MDCK cells and are, therefore, not
cell-type specific and do not seem to be dependent on marked receptor
overexpression.
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), a similar direct
relationship between specifically bound 125I-sCT and basal
cAMP production was found with wild-type hCTR-2 and hCTR-2/H184R. No
differences from the wild-type receptor were noted in any of the
mutants with respect to binding or sCT stimulations of cAMP and IPs
(data not shown). Thus, mutations of the highly conserved His in the
first intracellular loop seems to have different effects in different
receptors of the CTR subfamily.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-acetyl-sCT-(8–32)amide (23),
is a neutral antagonist with no inverse agonist activity; that is, it
does not inhibit the constitutive activity of hCTRs (see below).
The constitutive activity of hCTRs was demonstrated with regard to the
Gs protein-adenylyl cyclase-cAMP system. There was no
concomitant stimulation of the Gq
protein-phosphoinositide-specific phospholipase C-inositol 1,4,5-
trisphosphate system. This result is similar to the majority of
observations made with constitutively active GPCRs that signal through
both systems, for example, mutated receptors for TSH (10), LH (11), and
VP (26). Recent modifications of the classical model (by which GPCRs
signal) have been made to accommodate the observation that some
receptors, either mutant or wild-type, do initiate signals
constitutively, that is, in the absence of agonist binding. The
principal change has been to incorporate an equilibrium between R and
R1 conformations of the receptor in the absence of agonist. For GPCRs,
R1 couples to G protein to initiate the signal (18). According to the
revised theory, agonist serves to shift the equilibrium to favor R1
because agonist binds with higher affinity to R1 than to R. Conversely,
a shift in equilibrium favoring R is predicted to occur when receptor
is occupied by a ligand with inverse agonist (or negative antagonist)
activity, that is, a ligand that inhibits the signaling activity of a
constitutively active receptor. No change in the equilibrium between R
and R1 occurs when receptors are bound by neutral antagonists because
neutral antagonists are predicted to bind to both R and R1 with equal
affinity and only inhibit activation by agonists.
N-acetyl-sCT-(8–32)amide was shown to be a
neutral antagonist. The fact that receptors with double signaling
potential usually show agonist-independent activity only with respect
to the cAMP signaling cascade is consistent with the hypothesis
that the affinity of the activated conformer (R1) is greater for
Gs than Gq. Therefore, a small shift in the
equilibrium between R and R1 towards R1, as probably occurs in
constitutively active receptors, will permit activation of a sufficient
number of Gs proteins to increase adenylyl cyclase activity
and elevate cAMP levels but not to activate Gq. The lower
affinity of R1 for Gq would require a much greater shift of
R to R1 to stimulate the phospholipase C-signaling pathway. The
observed 10-fold higher potency of CT in stimulating cAMP production,
compared with IP second messengers (6), is consistent with this
hypothesis.
Constitutive activity of GPCRs is now well established, and there are a number of examples of mutated receptors that acquire this phenotype and cause disease in humans. However, only a relatively small number of native GPCRs have been found to exhibit constitutive activity. Indeed, several authors have pointed out that apparent constitutive activity of wild-type receptors may result from their overexpression in tissue culture (27) or transgenic animals (28, 29). The COS cell system does lead to overexpression of receptors and we, therefore, thought it was important to analyze hCTRs in a cell system that expresses hCTRs at levels that are found in cells that express hCTRs endogenously (25). We think this level may more nearly approximate that found in normal cells in situ. We, therefore, used the MDCK cell system also. We found that both hCTR-1 and hCTR-2 exhibited agonist-independent activity in both MDCK cells stably expressing approximately 15,000 receptors per cell and in COS-1 cells that transiently express as many as 2,000,000 receptors per cell. Thus, we have demonstrated that constitutive activity of hCTRs is not cell-type specific, nor is it a consequence of marked overexpression of receptors.
Previously it was shown that a 16-amino acid insertion in the first intracellular loop of hCTR-1 affects its signaling properties, in that hCTR-1 can activate adenylyl cyclase, leading to formation of cAMP; whereas it can not activate phospholipase C to generate IP second messengers, whereas hCTR-2 can signal through both pathways (3, 6). As a mutation of PTH/PTHrP receptors in a histidine residue that is conserved in the first intracellular loop in all members of the hCTR subfamily of GPCRs produced a constitutively active receptor, we examined whether this residue, His-184 in hCTR-2, also would have an impact on receptor signaling. We found that mutation of His-184 did not affect signaling by hCTR-2. These results demonstrate that even highly conserved specific residues may serve different functions in close members of GPCR subfamilies.
hCTRs have been found in several locations in human tissues, including the ovary, bone, renal tubular cells, and the central nervous system (30). We do not know whether the constitutive activity exhibited by hCTRs has a physiological role in any tissue. Two possibilities, however, can be considered. It is possible that basal hCTR activity, which is not dependent on the presence of CT in the environment of a cell, may be important for modest but sustained activation of cell processes in a specific tissue(s). Another possibility is dependent on the observation that constitutively active, mutant receptors display a lowerfold increase in second-messenger formation than native, inactive receptors and exhibit characteristics that are consistent with them being chronically desensitized and downregulated. These characteristics make receptors that are constitutively active better fitted for ungraded all-or-none types of responses (27). It is possible that ungraded responses to acute exposure to CT are involved in CT action in a specific tissue(s).
In summary, we have shown that hCTRs are constitutively active by
demonstrating that they mediate increased basal cAMP production in
COS-1 and MDCK cells that is of a magnitude sufficient to induce gene
expression. We used this system to categorize the peptide analogue
N-acetyl-sCT-(8–32)amide as a neutral
antagonist of hCTRs. Lastly, we found no changes in the signaling
properties of hCTRs in which a highly conserved His residue in its
first intracellular loop was mutated, even though a similar mutation in
the PTH/PTHrP receptor affected signaling.
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Acknowledgments |
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Footnotes |
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Received October 24, 1996.
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References |
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2-adrenergic
receptor. J Biol Chem 268:16483–164871B-adrenergic receptor in
transgenic mice induces cardiac hypertrophy. Proc Natl Acad Sci USA 91:10109–10113This article has been cited by other articles:
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  A. Couvineau, J.-J. Lacapere, Y.-V. Tan, C. Rouyer-Fessard, P. Nicole, and M. Laburthe Identification of Cytoplasmic Domains of hVPAC1 Receptor Required for Activation of Adenylyl Cyclase: CRUCIAL ROLE OF TWO CHARGED AMINO ACIDS STRICTLY CONSERVED IN CLASS II G PROTEIN-COUPLED RECEPTORS J. Biol. Chem., June 27, 2003; 278(27): 24759 - 24766. [Abstract] [Full Text] [PDF]
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A. M. Cypess, C. G. Unson, C.-R. Wu, and T. P. Sakmar Two Cytoplasmic Loops of the Glucagon Receptor Are Required to Elevate cAMP or Intracellular Calcium J. Biol. Chem., July 2, 1999; 274(27): 19455 - 19464. [Abstract] [Full Text] [PDF]
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 A.-O. Colson, J. H. Perlman, A. Jinsi-Parimoo, D. R. Nussenzveig, R. Osman, and M. C. Gershengorn A Hydrophobic Cluster between Transmembrane Helices 5 and 6 Constrains the Thyrotropin-Releasing Hormone Receptor in an Inactive Conformation Mol. Pharmacol., December 1, 1998; 54(6): 968 - 978. [Abstract] [Full Text]
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 D. R. Nussenzveig, M. D. F. C. Matos, and C. N. Thaw Human calcitonin receptor is directly targeted to and retained in the basolateral surface of MDCK cells Am J Physiol Cell Physiol, November 1, 1998; 275(5): C1264 - C1276. [Abstract] [Full Text] [PDF]
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 E. Geras-Raaka, L. Arvanitakis, C. Bais, E. Cesarman, E. A. Mesri, and M. C. Gershengorn Inhibition of Constitutive Signaling of Kaposi's Sarcoma-associated Herpesvirus G Protein-Coupled Receptor by Protein Kinases in Mammalian Cells in Culture J. Exp. Med., March 2, 1998; 187(5): 801 - 806. [Abstract] [Full Text] [PDF]
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 E. Schipani, G. S. Jensen, J. Pincus, R. A. Nissenson, T. J. Gardella, and H. Jüppner Constitutive Activation of the Cyclic Adenosine 3',5'-Monophosphate Signaling Pathway by Parathyroid Hormone (PTH)/PTH-Related Peptide Receptors Mutated at the Two Loci for Jansen's Metaphyseal Chondrodysplasia Mol. Endocrinol., June 1, 1997; 11(7): 851 - 858. [Abstract] [Full Text]
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) and
pFLAG-hCTR-2/H184R (
), in which His at position-184 was changed to
Arg, amounts ranged from 2–2000 ng/ml. A single regression line
defined the direct relationship between specific 125I-sCT
binding and cAMP production in both cell populations. These data are
from a representative of four experiments.