Constitutive Activity of Native Thyrotropin-Releasing Hormone Receptors Revealed Using a Protein Kinase C-Responsive Reporter Gene
Constitutive Activity of Native Thyrotropin-Releasing Hormone Receptors Revealed Using a Protein Kinase C- Responsive Reporter Gene
0 Division of Molecular Medicine, Department of Medicine, Cornell University Medical College and The New York Hospital , New York, New York 10021 , USA
The native TRH receptor (TRH-R), which is a G protein-coupled receptor that signals via the phosphoinositide transduction pathway, has been assumed to be inactive in the absence of agonist. In contrast, a mutant mouse TRH-R (C335Stop TRH-R) was shown previously to exhibit constitutive (or agonist-independent) signaling activity. In this report, we measured signaling activity of TRH-Rs using a protein kinase C-responsive reporter gene instead of formation of inositol phosphate second messenger molecules. Using this more sensitive system, we show that native mouse TRH-Rs exhibit agonist-independent signaling activity that is directly proportional to the number of receptors expressed in COS-1 cells and is inhibited by negative an-
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ONSTITUTIVE (or agonist-independent) signaling
activity of G protein-coupled receptors (GPCRs) has
been well documented (
1, 2
). The finding that certain
receptors exhibit constitutive activity has led to a modification of
traditional receptor theory (
3, 4
). It is now thought that
receptors can exist in at least two conformations (or
populations of conformations), inactive (R) and active (R*), and that
an equilibrium exists between these two states that markedly
favors R over R* in the majority of receptors in the absence
of agonist. It has been proposed that there is a shift in
equilibrium in some GPCRs that allows a sufficient number of
receptors to be in the active R* state and initiate signaling in
the absence of agonist. In most instances, these have been
mutated receptors that were produced in the laboratory or
found naturally in certain disease states (
5
). A number of
wild-type (or native) GPCRs have been shown to exhibit
agonist-independent activity (
6 ? 8
). Even with mutant
GPCRs, constitutive activity has been more readily shown
with receptors that couple to the adenylyl cyclase-cAMP
cascade than with receptors that couple to the
phosphoinositide-inositol 1,4,5-trisphosphate (IP3)/diacylglycerol
(DAG) pathway (
9
). In several instances, for example,
receptors for thyroid-stimulating hormone that can signal via
both the cAMP and IP3/DAG pathways, the majority of
constitutively active mutant receptors studied have
exhibited agonist-independent activity with regard to cAMP but
not via IP3/DAG signaling (
10
).
TRH-R is a GPCR that signals via the phosphoinositide
tagonist benzodiazepine drugs. As expected, the basal signaling
activity of native TRH-Rs is lower than C335Stop TRH-Rs. Constitutive
activity of native TRH-Rs is not peculiar to COS-1 cells in which
receptor density is markedly elevated, because it can also be
demonstrated in Madin Darby canine kidney cells stably expressing
mouse TRH-Rs and GH4C1 cells endogenously expressing rat
TRHRs. These findings support the thesis that native TRH-Rs oscillate
between active and inactive states. We suggest that demonstration of
constitutive activity of native receptors may depend on the sensitivity
of the signaling assay employed. (Endocrinology 138: 1471?1475,
1997)
transduction pathway (
11, 12
). In previous studies (
13, 14
),
we found no evidence for constitutive activity of native
TRH-R in experiments in which we measured generation of
inositol phosphate (IP) second messengers. In this report, we
present evidence of agonist-independent signaling activity
via the IP3/DAG pathway of native TRH-Rs using a sensitive
reporter system in which we measure induction of firefly
luciferase via a protein kinase C (PKC)-responsive promoter
(
15
).
Experimental Procedures
Materials
DMEM, HBBS, FBS, and lipofectamine were purchased from GIBCO/
BRL (Grand Island, NY), Nu-serum was from Collaborative Research
(Bedford, MA). [3H]Methyl-TRH was purchased from DuPont New
England Nuclear (Boston, MA), and myo-[3H]inositol was from
Amersham (Arlington Heights, IL). COS-1 were obtained from American
Type Culture Collection (Rockville, MD) and Madin-Darby canine
kidney (MDCK) type II cells were a gift from Dr. Enrique Rodriguez-Boulan
(Cornell University Medical College). Plasmid containing an activating
protein-1 (AP-1)-fos-Luc reporter gene construct was a gift from Dr. Paul
Deutsch (previously of Cornell University Medical College). All other
chemicals were obtained from Sigma Chemical Co. (St. Louis, MO).
Cell culture and transfection
COS-1 cells, GH4C1 cells endogenously expressing rat pituitary
TRHRS, and MDCK type II cells stably expressing mouse TRH-Rs were
grown in DMEM supplemented with 5% Nu-serum (COS-1 and GH4C1
cells) or calf serum (MDCK cells) in a humidified atmosphere of 5% CO2
at 37 C. Cells were seeded in six-well plates at a density of 2?3 3 105/well
on the day before transfection. Cells were transfected using (...truncated)