Constitutive Activity of Native Thyrotropin-Releasing Hormone Receptors Revealed Using a Protein Kinase C-Responsive Reporter Gene

Endocrinology, Apr 1997

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 antagonist 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 TRH-Rs. 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.

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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- - 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)


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Jinsi-Parimoo, Arti, Gershengorn, Marvin C.. Constitutive Activity of Native Thyrotropin-Releasing Hormone Receptors Revealed Using a Protein Kinase C-Responsive Reporter Gene, Endocrinology, 1997, pp. 1471-1475, Volume 138, Issue 4, DOI: 10.1210/endo.138.4.5059