Dimerization and Phosphorylation of Thyrotropin-releasing Hormone Receptors Are Modulated by Agonist Stimulation

Zhu, Chenming; Cook, Lloyd Allen; Hinkle, Patricia M.

doi:10.1074/jbc.m204221200

Cited by 46 publications

(45 citation statements)

References 71 publications

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“…When GRK2 was overexpressed, TRH-dependent phosphorylation was increased (Fig. 6A), confirming previous evidence that TRH receptor phosphorylation is primarily because of GRKs (14,17,20). Multiple lines of evidence show that the TRH-dependent phosphorylation observed with the coexpression of D71A and 4Ala-TRH receptors is not because of second messenger-activated kinases.…”

Section: Resultssupporting

confidence: 87%

“…Construction of the TRHR-FKBP-HA, 2HA-TRH receptor with double N-terminal HA epitopes and 2Flag-TRH receptor with prolactin signal sequence and double N-terminal Flag epitopes has been described (14,16). D71A and D71A/S355A mutant receptors were made from 2Flag-TRH receptors and 4Ala (S355A/S360A/S364A/T365A) mutant receptors from 2HA-TRH receptor by using the QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA).…”

Section: Methodsmentioning

confidence: 99%

“…Inhibitors of receptor homodimerization and dimerization-defective mutants are not available for most GPCRs, including the TRH receptor. Although dimerization of the TRH receptor has been demonstrated by several approaches and TRH has been found to increase the fraction of receptors behaving as dimers (14,15), the physiological ratio of the monomer:dimer:higher oligomer in cells is unknown. Solubilized TRH receptors run at the size of monomers and dimers on SDS/PAGE, and receptors with different epitope tags coimmunoprecipitate when coexpressed (14).…”

Section: T He Thyrotropin (Ish)-releasing Hormone (Trh) Receptormentioning

confidence: 99%

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Dimerization of the thyrotropin-releasing hormone receptor potentiates hormone-dependent receptor phosphorylation

Song

Jones

Hinkle

2007

Proc. Natl. Acad. Sci. U.S.A.

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The G protein-coupled thyrotropin (TSH)-releasing hormone (TRH) receptor forms homodimers. Regulated receptor dimerization increases TRH-induced receptor endocytosis. These studies test whether dimerization increases receptor phosphorylation, which could potentiate internalization. Phosphorylation at residues 355-365, which is critical for internalization, was measured with a highly selective phospho-site-specific antibody. Two strategies were used to drive receptor dimerization. Dimerization of a TRH receptor-FK506-binding protein (FKBP) fusion protein was stimulated by a dimeric FKBP ligand. The chemical dimerizer caused a large increase in TRH-dependent phosphorylation within 1 min, whereas a monomeric FKBP ligand had no effect. The dimerizer did not alter phoshorylation of receptors lacking the FKBP domain. Dimerization of receptors containing an N-terminal HA epitope also was induced with anti-HA antibody. Anti-HA IgG strongly increased TRH-induced phosphorylation, whereas monomeric Fab fragments had no effect. Anti-HA antibody did not alter phosphorylation in receptors lacking an HA tag. Furthermore, two phosphorylation-defective TRH receptors functionally complemented one another and permitted phosphorylation. Receptors with a D71A mutation in the second transmembrane domain do not signal, whereas receptors with four Ala mutations in the 355-365 region signal normally but lack phosphorylation sites. When D71A-and 4Ala-TRH receptors were expressed alone, neither underwent TRH-dependent phosphorylation. When they were expressed together, D71A receptor was phosphorylated by G protein-coupled receptor kinases in response to TRH. These results suggest that the TRH receptor is phosphorylated preferentially when it is in dimers or when preexisting receptor dimers are driven into microaggregates. Increased receptor phosphorylation may amplify desensitization. T he thyrotropin (ISH)-releasing hormone (TRH) receptorbelongs to the superfamily of seven transmembrane helix G protein-coupled receptors (GPCRs). TRH is a tripeptide that functions as a hormone regulating TSH and prolactin secretion. TRH receptors signal through G q and G 11 , leading to the production of inositol (1, 4, 5) triphosphate and the release of intracellular calcium. After TRH binds, the TRH receptor is rapidly desensitized (1). Desensitization of GPCRs is initiated when receptors are phosphorylated by G protein-coupled receptor kinases (GRKs) or second messenger-activated kinases. Phosphorylated receptors recruit ␤-arrestins and, as a result, become uncoupled from G proteins. Once docked on activated GPCRs, ␤-arrestins also bind to clathrin and adapter proteins, causing receptor internalization (2).A growing body of evidence suggests that many GPCRs, including the TRH receptor, form dimers or oligomers (3-6). Atomic force microscopy reveals higher order oligomers of rhodopsin (7). Receptor heterodimerization also has been shown to modulate ligand binding, signaling, and trafficking. Ligandbinding properties are altered by heterodimerization of ...

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Section: Resultssupporting

confidence: 87%

Section: Methodsmentioning

confidence: 99%

Section: T He Thyrotropin (Ish)-releasing Hormone (Trh) Receptormentioning

confidence: 99%

See 1 more Smart Citation

Dimerization of the thyrotropin-releasing hormone receptor potentiates hormone-dependent receptor phosphorylation

Song

Jones

Hinkle

2007

Proc. Natl. Acad. Sci. U.S.A.

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“…The HA-tagged TRH receptor is described in ref. 23. Peptides and rabbit antibodies to MRAP were prepared by New England Peptide.…”

Section: Methodsmentioning

confidence: 99%

Melanocortin-2 receptor accessory protein MRAP forms antiparallel homodimers

Sebag

Hinkle

2007

Proc. Natl. Acad. Sci. U.S.A.

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154

200

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The melanocortin-2 (MC2) receptor accessory protein (MRAP) is required for trafficking of the G protein-coupled MC2 receptor to the plasma membrane. The mechanism of action and structure of MRAP, which has a single transmembrane domain, are unknown. Here, we show that MRAP displays a previously uncharacterized topology. Epitopes on both the N-and C-terminal ends of MRAP were localized on the external face of CHO cells at comparable levels. Using antibodies raised against N-and C-terminal MRAP peptides, we demonstrated that both ends of endogenous MRAP face the outside in adrenal cells. Nearly half of MRAP was glycosylated at the single endogenous N-terminal glycosylation site, and over half was glycosylated when the natural glycosylation site was replaced by one in the C-terminal domain. A mutant MRAP with potential glycosylation sites on both sides of the membrane was singly but not doubly glycosylated, suggesting that MRAP is not monotopic. Coimmunoprecipitation of differentially tagged MRAPs established that MRAP is a dimer. By selectively immunoprecipitating cell surface MRAP in one or the other orientation, we showed that MRAP homodimers are antiparallel and form a stable complex with MC2 receptor. In the absence of MRAP, MC2 receptor was trapped in the endoplasmic reticulum, but with MRAP, the MC2 receptor was glycosylated and localized on the plasma membrane, where it signaled in response to ACTH. MRAP acted specifically, because it did not increase surface expression of other melanocortin, ␤2-adrenergic, or TSH-releasing hormone receptors. MRAP is the first eukaryotic membrane protein identified with an antiparallel homodimeric structure.ACTH ͉ G protein-coupled receptor ͉ membrane ͉ orientation T he G protein-coupled receptor (GPCR) family is the largest group of membrane proteins in the human genome. GPCRs respond to a wide array of signals and regulate numerous intracellular signaling pathways. They are characterized by an extracellular amino terminus that is usually glycosylated, seven transmembrane domains, and a cytoplasmic carboxyl terminus. After synthesis in the endoplasmic reticulum, GPCRs must move to the plasma membrane before they can signal in response to extracellular ligands.In the adrenal gland, the melanocortin-2 (MC2) receptor, also referred to as the corticotropin (ACTH) receptor, is activated by the pituitary hormone ACTH and promotes glucocorticoid biosynthesis. The MC2 receptor is expressed primarily in the adrenal cortex and is positively coupled to adenylyl cyclase. The MC2 receptor is a member of the class A rhodopsin-like family and the smallest known GPCR. Individuals harboring inactivating mutations in the MC2 receptor suffer from familial glucocorticoid deficiency, or hereditary unresponsiveness to ACTH (1). Recently, it was discovered that familial glucocorticoid deficiency can also arise from mutations in an accessory protein required for ACTH signaling, the MC2 receptor accessory protein (MRAP) (2). Lack of functional MRAP, like the lack of an MC2 receptor, causes ACTH re...

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“…The phosphorylation by CK2 can affect transcription factors by changing the DNA-binding activity as it is observed for c-Jun 25 and Sp1, 26 by modulating their transcriptional activities such as for MyoD, 27 HIV-1, 22 or IRF-1, 28 and by affecting protein stability, as observed for IjBa, 29,30 PTEN 31 and connexin 45.6. 32 In this work, by using classical selective inhibitors of this kinase such as apigenin, [33][34][35][36] DRB (5,6-dichloro-1-b-D-ribofuranosylbenzimidazole) 22,37,38 and TBB (4,5,6,7-tetrabromobenzotriazole), [39][40][41] we investigated the effect of hypoxia on CK2 activity and the role that this kinase may play to regulate HIF-1 activity.…”

mentioning

confidence: 99%

Role for casein kinase 2 in the regulation of HIF‐1 activity

Mottet

Ruys

Demazy

et al. 2005

Intl Journal of Cancer

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Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that plays a major role in cellular adaptation to hypoxia. The mechanisms regulating HIF-1 activity occurs at multiple levels in vivo. The HIF-1a subunit is highly sensible to oxygen and is rapidly degraded by the proteasome 26S in normoxia. Activation in hypoxia occurs through a multistep process including inhibition of HIF-1a degradation, but also increase in the transactivation activity of HIF-1. Several data indicate that phosphorylation could play a role in this regulation. In this report, we investigated the role of casein kinase 2 (CK2), an ubiquitous serine/ threonine kinase, in the regulation of HIF-1 activity. Hypoxia was capable of increasing the expression of the b subunit of CK2, of inducing a relocalization of this subunit at the plasma membrane, of inducing nuclear translocation of the a subunit and of increasing CK2 activity. Three inhibitors of this kinase, DRB (5,6-dichloro-1-b-D-ribofuranosyl-benzimidazole), TBB (4,5,6,7-tetrabromotriazole) and apigenin, as well as overexpression of a partial dominant negative mutant of CK2a, were shown to inhibit HIF-1 activity as measured by a reporter assay and through hypoxiainduced VEGF and aldolase expression. This does not occur at the stabilization process since they did not affect HIF-1a protein level. DNA-binding activity was also not inhibited. We conclude that CK2 is an important regulator of HIF-1 transcriptional activity but the mechanism of this regulation remains to be determined. Since HIF-1 plays a major role in tumor angiogenesis and since CK2 has been described to be overexpressed in tumor cells, this new pathway of regulation can be one more way for tumor cells to survive. ' 2005 Wiley-Liss, Inc.Key words: casein kinase 2; HIF-1; neoangiogenesis HIF-1 (hypoxia-inducible factor-1) is a key regulator of cell response to reduced oxygen level. In response to hypoxic conditions, HIF-1 increases the expression of downstream target genes such as erythropoietin (EPO), vascular endothelial growth factor (VEGF) and glycolytic enzymes (aldolase A, enolase-a) and mediates adaptation of cells to decreased oxygen level. 1 The role of HIF-1 in the regulation of tumor growth by hypoxia via the initiation of angiogenesis is certainly the best example of such an adaptive response. 2 Oncogene activation and tumor-suppressor inactivation result in deregulated cellular proliferation. However, most tumors grown larger than 1 mm 3 contain regions of low oxygen tension (hypoxia) due to an imbalance between oxygen supply and consumption. Formation of new blood vessels or ''neoangiogenesis'' is thus essential for further tumor growth. It is also important to allow tumor cell dissemination at distant sites, i.e., metastasis.Several studies using HIF-1 mutant cells have shown that HIF-1 has a profound effect on tumor biology. For example, tumors grown from HIF-1a-defective embryonic stem cells display abnormal vascularity and reduced growth rate. 3 Moreover, HIF-1 is upregulated in a broad r...

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Dimerization and Phosphorylation of Thyrotropin-releasing Hormone Receptors Are Modulated by Agonist Stimulation

Cited by 46 publications

References 71 publications

Dimerization of the thyrotropin-releasing hormone receptor potentiates hormone-dependent receptor phosphorylation

Dimerization of the thyrotropin-releasing hormone receptor potentiates hormone-dependent receptor phosphorylation

Melanocortin-2 receptor accessory protein MRAP forms antiparallel homodimers

Role for casein kinase 2 in the regulation of HIF‐1 activity

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