Cloning and expression of the thyrotropin-releasing hormone receptor from GH3 rat anterior pituitary cells

Functional thyrotropin-releasing hormone (TRH) receptors have been expressed in Xenopus laevis oocytes following the microinjection of total and poly(A)+ RNA from GH3 rat anterior pituitary tumour cells. Under voltage-clamp conditions, application of the peptide induced a biphasic Ca(2+)-dependent chloride current. The amplitude of the initial, fast, component of the response was dependent on the concentration of the hormone and on the amount of mRNA injected. Size fractionation of poly(A)+ RNA on a continuous sucrose gradient and Northern blot analysis indicated that the receptor was encoded by an mRNA of approx. 3.5 kb. A 3.28 kbp cDNA encoding the TRH receptor has been cloned and sequenced. Full functionality of the predicted 412-amino-acid receptor protein was demonstrated by functional expression of cell surface receptors in Xenopus oocytes after both cytoplasmic injection of sense RNA transcribed in vitro from this cDNA and nuclear injection of the cDNA under the control of the Herpes simplex virus thymidine kinase promoter. The predicted protein contains seven putative membrane-spanning domains and shows significant sequence identify with some G-protein-coupled receptors. RNA blot analysis indicates that the mRNA for the TRH receptor is exclusively expressed in the pituitary gland. Expression studies performed with clones in which the 3′ region of the mRNA has been successively shortened indicate that the 3′ terminal region is not an important determinant for efficient functional expression in oocytes.

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1,25-Dihydroxyvitamin D3-induced upregulation of the thyrotropin-releasing hormone receptor in clonal rat pituitary GH3 cells

Journal of Endocrinology ◽

10.1677/joe.0.1470397 ◽

1995 ◽

Vol 147 (3) ◽

pp. 397-404 ◽

Cited By ~ 5

Author(s):

L M Atley ◽

N Lefroy ◽

J D Wark

Keyword(s):

Vitamin D ◽

Fold Increase ◽

Thyrotropin Releasing Hormone ◽

Gh3 Cells ◽

Pituitary Hormone ◽

Pituitary Cells ◽

Hormone Production ◽

Releasing Hormone ◽

Rat Pituitary ◽

Trh Receptor

Abstract 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) is active in primary dispersed and clonal pituitary cells where it stimulates pituitary hormone production and agonist-induced hormone release. We have studied the effect of 1,25-(OH)2D3 on thyrotropin-releasing hormone (TRH) binding in clonal rat pituitary tumour (GH3) cells. Compared with vehicle-treated cells, 1,25-(OH)2D3 (10 nmol/l) increased specific [3H]MeTRH binding by 26% at 8 h, 38% at 16 h, 35% at 24 h and reached a maximum at 48 h (90%). In dose–response experiments, specific [3H]MeTRH binding increased with 1,25-(OH)2D3 concentration and reached a maximum at 10 nmol/l. Half-maximal binding occurred at 0·5 nmol 1,25-(OH)2D3/l. The vitamin D metabolite, 25-OH D3, increased [3H]MeTRH binding but was 1000-fold less potent than 1,25-(OH)2D3. In equilibrium binding assays, treatment with 10 nmol 1,25-(OH)2D3/l for 48 h increased the maximum binding from 67·4 ± 8·8 fmol/mg protein in vehicle-treated cells to 96·7 ± 12·4 fmol/mg protein in treated cells. There was no difference in apparent Kd (1·08 ± 0·10 nmol/l for 1,25-(OH)2D3-treated and 0·97 ± 0·11 nmol/l for vehicle-treated cells). Molecular investigations revealed that 10 nmol 1,25-(OH)2D3/l for 24 h caused an 8-fold increase in TRH receptor-specific mRNA. Actinomycin D (2 μg/ml, 6 h) abrogated the 1,25-(OH)2D3-induced increase in [3H]MeTRH binding. Cortisol also increased [3H]MeTRH binding but showed no additivity or synergism with 1,25-(OH)2D3. TRH-stimulated prolactin release was not enhanced by 1,25-(OH)2D3. We conclude that the active vitamin D metabolite, 1,25-(OH)2D3, caused a time- and dose-dependent increase in [3H]MeTRH binding. The effect was vitamin D metabolite-specific and resulted from an upregulation of the TRH receptor. Further studies are needed to determine the functional significance of this novel finding. Journal of Endocrinology (1995) 147, 397–404

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Characteristics of the Ca2+ spike and oscillations induced by different doses of thyrotropin-releasing hormone (TRH) in individual pituitary cells and nonexcitable cells transfected with TRH receptor complementary deoxyribonucleic acid.

Endocrinology ◽

10.1210/endo.135.3.8070350 ◽

1994 ◽

Vol 135 (3) ◽

pp. 1084-1092 ◽

Cited By ~ 6

Author(s):

E J Nelson ◽

P M Hinkle

Keyword(s):

Deoxyribonucleic Acid ◽

Thyrotropin Releasing Hormone ◽

Pituitary Cells ◽

Releasing Hormone ◽

Complementary Deoxyribonucleic Acid ◽

Trh Receptor ◽

Different Doses

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Dopamine has no Effect on Thyrotropin-Releasing Hormone Mobilization of Calcium from Intracellular Stores in Rat Anterior Pituitary Cells

Molecular Endocrinology ◽

10.1210/mend-2-10-966 ◽

1988 ◽

Vol 2 (10) ◽

pp. 966-972 ◽

Cited By ~ 11

Author(s):

Greg J. Law ◽

Jonathan A. Pachter ◽

Priscilla S. Dannies

Keyword(s):

Anterior Pituitary ◽

Thyrotropin Releasing Hormone ◽

Pituitary Cells ◽

Releasing Hormone ◽

Anterior Pituitary Cells

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Thyrotropin-Releasing Hormone Gene Expression in Cultured Anterior Pituitary Cells: Role of Gender

Neuroendocrinology ◽

10.1159/000126815 ◽

1995 ◽

Vol 61 (1) ◽

pp. 77-84 ◽

Cited By ~ 2

Author(s):

Thomas O. Bruhn ◽

Jan M.M. Rondeel ◽

Thomas G. Bolduc ◽

Ivor M.D. Jackson

Keyword(s):

Gene Expression ◽

Anterior Pituitary ◽

Thyrotropin Releasing Hormone ◽

Pituitary Cells ◽

Releasing Hormone ◽

Anterior Pituitary Cells

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Okadaic acid and calyculin A enhance the effect of thyrotropin-releasing hormone on GH3 rat anterior pituitary cells excitability

FEBS Letters ◽

10.1016/0014-5793(92)81362-p ◽

1992 ◽

Vol 311 (1) ◽

pp. 41-45 ◽

Cited By ~ 10

Author(s):

Luis M. Delgado ◽

Pilar de la Peña ◽

Donato del Camino ◽

Francisco Barros

Keyword(s):

Okadaic Acid ◽

Anterior Pituitary ◽

Thyrotropin Releasing Hormone ◽

Pituitary Cells ◽

Calyculin A ◽

Releasing Hormone ◽

Anterior Pituitary Cells

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Dopamine Inhibits the Arachidonate and Prolactin Release Stimulated by Thyrotropin-Releasing Hormone through an Islet-Activating Protein-Sensitive GTP-Binding Protein in Anterior Pituitary Cells

Neuroendocrinology ◽

10.1159/000125542 ◽

1990 ◽

Vol 52 (1) ◽

pp. 75-81 ◽

Cited By ~ 10

Author(s):

Masahide Ohmichi ◽

Kenji Hirota ◽

Koji Koike ◽

Kozo Kadowaki ◽

Masaaki Yamaguchi ◽

...

Keyword(s):

Anterior Pituitary ◽

Binding Protein ◽

Thyrotropin Releasing Hormone ◽

Pituitary Cells ◽

Gtp Binding Protein ◽

Prolactin Release ◽

Releasing Hormone ◽

Gtp Binding ◽

Anterior Pituitary Cells

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Action of thyrotropin-releasing hormone on mammotrophs and thyrotrophs

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1981.241.4.e298 ◽

1981 ◽

Vol 241 (4) ◽

pp. E298-E304

Author(s):

G. Snyder ◽

Z. Naor ◽

C. P. Fawcett ◽

S. M. McCann

Keyword(s):

Anterior Pituitary ◽

Thyrotropin Releasing Hormone ◽

Female Rats ◽

Pituitary Cells ◽

Cell Distribution ◽

Camp Content ◽

Releasing Hormone ◽

Gravity Sedimentation ◽

Unit Gravity Sedimentation ◽

Region Ii

Anterior pituitary cells from 15-day female rats were separated by unit gravity sedimentation into four populations (designated regions I-IV) based on the profile of cell distribution and the resulting content of radioimmunoassayable (RIA) hormones. The cells in regions II and IV released thyrotropin (TSH) in response to thyrotropin-releasing hormone (TRH, 5 ng/ml); however, those in region IV released only approximately 5% of their RIA content, whereas those in region II released approximately 26% in response to the same stimulus. Concomitant elevation of cAMP and of cGMP occurred in region II cells but only cGMP was elevated in region IV cells. Mammotrophs were localized in region III. They responded to TRH by releasing prolactin (PRL) and exhibiting increased cAMP content. These data provide support for the existence of two functionally distinct populations of thyrotrophs in 15-day-old female rats. The data also imply that cAMP is involved in TRH induced PRL release, whereas cGMP is involved in TRH-induced TSH release.

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