Isolation, regulation, and DNA-binding properties of three Drosophila nuclear hormone receptor superfamily members.

We have designed a rapid cloning and screening strategy to identify new members of the nuclear hormone receptor superfamily that are expressed during the onset of Drosophila metamorphosis. Using this approach, we isolated three Drosophila genes, designated DHR38, DHR78, and DHR96. All three genes are expressed throughout third-instar larval and prepupal development. DHR38 is the Drosophila homolog of NGFI-B and binds specifically to an NGFI-B response element. DHR78 and DHR96 are orphan receptor genes. DHR78 is induced by 20-hydroxyecdysone (20E) in cultured larval organs, and its encoded protein binds to two AGGTCA half-sites arranged as either direct or palindromic repeats. DHR96 is also 20E-inducible, and its encoded protein binds selectively to the hsp27 20E response element. The 20E receptor can bind to each of the sequences recognized by DHR78 and DHR96, indicating that these proteins may compete with the receptor for binding to a common set of target sequences.     

Corticotropin releasing hormone, receptor regulation and the stress response.

Corticotropin releasing hormone (CRH) coordinates behavioral, autonomic and hormonal responses to stress, including activation of the hypothalamic-pituitary-adrenal (HPA) axis with stimulation of adrenocorticotropin (ACTH) and glucocorticoids. Differential changes of expression of CRH and vasopressin(VP) in the parvicellular hypothalamic paraventricular nucleus (PVN), as well as regulation of CRH and VP receptors, are critical for the responsiveness of the HPA axis during stress. Pituitary CRH receptor (CRH-R)expression and content is controlled by the coordinated action of CRH, VP and glucocorticoids. Marked changes in hypothalamic and pituitary CRH-R expression support a key regulatory role for CRH in the HPA axis and the integrated stress response.     

Ovarian follicular development in the rat: hormone receptor regulation by estradiol, follicle stimulating hormone and luteinizing hormone.

The effects of estradiol, FSH and LH on ovarian follicular development and granulosa cell differentiation were examined in the immature rat hypophysectomized on day 24 of age. Administration of estradiol to hypophysectomized rats for 4 days stimulated the growth of large preantral follicles with a concomitant 1.5-fold increase in FSH receptor content and a 4-fold decrease in LH receptor content in the granulosa cells. When highly purified hFSH was administered alone, receptor content for FSH increased progressively for 4 days while receptor for LH remained essentially unchanged. However, when rats were pretreated with estradiol, the response of follicles to FSH was markedly enhanced as indicated by the appearance of large, antral follicles and elevated receptor content for both FSH and LH. Receptor content for FSH increased markedly in response to hFSH following only one day of estradiol pretreatment, while receptor content for LH increased most rapidly in response to hFSH after 3 days of estradiol pretreatment. LH administered to rats possessing large preovulatory follicles caused luteinization of granulosa cells and a marked decline in receptor content for both gonadotropins within 24 h. Receptor content remained low even 48 h after LH administration when granulosa cells were fully luteinized. These results indicated that follicular development and granulosa cell differentiation are dependent on steroid-protein hormone regulation of hormone specific receptors.     

Affinity labeling of rat liver thyroid hormone nuclear receptor.

The thyroid hormone receptor from rat liver nuclei has been covalently labeled with the N-bromoacetyl derivatives of L-thyroxine (T4) and 3,3',5-triiodo-L-thyronine (T3). Displacement binding studies showed that, in the presence of 100-fold molar excess of unlabeled N-bromoacetyl-T3 or T4, binding of [125I]T3 or [125I]T4 was nearly totally inhibited. Heat inactivation of the receptor (55 degrees C for 15 min) resulted in parallel losses in the binding of T3 (95%) and N-bromoacetyl-T3 (93%). These results indicated that T3 and T4 and their bromoacetyl derivatives compete for the same binding site. The nuclear receptor showed identical behavior in high-pressure liquid chromatography (HPLC) whether bound to T3 or T4 or covalently labeled with their bromoacetyl derivatives. HPLC provided a single-step 100-fold purification of the nuclear receptor. Na-DodSO4 gel electrophoresis of the nuclear receptor labeled with N-bromoacetyl derivatives of [125I]T3 or [125I]T4 showed one major radioactive component with a molecular weight of 56,000. Furthermore, in the absence of denaturant, the nuclear receptor either bound to [125I]T3 or covalently labeled with N-bromoacetyl-[125I]T3 showed identical mobility. These results suggested that the nuclear receptor is a single polypeptide chain and binds either T3 or T4. Nuclear receptors covalently linked with N-bromoacetyl derivatives of [125I]T3 or [125I]T4 may be useful as a marker for the preparative purification of receptor.     

Putative nuclear triiodothyronine receptors in tadpole erythrocytes: regulation of receptor number by thyroid hormone

Putative thyroid hormone (TH) receptors have been detected in the nuclei of red blood cells (RBCs) from Rana catesbeiana tadpoles, and their binding characteristics have been examined. Nuclear T3 saturation analyses were carried out in vitro in intact RBCs suspended in phosphate-buffered amphibian Ringer. After incubation with T3, intact nuclei were obtained by centrifugation after lysing the cells in a sucrose-Tris-HCl buffer containing 0.2% saponin and then adding Triton X-100 (final concentration 0.125%) to the lysed mixture to reduce nonspecific binding to less than 10% of total binding. Scatchard analysis of equilibrium binding data revealed that RBCs from premetamorphic (first year) tadpoles contained 502 +/- 39 (SE) T3 binding sites per nucleus; in prometamorphic (second year) tadpoles the number had increased to 844 +/- 39. Development was also accompanied by some increase in the affinity of these sites; dissociation constant (Kd) = 1.8 +/- 0.39 X 10(-11) M and 0.95 +/- 0.108 X 10(-11) M in pre- and prometamorphic tadpoles, respectively. The number of sites per nucleus in both pre- and prometamorphic tadpole RBCs was greatly increased by pretreatment in vivo with either T3 or T4 for 6-10 days; a comparable number of sites per nucleus (2225 +/- 65) was observed after maximal stimulation by either hormone at both stages of development. Significant increases in receptor number were observed 10 days after injection of 0.03 nmol T3 or 0.06 nmol T4 into tadpoles weighing 13-15 g; maximal effects were obtained with 0.1 nmol T3 or 1.0 nmol T4. On the basis of these observations and the evidence that more TH is present in pro- than in premetamorphic tadpoles, it is suggested that the spontaneous increase in receptor number in RBC nuclei associated with progression from the pre- to prometamorphic phase is due, at least in part, to increased levels of endogenous TH.