Radioligand Assays for Oestradiol and Progesterone Conjugated to Protein Reveal Evidence for a Common Membrane Binding Site in the Medial Preoptic Area‐Anterior Hypothalamus and Differential Modulation by Cholera Toxin and GTP<i>γ</i>S

Caldwell, Jack D.; Walker, Cheryl H.; Rivkina, A; Pedersen, Cort A.; Mason, George A.

doi:10.1046/j.1365-2826.1999.00356.x

Cited by 18 publications

(20 citation statements)

References 36 publications

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“…4B). These results, summarized in Table 1, strongly suggest the involvement of G proteins in signal transduction via membrane‐bound progesterone receptors in the fungus and are in accordance with observations by Caldwell et al[10] who, using GTPγS and toxins, demonstrated that G proteins were associated with progesterone and estradiol membrane binding sites in the medial preoptic area–anterior hypothalamus of rat.…”

Section: Resultssupporting

confidence: 91%

Membrane-bound progesterone receptors coupled to G proteins in the fungusRhizopus nigricans

Lenasi

Bavec

Zorko

2002

FEMS Microbiology Letters

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Steroid binding sites with high affinity for progesterone (Kd=40+/-14 nM determined by binding, and Kd=71+/-22 nM determined by displacement studies) and lower affinity for 21-hydroxyprogesterone and for testosterone, but no affinity for estradiol-17beta, onapristone and alpha-naphthoflavone were detected in the enriched plasma membrane fraction of the fungus Rhizopus nigricans. The amount of steroid binding sites is in accordance with the value of B(max)=744+/-151 fmol (mg protein)(-1). In the membrane fraction, progesterone induced about 30% activation of G proteins over basal level, as determined by GTPase activity (EC50=32+/-8 nM) and by the guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding rate (EC50=61+/-21 nM). The affinity of receptors for progesterone was substantially decreased in the presence of GTPgammaS and of cholera toxin. Our results suggest the existence of progesterone receptors in the membrane of Rhizopus nigricans and their coupling to G proteins.

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Section: Resultssupporting

confidence: 91%

Membrane-bound progesterone receptors coupled to G proteins in the fungusRhizopus nigricans

Lenasi

Bavec

Zorko

2002

FEMS Microbiology Letters

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“…SHBG (human SHBG; Lot # 596402 with a ratio of total protein to SHBG of 1.43, giving a purity of ∼70%; Cortex Biochem Inc., San Leandro, Calif., USA) was iodinated by the chloramine T method previously described for conjugated steroids in Caldwell et al [42]. Briefly, 50 µl of 1 mg/ml SHBG in 40 m M Trizma was added to 25 µl chloramine T (0.8 mg/ml) in the vial of Na- 125 I (1 mCi from ICN Pharmaceuticals, Costa Mesa, Calif., USA) for 12 min at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Internalization of Sex Hormone-Binding Globulin into Neurons and Brain Cells in vitro and in vivo

et al. 2007

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Background: Sex hormone-binding globulin (SHBG) is a 94-kDa homodimer that binds steroids and is made in the hypothalamus. We have demonstrated that infusions of SHBG into the hypothalami of rats increase their female sexual receptivity except when SHBG is coupled to dihydrotestosterone (DHT) suggesting that SHBG has an active function in behavioral neuroendocrinology. Methods: This study examines the possibility that SHBG is internalized by neuronal and/or non-neuronal brain cells as one possible mode of action using in vitro and in vivo techniques. Results: First, analysis of the uptake of radiolabeled SHBG (¹²⁵I-SHBG) found ¹²⁵I-SHBG uptake in HT22 hippocampal cells stably transfected with cDNA for ERβ (HT22-ERβ). The addition of DHT to ¹²⁵I-SHBG significantly inhibited ¹²⁵I-SHBG uptake in HT22-ERβ cells but not in HT22-ERα or HT22 wild-type cells. SHBG internalization was specific as it did not occur in either the human neuroblastoma cell line SK-N-SH or the glioma cell line C6. Second, SHBG was labeled with a fluor (Alexa-555^TM), and infused into the lateral cerebroventricles of ovariectomized rats. Optimal SHBG uptake was seen 10 min after these infusions. SHBG uptake was seen in specific parts of the choroid plexus and periventricular cells as well as into cells in the paraventricular nucleus, the medial forebrain bundle, and the habenula. Conclusions: These studies suggest that SHBG is internalized by brain cells, which may be affected by the presence of ERβ. The gonadal steroids have numerous effects in brain and the discovery that the steroid-binding protein SHBG is taken up into neurons and brain cells may demand a change in thinking about how steroids are delivered to brain cells to affect neurophysiology.

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“…The MH was dissected out of the MH section by making a rectangular cut with its lateral sides made by vertical sagittal cuts from the medial extent of the optic tracts and with its dorsal edge 1 mm dorsal to the dorsal extent of the third ventricle. These are the same dissections that we have previously used to isolate the MPOA-AH and MH [30, 42, 43, 45, 46]. …”

Section: Methodsmentioning

confidence: 99%

Estradiol Control of Expression and Levels of Estradiol-Binding Proteins in the Medial Preoptic Area, Medial Hypothalamus and Pituitary

et al. 2003

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The brains of mammals have at least three estradiol-binding proteins: estradiol receptor-α (ERα), ERβ, and sex hormone-binding globulin (SHBG). In this study we compare the effects of estradiol treatment on the expression of mRNA for these three estradiol-binding proteins in two reproductively important brain areas, the medial preoptic area-anterior hypothalamus (MPOA-AH) and medial hypothalamus (MH) as well as in the hippocampus in ovariectomized rats, using the reverse transcriptase-polymerase chain reaction (RT-PCR). We also used surface-enhanced laser desorption ionization time of flight (SELDI-TOF) mass spectrometry (MS) to analyze the effects of estradiol in ovariectomized rats on SHBG levels in the MPOA-MH as well as the neurohypophysis. In vivo estradiol treatment in ovariectomized rats eliminated or significantly reduced expression of all three estradiol-binding proteins in both the MPOA-AH and MH. This change in ERα, ERβ, and SHBG expression did not occur in the hippocampus. Both Northern blot and DNA sequence analysis confirmed the results of the RT-PCR for SHBG. SELDI-TOF MS analysis demonstrated that in vivo estradiol treatments resulted in dramatically decreased levels of SHBG in the hypothalamus and that a reduction in SHBG mRNA by estradiol treatment also resulted in a reduction in SHBG protein levels. Estradiol treatment also eliminated detectable SHBG from the neurohypophysis, suggesting that estradiol controls SHBG levels in this release site. That in vivo estradiol treatments had the same inhibitory effects on mRNA levels for SHBG and both ERs suggests similar translational control mechanisms for all three steroid-binding proteins in the brain. That estradiol treatments also reduced pituitary SHBG suggests that such treatment releases SHBG from the neurohypophysis.

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Radioligand Assays for Oestradiol and Progesterone Conjugated to Protein Reveal Evidence for a Common Membrane Binding Site in the Medial Preoptic Area‐Anterior Hypothalamus and Differential Modulation by Cholera Toxin and GTPγS

Cited by 18 publications

References 36 publications

Membrane-bound progesterone receptors coupled to G proteins in the fungusRhizopus nigricans

Membrane-bound progesterone receptors coupled to G proteins in the fungusRhizopus nigricans

Internalization of Sex Hormone-Binding Globulin into Neurons and Brain Cells in vitro and in vivo

Estradiol Control of Expression and Levels of Estradiol-Binding Proteins in the Medial Preoptic Area, Medial Hypothalamus and Pituitary

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