Regulation Of Receptor Concentration Research Articles

Receptores a estrógenos y progesterona

La estrogenoterapia está en aumento y ello obliga a un adecuado conocimiento del mecanismo de acción de los esteroides, siendo al momento conocidos su transporte y metabolismo. Por ello discutiremos los aspectos moleculares de la acción de los estrógenos a nivel de los receptores nucleares, precisando la estructura y funcionamiento de los mismos. Se revisa los factores de transcripción, describiendo sus dominios de unión al DNA y de activación. Se hace precisiones de la transcripción, el papel de la polimerasa II y los factores generales de transcripción, que forman el complejo de iniciación de transcripción. Se culmina exponiendo cómo es la regulación de la concentración del receptor a través del mecanismo de relleno y puntualizando algunas implicancias clínicas. La estrogenoterapia está en aumento y es conveniente que su uso sea consecuencia de un adecuado conocimiento del mecanismo de acción del estrógeno en su célula blanco; este conocimiento biomolecular permite comprender la respuesta individual o grupal a esa terapia. Los receptores hormonales a estrógenos y progesterona han sido encontrados y estudiados en todo el organismo1-3 y son parte de la regulación del med¡o interno; su comprensión explica la fisiología de hormonas esteroides.

Ruminants express a prolactin receptor of Mr 33 000—36 000 in the mammary gland throughout pregnancy and lactation

Developmental variation in the expression of the prolactin receptor in the ruminant mammary gland was investigated. Affinity chromatography revealed that bovine prolactin and human GH each bound to the same mammary gland proteins, yielding fractions enriched in binding activity and a protein of M(r) 36,000, assumed to be a bovine prolactin receptor. Affinity cross-linking of 125I-labelled human GH to mammary microsomes confirmed that the M(r) 36,000 protein was a bovine prolactin receptor. Binding assays of receptors in microsomes from the mammary tissue of cows and ewes at various stages of the lactational/reproductive cycle indicated developmental regulation of receptor concentration, but not receptor type, as no other bovine prolactin receptor type was detected by affinity cross-linking. These results suggest that differences in the response to prolactin in the mammary gland at various developmental stages in ruminants are not due to the expression of different forms of the prolactin receptor, and the lack of a prolactin effect on established lactation in ruminants is not due to the absence of the M(r) 36,000 form of the prolactin receptor.

Calcitonin Receptors on Circulating Normal Human Lymphocytes*

Circulating human lymphocytes possess specific and functional receptors for calcitriol and PTH. We sought to determine if they also possessed receptors for calcitonin (CT), the third classical calciotropic hormone. We isolated blood mononuclear cells from healthy volunteers to separate monocytes, total lymphocytes, and T-lymphocytes; the purity of the three cell populations was more than 90%, 95%, and 85%, respectively. Salmon CT (sCT) was labeled by the chloramine-T method (SA, 254 muCi/micrograms) without loss of biological activity. We found saturable (16 h at 8 C), specific, high affinity binding sites for [125I]sCT on unstimulated lymphocytes. As for CT receptors on other cells, binding of [125I]sCT was poorly reversible. Binding specificity was demonstrated by the total absence of competing effect of several unrelated hormones; human CT and CT gene-related peptide competed much less efficiently than sCT for the binding sites, whereas PDN-21 had no effect. When plotted according to the method of Scatchard, binding data on the mixed population of T- and B-lymphocytes showed an apparent Kd (mean +/- SD) of 2.9 +/- 1.0 x 10(-10) M (n = 34), with an estimation of 91-8338 (median, 1971) binding sites/cell. The data were repeatedly compatible with an aspect of positive cooperativity between the binding sites, as confirmed by a Hill coefficient greater than 1 (1.18 +/- 0.13). However, this aspect of positive cooperativity in CT binding was not observed on isolated T-lymphocytes (Hill coefficient, 0.96 +/- 0.08; n = 9; P less than 0.001 vs. the mixed population of lymphocytes). CT did not induce a significant increase in cAMP levels, but regulation of receptor concentration was demonstrated by the finding of down-regulation of CT-binding sites after sCT or human CT preincubation. In summary, we have found saturable, specific, high affinity receptors for CT on unstimulated normal human T-lymphocytes, which could, thus, be target sites for CT action on bone metabolism or on the immune system.

Characterization of a specific insulin-like growth factor-I/somatomedin-C receptor on high density, primary monolayer cultures of bovine articular chondrocytes: regulation of receptor concentration by somatomedin, insulin and growth hormone

In order to obtain a phenotypically stable cell population of chondrocytes, high density primary monolayer cultures of bovine articular chondrocytes were established. Using these cultures, a specific insulin-like growth factor-I/somatomedin-C (IGF-I/SM-C) receptor was demonstrated and characterized. At 15 degrees C steady-state binding was attained by 5 h, and averaged 25% per 2.2 X 10(6) cells. Fifty per cent displacement of 125I-labelled IGF-I/SM-C by unlabelled IGF-I/SM-C occurred at concentrations of only 2.3 ng/ml, whereas IGF-II and porcine insulin were approximately 15- and 1000-fold less potent respectively. Scatchard analysis gave a linear plot, with a calculated association constant of 2.26 X 10(9) l/mol and a receptor number of 15 400 sites per cell. Preincubation of chondrocyte monolayers with either IGF-I SM-C or porcine insulin at 37 degrees C for 20 h resulted in reduction of 125I-labelled IGF-I/SM-C binding in a dose-dependent manner, although higher concentrations were required with insulin. More than 40% down-regulation of the receptor occurred with IGF-I/SM-C at concentrations of 10 nmol/l and nearly 70% reduction at 50 nmol/l. Interestingly, after preincubation with either human (h) or bovine (b) GH, 40% down-regulation of 125I-labelled IGF-I/SM-C binding was observed at concentrations of 10 mumol/l. Local production of IGF-I/SM-C by chondrocytes in response to GH stimulation may have occurred, but, because only 120 pmol IGF-I/SM-C and less than 30 pmol IGF-I/SM-C per litre were recovered from serum-free conditioned media preincubated with bGH and hGH respectively, this was not established.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased Cytosolic Androgen Receptor Binding in Rat Striated Muscle Following Denervation and Disuse

We investigated the effects of denervation and disuse on cytosolic androgen receptor binding by rat striated muscle. Denervation of the extensor digitorum longus and tibialis anterior muscles caused a 40-50% increase in cytosolic androgen receptor concentration with no change in apparent binding affinity. This effect was evident at 6 h postdenervation, maximal at 24 h, and declined to 120% of the control level 72 h after denervation. A 40% increase in cytosolic androgen receptor concentration was also noted 24 hr after denervation of the hormone-sensitive levator ani muscle. The effect of denervation on androgen receptors was not blocked by in vivo injection of cycloheximide; therefore, de novo receptor synthesis probably is not involved in the observed increase. Disuse, produced by subperineurial injection of tetrodotoxin into the tibial and common peroneal branches of the sciatic nerve, mimicked the effect of denervation on androgen receptor binding, suggesting that neuromuscular activity is important in regulation of receptor concentration. Possible mechanisms subserving this effect are discussed.

Characterization of insulin-like growth factor I receptors on cultured rat bone cells: regulation of receptor concentration by glucocorticoids.

A specific receptor for insulin-like growth factor I (IGF-I) has been demonstrated in cultured fetal rat osteoblast-like bone cells. Specific binding of [125I]IGF-I to bone cells incubated at 15 C reached a steady state by 5 h. Half-maximal inhibition of [125I]IGF-I binding by unlabeled IGF-I was observed at 7 ng/ml. Multiplication-stimulating activity, insulin, and proinsulin were less effective than unlabeled IGF-I in competing for receptor occupancy. Scatchard analysis showed a curvilinear plot, with a Ka similar to that observed in human fibroblasts. Incubation of cell monolayers with glucocorticoids resulted in a concentration-dependent increase in [125I]IGF-I binding. This increase in [125I]IGF-I binding was dependent on cell density. After a 2-day exposure to dexamethasone, no increase in binding was observed in sparsely plated cells; however, an increase in binding was observed after 3 days in culture (log phase) and was maximal by 5 days (peak log phase). These data indicate that rat bone cells possess a specific receptor for IGF-I with binding characteristics similar to those reported in human fibroblasts, and that IGF-I receptor concentrations are increased by exposure to glucocorticoids. A role for glucocorticoids and IGF-I in rat bone proliferation is suggested by these findings.

Characterization of the Somatomedin-C/Insulin-Like Growth Factor I (SM-C/IGF-I) Receptor on Cultured Human Fibroblast Monolayers: Regulation of Receptor Concentrations by SM-C/IGF-I and Insulin*

We have characterized 125I-labeled somatomedin-C/insulin-like growth factor 1 ([125I]SM-C/IGF-I) binding to cultured human fibroblast monolayers and have investigated the modulation of receptor concentrations by SM-C/IGF-I and insulin. Specific binding of [125I]SM-C/IGF-I to confluent monolayers at 15 C reached a steady state in 2.5 h and averaged 11.37 ± 0.71% (mean ± SEM)/1.5 × 106 cells. At 30 C, binding occurred more rapidly, peaking at 30 min, but averaged only 4%. Binding was reversible at both temperatures, and degradation of radioligand by fibroblast monolayers was negligible. Half-maximal inhibition of [125I]SM-C/IGF-I binding was observed at SM-C/IGF-I concentrations of 7 ng/ml. Compared to SM-C/IGF-I, the relative potencies of other hormones was: IGF-II, 1:10; multiplication-stimulating activity, 1:17, porcine insulin, 1:300; porcine proinsulin, 1:1000; and human GH nonreactive. Scatchard analysis was consistent with a single class of receptors, with a Ka of 1.07 × 109 M−1 and 20,000 sites/cell...

Mechanism of thyroid hormone inhibition of thyrotropin-releasing hormone action.

The addition of thyroid hormone to cultures of GH3 or GH4C1 pituitary tumor cells maintained in medium with hypothyroid serum decreased the concentration of specific receptors for TRH. The relationship between thyroid hormone effects on TRH receptors and TRH responses was examined by testing the concentration dependence, time course, and specificity of these changes. The concentrations of T3 giving half-maximal decreases in [3H]TRH binding and inhibition of the PRL response to TRH were 0.20 and 0.24 nM, respectively. TRH stimulated the rate of [3H]uridine uptake by 50% in cultures incubated without added T3 but did not increase [3H]uridine uptake in cells incubated with thyroid hormone. The PRL response to TRH was substantially inhibited 12 h after the addition of T3, and the uridine uptake response was completely blocked in 8 h. Two other stimuli of PRL secretion, sodium butyrate and isobutylmethylxanthine, were effective in the presence or absence of T3. Thyroid hormone did not reduce the specific binding of either [125I-Tyr1]somatostatin or [125I]iodoepidermal growth factor. Somatostatin decreased the secretion of GH and PRL by pituitary tumor cells grown with or without T3. The data show that the effects of thyroid hormones on TRH receptors are specific and suggest that regulation of receptor concentrations may be the direct cause of thyroid hormone regulation of pituitary responsiveness to TRH.

Regulation of receptor concentration by homologous hormone. Effect of human growth hormone on its receptor in IM-9 lymphocytes.

When cultured human lymphocytes of the IM-9 line were exposed to human growth hormone (hGH) at 37 degrees, washed for 2 hours, and incubated with 125I-hGH, the binding of 125I-hGH was reduced. The magnitude of the reduction in binding was dependent on the concentration of growth hormone present as well as the duration of the exposure. As little as 2 X 10(-11) M (0.5 ng/ml) growth hormone had a discernible effect. Growth hormone at 2 X 10(-10) M (5.0 ng/ml), which is a low resting concentration of hormone in vivo and occupies about 20% of the receptors at steady state at 30 degrees, produced a 50% reduction in binding while 20 mg/ml, which occupies about 50% of the receptors under steady state conditions, produced an 80% loss of receptors. Further increases in growth hormone concentration produced little further effect on receptor loss. Thus, the loss of receptors at a given concentration of growth hormone (up to 20 ng/ml) in the preincubation at 37 degrees was greater than the occupancy produced by that concentration of growth hormone receptors under steady state conditions at 30 degrees. Analysis of the data indicated that the decrease in binding of 125I-hGH was due to a loss of receptors per cell without any change in affinity of receptor for hormone or in cell number. The concentration of insulin receptors on these cells was affected by the insulin concentration in the medium, and the concentration of growth hormone receptors was affected by growth hormone, but neither hormone had any effect on the heterologous receptors. Exposure of the cells to cycloheximide (0.1 mM) produced a progressive but smaller loss of growth hormone receptors, and the effect of cycloheximide was additive to the receptor loss induced by growth hormone, suggesting that cycloheximide inhibited synthesis of receptors while growth hormone accelerated loss of receptors. When growth hormone was removed from the medium, receptor concentrations were restored rapidly; half of the loss was restored by 6 to 8 hours and the full complement of receptors was restored by 24 hours following removal of the hormone. If the growth hormone was removed and replaced with cycloheximide, the return of the receptors was delayed until the cycloheximide was removed. Thus restoration of the receptors appeared to require the synthesis of new proteins. These data indicate that in the IM-9 lymphocytes the concentration of growth hormone receptors is very sensitive to regulation by growth hormone and also add further support to the suggestion that hormones in general actively regulate the concentration of their own receptors.