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1.
The intrinsic tryptophan fluorescence of membranes prepared from the GH 3 strain of hormone-producing pituitary cells was monitored by spectrofluorometry. Membranes of GH 3 cells have specific receptors which bind thyrotropin-releasing hormone (TRH). When TRH binds to GH 3 membranes there is quenching of tryptophan fluorescence. The kinetics of the change in fluorescence of GH 3 membranes and of TRH binding are similar. In addition, the concentration of TRH required to produce a half-maximum change in fluorescence is 10 nM, and that required for half-maximum binding of TRH to receptors is 11 nM. Inactive TRH analogs which do not bind to TRH receptors likewise do not alter GH 3 membrane fluorescence, and a pituitary cell strain which lacks TRH receptors does not change membrane fluorescence on incubation with TRH. We conclude that the TRH-receptor interaction in GH 3 membranes is associated with a change in membrane conformation that is readily measured by differential spectrofluorometry. 相似文献
2.
The presence of the pertussis toxin (PTX) insensitive GTP-binding proteins (G-proteins) G q and/or G 11 has been demonstrated in three different prolactin (PRL) and growth hormone (GH) producing pituitary adenoma cell lines. Immunoblocking of their coupling to hormone receptors indicates that G q and/or G 11 confer throliberin (TRH) responsive phospholipase C (PL-C) activity in these cells. The contention was substantiated by immunoprecipitation analyses snowing that anti G q/11-sera coprecipitated PL-C activity. In essence, only G q/11 (but neither G i2, G i3 nor G o) seems to mediate the TRH-sensitive PL-C activity, while G o may be coupled to a basal or constitutive PL-C activity. Immunoblocking studies imply that the B-complex also, to some extent, may stimulate GH 3 pituitary cell line PL-C activity. Finally, the steady state levels of G q/11 mRNA and protein were downregulated upon long term exposure of the GH3 cells to TRH (but not to vasoactive intestinal peptide = VIP). 相似文献
3.
We monitored the radioligand-binding characteristics of thyrotropin-releasing hormone (TRH) receptors, functional activity of G q/11α proteins, and functional status of the whole signaling cascade in HEK293 expressing high levels of TRH receptors and G 11α. Our analyses indicated that disruption of plasma membrane microdomains by cholesterol depletion did not markedly influence the binding parameters of TRH receptors, but it altered efficacy of signal transduction. The functional coupling between TRH receptor and G q/11α was assessed by agonist-stimulated [ 35S]GTPγS binding, and results of these measurements pointed out to significantly lower potency of TRH to mediate G protein activation in the plasma membrane fraction isolated from cholesterol-depleted cells; there was a shift in sensitivity by one order of magnitude to the higher concentrations. A markedly lower sensitivity to stimulation with TRH was also observed in our experiments dealing with determination of hormone-induced Ca 2+ response. These data suggest that the intact structure of plasma membranes is an important optimum signal transduction initiated by TRH receptors and mediated by G q/11α proteins. 相似文献
4.
The object of the present study was to determine the relative importance of Ca++ and cyclic nucleotides as “second messengers” in thyroliberin (TRH)-mediated prolactin (PRL) release in the GH 3 and GH 4 rat pituitary tumor cell lines. PRL, cyclic adenosine 3': 5'-monophosphate (cAMP), and cyclic guanosine 3': 5'-monophosphate (cGMP) were measured by radioimmunoassay (RIA) following TRH stimulation. TRH increased PRL release and cAMP levels in GH 3 and GH 4 cells, but cGMP increases were variable. Treatment with 1 mM theophylline increased PRL release and raised cAMP and cGMP. Addition of TRH to theophylline-pretreated cells produced further significant increases in PRL release without any additional increases in cAMP and cGMP. Co++, a Ca++ antagonist, abolished TRH-induced PRL release in a dose-dependent manner. The Co++ inhibition was partially reversed by Ca++ in GH 3 or GH 4 cells. Furthermore, the Ca++ ionophore A23187 stimulated PRL release. We conclude that Ca++ is the primary “second messenger” for TRH-mediated PRL release from GH 3 or GH 4 cells. 相似文献
5.
The GH 4C 1 strain of hormone-producing rat pituitary cells has specific receptors for the tripeptide thyrotropin-releasing hormone (TRH). Membranes prepared from GH 4C 1 cells show intrinsic tryptophan fluorescence which was quenched by low concentrations (10–100 nM) of TRH and Nτ-methyl TRH but not by biologically inactive analogs of TRH. Membranes from GH 4C 1 cells were subjected to thermal denaturation. A conformational transition was noted above 40°C and an irreversible denaturation was observed at 52°C. TRH-induced quenching of intrinsic fluorescence was lost completely in membranes previously incubated for 10 min at 30°C while loss of [ 3H]-TRH binding was only about 20% at this temperature. Collisional quenching by iodide revealed that about 38% of the tryptophanyl residues in GH 4C 1 membranes were exposed to solvent. Quenching by TRH occurred with a shift in wavelength maximum from 336 to 342 nm suggesting that few of the tryptophanyl residues quenched by the tripeptide are totally exposed. Membranes prepared from cells preincubated with 20 nM TRH for 48 h, in which TRH receptors were decreased to 30% of control values, showed no quenching of tryptophan fluorescence in response to freshly added TRH. We conclude that the TRH-receptor interaction in GH 4C 1 cells is associated with a change in membrane conformation that can be measured by differential spectrofluorometry of intrinsic tryptophan fluorescence. 相似文献
6.
Summary Chronic treatment (more than 3 d) of GH 3 cells, cloned rat pituitary cells producing prolactin, with 100 n M TRH resulted in a 41% reduction in the rate of cell growth in a medium containing 0.5% fetal bovine serum. These effects
of TRH appeared both in the medium containing a higher concentration of serum and in that containing six growth factors, i.e.
insulin, transferrin, parathyroid hormone, fibroblast growth factor, triiodothyronine, and multiplication-stimulating activity
(MSA) instead of serum. TRH stimulated prolactin production by GH 3 cells in a dose-dependent manner both in the serum-supplemented and serum-free media. On the other hand, TRH, at 1 n M, elicited a 130% stimulation in the cellular growth, whereas, at concentrations of more than 10 n M, it inhibited the growth significantly.
In the defined culture system, it was demonstrated that TRH stimulated prolactin production in the presence or absence of
six growth factors, whereas its inhibitory effects on cellular growth appeared only in the presence of MSA regardless of the
presence or absence of the other five factors. Furthermore, it was shown that a dose-dependent stimulatory effect of MSA on
the growth of GH 3 cells was suppressed by TRH. TRH exhibited only a stimulatory effect on cellular growth in the medium containing the five
factors other than MSA. In conclusion, TRH could inhibit cell growth of GH 3 in the presence of MSA in the defined medium or MSA-like factor(s) in the serum-supplemented medium. 相似文献
7.
The effect of vasoactive intestinal peptide (VIP) on prolactin (PRL) secretion from pituitary cells is reviewed and compared to the effect of thyrotropin releasing hormone (TRH). These two peptides induced different secretion profiles from parafused lactotrophs in culture. TRH was found to increase PRL secretion within 4 s and induced a biphasic secretion pattern, while VIP induced a monophasic secretion pattern after a lag time of 45–60 s.The secretion profiles are compared to changes in adenylate cyclase activity, production of inositol polyphosphates, changes in intracellular calcium concentrations and changes in electrophysiological properties of the cell membrane.Abbreviations AC
adenylate cyclase
- DG
diacyglycerol
- GH
growth hormone
- GTP
guanosine trisphosphate
- G i
GTP binding proteins that mediate inhibition of adenylate cyclase and that are pertussis toxin sensitive
- G s
GTP binding protein that mediates stimulation of adenylate cyclase
- GH cells
clonal rat pituitary tumor cells producing PRL and/or growth hormone
- GH 3 GH 4C 1 and GH 4B6
subclones of GH cells
- PKA
protein kinase A
- PKC
protein kinase C
- PLC
phospholipase C
- PRL
prolactin
- TPA
12-O-tetradecanoyl phorbol 13-acetate
- TRH
thyrotropin releasing hormone
- VIP
vasoactive intestinal peptide 相似文献
8.
Phosphatidylinositol (Ptd Ins) breakdown in response to thyrotropin-releasing hormone (TRH) was measured after preincubation of both normal rat anterior pituitary cells and GH 3 turnout cells with [ 3H]inositol by the determination of [ 3H]inositol phosphate accumulation in the presence of lithium (which inhibits myo-inositol phosphatase). The method employed, which was originally developed for use with tissue slices, was adapted for isolated cells in monolayer culture. In GH 3 cells, TRH stimulated the breakdown of phosphoinositide in a manner similar to that reported previously using alternative methods. Furthermore, in normal male anterior pituitary cells the dose-response profile for TRH stimulation of inositol phosphate accumuJation was found to correlate well with the dose-response profile for TRH stimulation of prolactin secretion. As this response was maintained in the absence of added calcium, the breakdown of phosphoinositide would appear to be implicated as an event preceding calcium mobilization. 相似文献
9.
In this study, we examined the binding of soluble TSP1 (and ox-LDL) to CD36-transfected cells and the mechanisms by which immobilized TSP1 mediated attachment and haptotaxis (cell migration towards a substratum-bound ligand) of these transfected cells. CD36 cDNA transfection of NIH 3T3 cells clearly induced a dramatic increase in binding of both soluble [ 125I]-TSP1 and [ 125I]-ox-LDL to the surface of CD36-transfected cells, indicating that there was a gain of function with CD36 transfection in NIH 3T3 cells. Despite this gain of function, mock- and CD36-transfected NIH 3T3 cells attached and migrated to a similar extent on immobilized TSP1. An anti-TSP1 oligoclonal antibody inhibited CD36-transfected cell attachment to TSP1 while function blocking anti-CD36 antibodies, alone or in combination with heparin, did not. A series of fusion proteins encompassing cell-recognition domains of TSP1 was then used to delineate mechanisms by which NIH 3T3 cells adhere to TSP1. Although CD36 binds soluble TSP1 through a CSVTCG sequence located within type 1 repeats, 18,19 CD36-transfected NIH 3T3 cells did not attach to immobilized type 1 repeats while they did adhere to the N-terminal, type 3 repeats (in an RGD-dependent manner) and the C-terminal domain of TSP1. Conversely, Bowes melanoma cells attached to type 1 repeats and the N- and C-terminal domains of TSP1. However, CD36 cDNA transfection of Bowes cells did not increase cell attachment to type 1 repeats compared to that observed with mock-transfected Bowes cells. Moreover, a function blocking anti-CSVTCG peptide antibody did not inhibit the attachment of mock- and CD36-transfected Bowes cells to type 1 repeats. It is suggested that CD36/TSP1 interaction does not occur upon cell–matrix adhesion and haptotaxis because TSP1 undergoes conformational changes that do not allow the exposure of the CD36 binding site. © 1998 John Wiley & Sons, Ltd. 相似文献
12.
The GnRH receptor is coupled to G proteins of the families G q and G 11. G q and G 11 coupling leads to intracellular signaling through the phospholipase C pathway. GnRHR coupling to other G proteins is controversial. This study provides evidence that G protein families G s, G i, G q and G 11 complete for binding with the GnRHR. We quantified interactions of over-expressed G proteins with GnRHR by a competitive binding approach, using measurements of second messengers, IP and cAMP. Transient co-transfection of HEK293 cells with human WT GnRHR and with stimulatory and inhibitory G proteins (G q, G 11 and G s, G i) led to either production or inhibition of total inositol phosphate (IP) production, depending on the G protein that was over-expressed. Studies were conducted in different human (COS7, HeLa) and rodent-derived (CHO-K1, GH 3) cell lines in order to confirm that G protein promiscuity observed with the GnRHR was not limited to a particular cell type. 相似文献
13.
Objectives: Extensive research has been dedicated to elucidating the mechanisms of signal transduction through different G protein-coupled receptors (GPCRs). However, relatively little is known about the regulation of receptor movement within the cell membrane upon ligand binding. In this study we focused our attention on the thyrotropin-releasing hormone (TRH) receptor that typically couples to G q/11 proteins. Methods: We monitored receptor diffusion in the plasma membrane of HEK293 cells stably expressing yellow fluorescent protein (YFP)-tagged TRH receptor (TRHR-YFP) by fluorescence recovery after photobleaching (FRAP). Results: FRAP analysis indicated that the lateral movement of the TRH receptor was markedly reduced upon TRH binding as the value of its diffusion coefficient fell down by 55%. This effect was prevented by the addition of the TRH receptor antagonist midazolam. We also found that siRNA-mediated knockdown of Gq/11α, Gβ, β-arrestin2 and phospholipase Cβ1, but not of Giα1, β-arrestin1 or G protein-coupled receptor kinase 2, resulted in a significant decrease in the rate of TRHR-YFP diffusion, indicating the involvement of the former proteins in the regulation of TRH receptor behavior. The observed partial reduction of the TRHR-YFP mobile fraction caused by down-regulation of Giα1 and β-arrestin1 suggests that these proteins may also play distinct roles in THR receptor-mediated signaling. Conclusion: These results demonstrate for the first time that not only agonist binding but also abundance of some signaling proteins may strongly affect TRH receptor dynamics in the plasma membrane. 相似文献
14.
G-proteins transduce signals along diverse pathways, but the factors involved in pathway selection are largely unknown. Here, we have studied the ability of G αq to select between two effectors—mammalian inositide-specific phospholipase C β (PLC β) and phosphoinositide-3-kinase (PI3K)—in human embryonic kidney 293 cells. These studies were carried out by measuring interactions between eCFP- and eYFP-tagged proteins using Forster resonance energy transfer in the basal state and during stimulation. Instead of association of G αq with effectors through diffusion and exchange, we found separate and stable pools of G αq-PLC β and G αq-PI3K complexes existing throughout the stimulation cycle. These separate complexes existed despite the ability of G αq to simultaneously bind both effectors as determined by in vitro measurements using purified proteins. Preformed G-protein/effector complexes will limit the number of pathways that a given signal will take, which may simplify predictive models. 相似文献
15.
Following agonist action, G-protein-coupled receptors may exhibit differential coupling to G-proteins or second messenger pathways, supporting the notion of agonist-directed trafficking. To explore these mechanisms, we have designed and transfected synthetic siRNA duplexes to knockdown different G α subunits in Chinese hamster ovary (CHO) cells expressing human (h)5-hydroxytryptamine 1A receptors (CHO-h5-HT 1A). siRNAs against G αi2 and G αi3 transfected alone or in combination caused a large decrease in the corresponding mRNA level (64-80%) and also at the protein level for G αi3 (60-70%), whereas a non-specific siRNA showed no effect. In membranes of CHO-h5-HT 1A, 5-HT stimulated guanosine-5′-O-(3-[ 35S]thio)-triphosphate ([ 35S]GTPγS) binding was differentially affected by transfection of siRNAs against G αi protein, siRNAs against G αi2 inducing a more important decrease in the efficacy of 5-HT than transfection of siRNAs against G αi3. The high potency component was abolished after transfection of siRNAs against G αi3 and the lower potency component was suppressed after transfection of siRNAs against G αi2. To directly investigate G αi3 activation we used an antibody-capture/scintillation proximity assay. (+)8-OH-DPAT yielded bell-shaped curves for G αi3 activation, a response that was abolished after transfection of siRNAs against G αi3 protein. Interestingly, (+)8-OH-DPAT yielded a sigmoidal response when only G αi3 protein was expressed. These data suggest that when efficacious agonists attain a high level of occupation of h5-HT 1A receptors, a change occurs that induces coupling to G αi2 protein and suppresses signalling through G αi3 subunits. 相似文献
17.
Cardiomyocytes have a complex Ca 2+ behavior and changes in this behavior may underlie certain disease states. Intracellular Ca 2+ activity can be regulated by the phospholipase C β–G αq pathway localized on the plasma membrane. The plasma membranes of cardiomycoytes are rich in caveolae domains organized by caveolin proteins. Caveolae may indirectly affect cell signals by entrapping and localizing specific proteins. Recently, we found that caveolin may specifically interact with activated G αq, which could affect Ca 2+ signals. Here, using fluorescence imaging and correlation techniques we show that G αq-G βγ subunits localize to caveolae in adult ventricular canine cardiomyoctyes. Carbachol stimulation releases G βγ subunits from caveolae with a concurrent stabilization of activated G αq by caveolin-3 (Cav3). These cells show oscillating Ca 2+ waves that are not seen in neonatal cells that do not contain Cav3. Microinjection of a peptide that disrupts Cav3-G αq association, but not a control peptide, extinguishes the waves. Furthermore, these waves are unchanged with rynaodine treatment, but not seen with treatment of a phospholipase C inhibitor, implying that Cav3-G αq is responsible for this Ca 2+ activity. Taken together, these studies show that caveolae play a direct and active role in regulating basal Ca 2+ activity in cardiomyocytes. 相似文献
18.
Specific high affinity binding sites for [ 3H]1α, 25-dihydroxy-vitamin D 3 were observed in nuclear fractions of rat pituitary adenoma GH 3 cells. Crude nuclear (P1) sites demonstrated a pharmacological specificity for vitamin D 3 metabolites and analogues that was in accord with the characteristics of 1α, 25-dihydroxyvitamin D 3 receptors in recognized target organs. GH 3 cells grown in serum-containing medium contained significant amounts of 1α, 25-dihydroxy-vitamin D 3 in a P1 extract, whereas no 1α, 25-dihydroxyvitamin D 3 was detectable in P1 extracts from cells cultured in the absence of serum. Binding of [ 3H]1α, 25-dihydroxyvitamin D 3 to the P1 fraction was unaffected by prior depletion of intracellular 1α, 25-dihydroxyvitamin D 3, suggesting that association of [ 3H]1α, 25-dihydroxyvitamin D 3 to nuclear sites is not attributable to translocation of a cytosolic hormone-receptor complex and molecular exchange. The results support the concept that 1α, 25-dihydroxyvitamin D 3 has a physiological role in mediating pituitary hormone secretion. 相似文献
19.
Summary The hybrid GH cell strain, 928-9b, isolated from PRL+ (prolactin [PRL] producing) GH 4Cl and PRL (PRL non-producing) FIBGH12CI cells, has specific TRH (thyroliberin) receptors, yet does not respond to this peptide hormone. Unlike the parent strain, GH 4Cl, TRH does not stimulate synthesis or release of PRL in the hybrid strain. In contrast, treatment of 928-9b cells with another peptide, EGF (epidermal growth factor), stimulates both release and synthesis of PRL. The number of EGF receptors in the hybrid strain (2.5 × 10 3/cell) and the affinity of these receptors for ligand (2.2 nM) are comparable to that of the parent strain, GH 4C 1. The EGF dose response curve is also essentially the same for parent and hybrid cells for the enhancement of PRL production. A 3-8-fold enhancement of PRL production is observed and 1/2 maximal enhancement occurs at approximately 5 × 10 11 M EGF for both strains. TRH does not have any potentiating effect on EGF-induced stimulation of PRL release or PRL synthesis in the hybrid strain. Although EGF and TRH have similar biological effects in responsive GH cells, binding of one hormone to its receptors does not modulate the binding of the heterologous hormone. These findings demonstrate that more than one effect of TRH is defective in 928-9b cells even though EGF responses are intact. This suggests that 1) TRH-stimulated PRL release and TRH-stimulated PRL production have a common intermediate step, and 2) TRH and EGF have a different mechanism of action in GH cells. 相似文献
20.
A series of novel ( E)-3-(3,4-dihydroxyphenyl)acrylylpiperazine derivatives had been synthesized and evaluated their biological activities as potential tubulin polymerization inhibitors. Among these compounds, compound 3q exhibited potent antiproliferative activities against three cancer cell lines in vitro, and antitubulin polymerization activity with IC 50 of 0.92 μM, which was superior to that of colchicine (IC 50 = 1.34 μM). Docking simulation was performed to insert compound 3q into the crystal structure of tubulin at colchicine binding site to determine the probable binding model. These results suggested that compound 3q may be a promising antitubulin agent for the potential treatment of cancer. 相似文献
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