Investigation of the effect of α-melanocyte-stimulating hormone on proliferation and early stages of differentiation of human induced pluripotent stem cells

We have studied the influence of α-melanocyte-stimulating hormone (α-MSH) on proliferation and early stages of differentiation of human induced pluripotent stem cells (iPSc). We have demonstrated that α-MSH receptor genes are expressed in undifferentiated iPSc. The expression levels of MCR1, MCR2, and MCR3 increased at the embryoid body (EB) formation stage. The formation of neural progenitors was accompanied by elevation of MCR2, MCR3, and MCR4 expression. α-MSH had no effect on EB generation and iPSc proliferation at concentrations ranging from 1 nM to 10 μM. At the same time, α-MSH increased the generation of neural rosettes in human iPSc cultures more than twice.     

Metabolic clearance rate of adrenocorticotropin in the rat.

The metabolic clearance rate (MCR) of adrenocorticotropin (ACTH) was estimated after the intravenous infusion of graded rates of the hormone (40-2560 muU/min per 100 g body weight) in rats pretreated with chlorpromazine, morphine, and Nembutal, a preparation which proved effective in blocking endogenous ACTH release. The hormone was infused over a period of 45 min, at which time the plasma ACTH concentration had reached a steady state. A specific and sensitive bioassay, based on the corticosterone production of dispersed adrenal cells, was used to measure the plasma ACTH concentration. With increasing infusion rates of ACTH, a threefold decrease in the MCR of ACTH was observed. Previous studies of our group have shown that the MCR of corticosterone increases as a function of the infusion rate of the steroid. It appears, therefore, that the metabolism of these two hormonal links of the hypothalamo-pituitary-adrenocortical axis vary in opposite fashions as a function of the secretion rate of the hormone.     

Phosphorylation on tyrosine of in vitro synthesized human estrogen receptor activates its hormone binding

Hormone binding controls the activity of estradiol receptor. The in vitro synthesized human receptor binds hormone with high affinity and low efficiency (1-4% of the maximal binding). We now report that phosphorylation on tyrosine of the synthetic receptor by an extensively purified calf uterus kinase increases hormone binding towards maximal levels without change in affinity. This is the first direct demonstration that a newly synthesized hormone receptor acquires ligand binding through phosphorylation. The use of in vitro synthesized proteins as substrates for enzymes which cause functional modifications of proteins is very promising because it is easy to identify the modified domains and residues by using deleted and point mutated proteins. Experiments with two estradiol receptor deletion mutants, one which lacks the N-terminal half of the receptor and binds hormone independently from the N-terminal half of the receptor, the other which lacks the C-terminal half of the receptor and contains the domain required to recognize the estradiol responsive elements, show that tyrosine phosphorylation occurs exclusively within or near the hormone binding domain of the receptor.     

Analysis and Application of an Equilibrium Model forin vitroBioassay Systems with Three Components: Receptor, Hormone and Hormone-Binding-Protein

A simple theoretical framework is presented for bioassay studies using three componentin vitrosystems. An equilibrium model is used to derive equations useful for predicting changes in biological response after addition of hormone-binding-protein or as a consequence of increased hormone affinity. Sets of possible solutions for receptor occupancy and binding protein occupancy are found for typical values of receptor and binding protein affinity constants. Unique equilibrium solutions are dictated by the initial condition of total hormone concentration. According to the occupancy theory of drug action, increasing the affinity of a hormone for its receptor will result in a proportional increase in biological potency. However, the three component model predicts that the magnitude of increase in biological potency will be a small fraction of the proportional increase in affinity. With typical initial conditions a two-fold increase in hormone affinity for its receptor is predicted to result in only a 33% increase in biological response. Under the same conditions an 11-fold increase in hormone affinity for receptor would be needed to produce a two-fold increase in biological potency. Some currently used bioassay systems may be unrecognized three component systems and gross errors in biopotency estimates will result if the effect of binding protein is not calculated. An algorithm derived from the three component model is used to predict changes in biological response after addition of binding protein toin vitrosystems. The algorithm is tested by application to a published data set from an experimental study in anin vitrosystem (Limet al., 1990,Endocrinology127,1287–1291). Predicted changes show good agreement (within 8%) with experimental observations.     

Glucagon receptors on isolated hepatocytes and hepatocyte membrane vesicles. Discrete populations with ligand- and environment-dependent affinities

Analysis of glucagon and deshistidine glucagon binding to isolated canine hepatocytes and to hepatocyte membrane vesicles (formed by budding of hepatocytes in hypotonic medium) reveals two separate populations of hormone binding sites. Mathematical modeling further shows that the high affinity population represents 1% of the total in all four cases. Although calculated dissociation constants for hormone binding range from 0.2 to 400 nM, whether considering glucagon or deshistidine glucagon binding, or binding to the high affinity or low affinity receptor populations, receptor affinity increases 2- to 100-fold in the environment of the membrane vesicle; concomitant with this alteration in receptor affinity, receptor selectivity for the structure of the native hormone decreases 1.5- to 40-fold in hepatocyte-derived vesicles. Consideration of receptor affinity in relation to receptor number suggests that hepatocyte glucagon binding is distributed about equally between high and low affinity receptor populations at typical portal hormone levels. Nevertheless, consideration of receptor binding in relation to biological activity suggests that the activity of glucagon in inhibiting carbohydrate flux into glycogen is attributable to occupancy of the high affinity receptor population.     

Immunophotochemical analysis of mineralocortin by polyclonal antibodies against the native receptor from rat kidney

We have obtained a polyclonal antiserum by immunizing fawn Burgundy rabbits with the mineralocorticoid receptor (MCR) purified biochemically from rat kidneys. High titers of anti-MCR activity were obtained in radioimmunoassays within 3 weeks and increased with a booster shot. In Western blot analysis, the antibody revealed a major band of 94–98 kDa in renal cytosol from rat and beef kidneys. We also developed a fluorographic procedure where the MCR linked covalently to tritiated R-5020, following ultraviolet irradiation, gave imprints superimposable on the Western blot profile. The fluorographic pattern was specific since it was largely abolished in the presence of cold RU 26752 that is specific to MCR, or mineralocortin. The immune IgG precipitated rat renal MCR-[³H]RU 26752 complexes in a dose-dependent manner and also recognized MCR bound to the natural hormone aldosterone. During gel permeation chromatography on Sephacryl, the elution profile of [³H]RU 26752 shifted to high-molecular-weight regions in the presence of immune IgG. The receptor protein could be immunolocalized primarily to the principal cells of the collecting duct in rat kidney but the intercalated cells and glomeruli were not labeled, contrary to beef kidney where a uniform pattern of immunostaining was evident. These should permit large-scale purification of the MCR for detailed physicochemical studies and for screening of the MCR-positive tissues during various pathophysiological syndromes.     

Adaptation of methane formation and enzyme contents during growth of Methanobacterium thermoautotrophicum (strain ΔH) in a fed-batch fermentor

During growth of Methanobacterium thermoautotrophicum in a fed-batch fermentor, the cells are confronted with a steady decrease in the concentration of the hydrogen energy supply. In order to investigate how the organism responds to these changes, cells collected during different growth phases were examined for their methanogenic properties. Cellular levels of the various methanogenic isoenzymes and functionally equivalent enzymes were also determined. Cells were found to maintain the rates of methanogenesis by lowering their affinity for hydrogen: the apparent K _m ^H2 decreased in going from the exponential to the stationary phase. Simultaneously, the maximal specific methane production rate changed. Levels of H₂-dependent methenyl-tetrahydromethanopterin dehydrogenase (H₂-MDH) and methyl coenzyme M reductase isoenzyme II (MCR II) decreased upon entry of the stationary phase. Cells grown under conditions that favored MCR II expression had higher levels of MCR II and H₂-MDH, whereas in cells grown under conditions favoring MCR I, levels of MCR II were much lower and the cells had an increased affinity for hydrogen throughout the growth cycle. The use of thiosulfate as a medium reductant was found to have a negative effect on levels of MCR II and H₂-MDH. From these results it was concluded that M. thermoautotrophicum responds to variations in hydrogen availability and other environmental conditions (pH, growth temperature, medium reductant) by altering its physiology. The adaptation includes, among others, the differential expression of the MDH and MCR isoenzymes.     

Relationship between the affinity and proteolysis of the insulin receptor. Evidence that higher affinity receptors are preferentially degraded

125I-Insulin binding to rat liver plasma membranes initiated two processes that occurred with similar time courses: an increase of receptor affinity for hormone and degradation of the Mr 135,000 alpha subunit of the insulin receptor to a fragment of Mr 120,000. Inhibitors of serine proteinases prevented alpha subunit degradation without affecting the affinity change. This shows that the change of affinity is not produced by receptor proteolysis and that the intact alpha subunit of the insulin receptor can exist as a higher or lower affinity species. Hormone binding was much more rapid than receptor proteolysis and the initial rate of alpha subunit degradation was independent of the concentration of occupied lower affinity receptors. Only persistent hormone binding and the accumulation of higher affinity insulin-receptor complexes led to significant receptor proteolysis. As the incubation time between 125I-insulin and membranes increased, the rate at which hormone dissociated from Mr 135,000 complexes diminished, whereas hormone dissociated from Mr 120,000 complexes slowly after brief or extended incubations. These observations suggest that 125I-insulin binds to membranes to form low affinity complexes that are not substrates for proteolysis. A slow conformational change produces higher affinity hormone-receptor complexes that are selectively degraded. Thus, the conversion between states of affinity may play a role in the regulation of receptor proteolysis and, consequently, insulin action in cells.     

Role of the phenylalanine B24 side chain in directing insulin interaction with its receptor. Importance of main chain conformation

We have investigated (by use of semisynthetic insulin analogs and isolated canine hepatocytes) the role of invariant residue PheB24 in determining the affinity of insulin-receptor interactions. Our results confirm that replacement of PheB24 by D-Phe is not detrimental to ligand binding to receptor, show that D-Ala is well tolerated at position B24 (whereas Ala is not), and demonstrate that [GlyB24]insulin retains as much as 78% of the receptor binding potency of native insulin. Additional findings show that replacement of PheB24 by D-Pro or by alpha-aminoisobutyric acid results in analogs with severely decreased binding potency, and that the COOH-terminal domain containing residues B26-B30 plays a positive role in determining receptor binding potency in GlyB24-substituted insulin (whereas it plays a negative role in determining the receptor binding potency of its GlyB25-substituted counterpart). We interpret our results as identifying (a) a critical role for the insulin main chain near residue B24 in determining the affinity of receptor for ligand, (b) the importance of main chain flexibility in achieving a high affinity state of receptor-bound hormone, and (c) a potential interaction of the PheB24 side chain with receptor which initiates main chain structural changes in the natural hormone, but which does not itself confer affinity to ligand-receptor interactions.     

Hormone-induced changes in the in vitro DNA-binding activity of the chicken progesterone receptor

Previous analyses have indicated that steroid hormone receptors undergo an allosteric change in structure upon binding by the steroid ligand. This structural change was envisioned as an intramolecular unmasking of the protein's DNA-binding domain, thus allowing the receptor to function in gene regulation. We report an analysis of the effect of hormone on the DNA-binding activity of the chicken progesterone receptor. Using an isocratic elution of DNA affinity columns we show that unliganded receptor (aporeceptor) can bind a 23-basepair progesterone response element with high affinity and a high degree of sequence preference. Hormone causes a 1.5-fold increase in affinity for the PRE sequence and a 2-fold decrease in affinity for non-specific DNA. Kinetic analysis of the off-rate of receptor-DNA complexes is consistent with this minor effect of hormone. In addition, gel retardation analysis of receptor-progesterone response element complexes further substantiates that hormone is not required for sequence-specific DNA binding. These results indicate that hormone is not necessary for the progesterone receptor to fold into a conformation that recognizes specific gene regulatory sequences.     

Development of a thyroid hormone receptor targeting conjugate

Molecular conjugates of hormone receptor-ligands with molecular probes or functional domains are finding diverse applications in chemical biology. Whereas many examples of hormone conjugates that target steroid hormone receptors have been reported, practical ligand conjugates that target the nuclear thyroid hormone receptor (TRbeta) are lacking. TR-targeting conjugate scaffolds based on the ligands GC-1 and NH-2 and the natural ligand triiodothyronine (T3) were synthesized and evaluated in vitro and in cellular assays. Whereas the T3 or GC-1 based conjugates did not bind TRbeta with high affinity, the NH-2 inspired fluorescein-conjugate JZ01 showed low nanomolar affinity for TRbeta and could be used as a nonradiometric probe for ligand binding. A related analogue JZ07 was a potent TR antagonist that is 13-fold selective for TRbeta over TRalpha. JZ01 localizes in the nuclei of TRbeta expressing cells and may serve as a prototype for other TR-targeting conjugates.     

Fatty acyl-CoAs are potent inhibitors of the nuclear thyroid hormone receptor in vitro

We report that long-chain fatty acyl-CoAs are potent inhibitors of the thyroid hormone (T3) receptor isolated from rat liver nuclei. Both saturated and unsaturated fatty acyl-CoAs were similarly potent. Fifty per cent inhibition of T3 binding by the receptor was observed at an oleoyl-CoA concentration as low as 1.3 microM, and the affinity of oleoyl-CoA for the receptor (Ki) was estimated to be 0.45 microM. Fatty acyl-CoAs also promoted dissociation of the hormone bound to the receptor. The action of fatty acyl-CoAs was competitive for the hormone binding site, resulting in a reduction in the receptor's affinity for T3. These observations suggest that fatty acyl-CoAs modulate the binding of the thyroid hormone to its nuclear receptor, in vitro. Whether or not such events occur in vivo remains to be determined.     

Physiological and metabolic functions of melatonin

Melatonin is a lipophilic hormone, mainly produced and secreted at night by the pineal gland. Melatonin synthesis is under the control of postganglionic sympathetic fibers that innervates the pineal gland. Melatonin acts via high affinity G protein-coupled membrane receptors. To date, three different receptor subtypes have been identified in mammals: MT1 (Mel 1a) and MT2 (Mel 1b) and a putative binding site called MT3. The chronobiotic properties of the hormone for resynchronization of sleep and circadian rhythms disturbances has been demonstrated both in animal models or in clinical trials. Several other physiological effects of melatonin in different peripheral tissues have been described in the past years. In this way, it has been demonstrated that the hormone is involved in the regulation of seasonal reproduction, body weight and energy balance. This contribution has been focused to review some of the physiological functions of melatonin as well as the role of the hormone in the regulation of energy balance and its possible involvement in the development of obesity.     

Effect of helix-promoting strategies on the biological activity of novel analogues of the B-chain of INSL3

Insulin-like 3 (INSL3) is a novel circulating peptide hormone that is produced by testicular Leydig cells and ovarian thecal and luteal cells. In males, INSL3 is responsible for testicular descent during foetal life and suppresses germ cell apoptosis in adult males, whereas in females, it causes oocyte maturation. Antagonists of INSL3 thus have significant potential clinical application as contraceptives in both males and females. Previous work has shown that the INSL3 receptor binding region is largely confined to the B-chain central α-helix of the hormone and a conformationally constrained analogue of this has modest receptor binding and INSL3 antagonist activity. In the present study, we have employed and evaluated several approaches for increasing the α-helicity of this peptide in order to better present the key receptor binding residues and increase its affinity for the receptor. Analogues of INSL3 with higher α-helicity generally had higher receptor binding affinity although other structural considerations limit their effectiveness.     

The follicle-stimulating hormone (FSH) receptor in testis: interaction with FSH, mechanism of signal transduction, and properties of the purified receptor

The follicle-stimulating hormone (FSH) receptor purified from calf bovine testis membranes appears to be an oligomeric glycoprotein, consisting of 4 disulfide-linked monomers of molecular weight about 60,000 each. Polyclonal antibodies to the hormone binding sites of the receptor have been developed. FSH interaction with the receptor seems to involve multiple discrete binding regions, which include amino acids 34-37 and 49-52 of the human FSH beta subunit. The interaction between FSH and the membrane-bound receptor is reversible at low temperatures but becomes increasingly irreversible as the temperature increases. FSH interaction with the soluble receptor is reversible over a wider temperature range. The hydrophobic effect is a significant factor in the initial hormone receptor interaction in each system. FSH bound to membrane receptors on cultured immature rat Sertoli cells is internalized and degraded to the level of amino acids. Current evidence suggests that the membrane receptor may exist as free receptor, and complexed with G-protein. A functional receptor/G-protein/adenylate cyclase complex has been reconstituted in liposomes. The G-protein of testis membranes contains both high and low affinity guanosine triphosphate (GTP) binding sites. Both are capable of modulating FSH receptor binding, whereas only the high affinity sites seem to be required for activation of adenylate cyclase. Although testis membranes contain a phosphatidylinositide hydrolysis system, the latter is not directly influenced by FSH.     

Affinities of progestogen and estrogen receptors in rabbit uterus for synthetic progestogens

The affinities of progestogen and estrogen receptors of rabbit uterus for a number of synthetic progestogens in clinical use and some analogues have been measured. Progesterone, 17-hydroxyprogesterone caproate, the chlormadinone, megestrol and cyproterone acetates had similar affinities for the progestogen receptor. Medroxyprogesterone acetate and particularly DU-41164 possessed much higher affinities. 17-hydroxyprogesterone or other agents with a free 17-hydroxyl had much lower receptor affinities than the corresponding 17-esters.Of the nortestosterone derivatives tested, norethisterone equalled progesterone in affinity for the progestogen receptor while d-norgestrel and Wy-4355 were more active. Norethynodrel and ethynodiol diacetate had much lower receptor affinities than progesterone. These results are discussed in relation to possible metabolic bioactivation.Only norethynodrel and ethynodiol (free alcohol) showed marked affinity for the estrogen receptor.     

Purification and protein sequence analysis of rat liver prolactin receptor

Prolactin receptors were purified from rat liver membranes by single-step immunoaffinity chromatography using a specific monoclonal antibody to the rat liver prolactin receptor. Scatchard analysis of 125I-human growth hormone binding to the purified receptor revealed two classes of specific binding sites with Ka = 18.5 x 10(9) and 1.2 x 10(9) M-1. Considering that both classes of binding sites are responsible for high affinity prolactin binding, the partially purified receptor preparation had a binding activity of 1.69 nmol/mg protein, representing 1000-fold purification over microsomal receptors with a recovery of 52%. From three separate purifications, 6 mg of partially purified prolactin receptor were obtained with a purity of approximately 4 to 6.5%. Thus, the use of monoclonal antibody for affinity chromatography resulted in a large improvement of prolactin receptor purification compared to previous hormone affinity chromatography (300-fold purification, 15% recovery). The purified receptor was run on preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis, and a homogeneous preparation of prolactin receptor was obtained by electroelution from gel slices corresponding to Mr 38,000-43,000. Immunoblot analysis using a radiolabeled monoclonal antibody revealed two separate but closely located bands of Mr 42,000 and 40,000 in microsomal, partially purified, and electroeluted preparations. The homogeneous receptor protein was extensively digested with L-1-tosylamido-2-phenylethyl chloromethyl ketone trypsin, and 10 internal amino acid sequences of the rat liver prolactin receptor were determined by gas-phase sequence analysis. Oligonucleotide probes were prepared against two of these internal sequences, and a prolactin receptor cDNA was isolated from a rat liver library using one of these probes (Boutin, J. M., Jolicoeur, C., Okamura, H., Gagnon, J., Edery, M., Shirota, M., Banville, D., Dusanter-Fourt, I., Djiane, J., and Kelly, P. A. (1988) Cell 53, 69-77). The amino acid sequence deduced from the cDNA reveals three potential sites of N-linked glycosylation, two of which were confirmed during protein sequencing. The prolactin receptor was characterized by affinity labeling with 125I-human growth hormone. Cross-linking of microsomes revealed a single band for the hormone-receptor complex with Mr 62,000. On the other hand, cross-linking of Triton X-100-solubilized or partially purified receptor with labeled hormone resulted in the appearance of two bands with Mr 62,000 and 102,000, suggesting the existence of a subunit structure of the prolactin receptor, or alternatively, the existence of two types of prolactin receptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thyroid hormone and DNA binding properties of a mutant c-erbA beta receptor associated with generalized thyroid hormone resistance

We have previously reported a family, Kindred A, with autosomal dominant generalized thyroid hormone resistance in which affected members were found to have a mutation in the carboxy-terminal domain of the c-erbA beta thyroid hormone receptor. In the current study, the thyroid hormone and DNA-binding properties of this mutant receptor were determined using c-erbA beta protein synthesized in vitro. Both the wild-type human placental c-erbA beta and Kindred A receptors bound [125I]-triiodothyronine, although the Kindred A receptor had decreased affinity for the hormone. The affinity for triiodothyronine was 4.5 x 10(9) M-1 and 2.3 x 10(10) M-1 for the mutant and wild-type receptors, respectively. No abnormality of DNA-binding was detected with the Kindred A receptor using a sensitive avidin-biotin DNA-binding assay with DNA fragments containing thyroid hormone response elements. The Kindred A mutant receptor which displays abnormal triiodothyronine-binding but normal DNA-binding activities in vitro acts as a dominant negative inhibitor of thyroid hormone action in man.     

autoregulation of rat adenohypophyseal thyrotropin-releasing hormone receptor

The possible mechanism of attenuation of thyrotropin response to exogenous thyrotropin-releasing hormone after repeated administrations of the releasing hormone has been studied. To this end, the effect of prolonged hormone treatment on the binding of hormone to its receptor in the anterior pituitary gland has been evaluated. The data show that prolonged hormonal treatment resulted in a reduction in the number (B_max) but not the binding affinity (K_D) of the receptor. The effect was reversible and depended on the duration of treatment. This phenomenon of down regulation or the decrease in the receptor number was found not to be due to either the metabolism of releasing hormone or its ability to activate pituitary-thyroid-axis.