Sequence Of ACTH Research Articles

The melanocortin-2 receptor of the rainbow trout: Identifying a role for critical positions in transmembrane domain 4, extracellular loop 2, and transmembrane domain 5 in the activation of rainbow trout MC2R

The activation of either teleost or tetrapod melanocortin-2 receptor (MC2R) orthologs requires interaction between the HFRW motif and R/KKRRP motif in the primary sequence of ACTH, and two corresponding sites on the melanocortin 2 receptor. While the HFRW contact site on MC2R appears to involve residues in TM2, TM3, and TM6, several studies on human MC2R point to the EC2/TM5 region of MC2R as a possible location for the R/KKRRP contact site. In this study nineteen single-alanine mutants of rainbow trout (rt) MC2R were made beginning at V153 in TM4, at all positions in EC2 (extracellular loop 2), to F175 in TM5. For twelve of these alanine mutants (i.e., V153, G155, C162, D163, T165, V166, I167, H169, F170, H172, V173, L174), alanine substitution did not have a statistically significant effect on activation of the receptor. For four of these alanine mutations (i.e., V157, M158, F161, K168), while the negative shift in ligand sensitivity was statistically significant, the magnitude of the negative shift in activation was fivefold or less. However, for substitution at V159 in TM4 (negative shift in activation: 110 fold), F171 in TM5 (negative shift in activation: 48-fold), and F175 in TM5 (negative shift in activation: 100 fold), the effect on activation was both statistically significant and may be physiologically relevant. To support this conclusion, a triple alanine mutant of rtMC2R (V159/A, F171/A, F175/A), and this mutant receptor could not be activated by ACTH at concentrations as high as 10−6M. A Cell Surface ELISA analysis indicated that the trafficking of the triple alanine mutant rtMC2R to the plasma membrane was not impaired by the alanine substitutions. Collectively, these observations point to a critical role for TM4 and TM5 in the activation of the rainbow trout melanocortin-2 receptor.

Observations on the ligand selectivity of the melanocortin 2 receptor

The melanocortin 2 receptor (MC2R) is unique in terms of ligand selectivity and in vitro expression in mammalian cell lines as compared to the other four mammalian MCRs. It is well established that ACTH is the only melanocortin ligand that can activate the ACTH receptor (i.e., melanocortin 2 receptor). Recent studies have provided new insights into the presence of a common binding site for the HFRW motif common to all melanocortin ligands. However, the activation of the melanocortin 2 receptor requires an additional amino acid motif that is only found in the sequence of ACTH. This mini-review will focus on these two topics and provide a phylogenetic perspective on the evolution of MC2R ligand selectivity.

α-TC1.9 cells – A model system for analyzing the endoproteolytic processing of POMC

The mouse α-TC1.9 endocrine cell line was used to analyze the amino acid requirements for endoproteolytic processing at the paired basic amino acid cleavage site, K 141R 142 that is N-terminal to the ACTH sequence in the POMC proprotein of the anuran amphibian, Silurana tropicalis. Real-Time PCR analysis of non-transfected α-TC1.9 cells indicated that these cells endogenously express the pc2 (proprotein convertase 2) gene, but do not express the pc1/ 3 (proprotein convertase 1/3) gene or the pomc gene. In addition, immunocytochemical analysis and RIA analysis of non-transfected α-TC1.9 cells did not detect the presence of POMC-related products in these cells. For this study the open reading frame of a S. tropicalis POMC cDNA (wild-type) was placed into an expression vector and transiently transfected into α-TC1.9 cells. Two days after transfection the steady-state levels of α-MSH-related and β-endorphin-related end-products were nearly the same as the steady-state levels of these POMC-related end-products in extracts of the S. tropicalis intermediate pituitary. Transient transfection of either the R 142/A 142 pomc construct or the K 141/A 141 pomc construct completely blocked cleavage at this site and yielded a 6K immunoreactive product that had the ACTH(1–13)NH 2 sequence at the C-terminal end of the fusion protein. However, substitution of an alanine residue at R 137, Q 138, E 139, and N 140 had no effect on cleavage at the K 141R 142 cleavage site. Collectively, these results indicate that secondary structure N-terminal to the K 141R 142 does not appear to influence cleavage at this site. However, both K 141 and R 142 are required for the integrity of this cleavage site. Finally, this study indicates that α-TC1.9 cells should be useful for studying the amino acid requirements for the other endoproteolytic cleavage sites in the S. tropicalis POMC proprotein.

Interaction of adrenocorticotropin peptides with microheterogeneous systems — A fluorescence study

Adrenocorticotropin (ACTH) and α-melanocyte stimulating hormone (α-MSH) are peptides which present many physiological effects related to pigmentation, motor and sexual behavior, learning and memory, analgesia, anti-inflammatory and antipyretic processes. The 13 amino acid residues of α-MSH are the same initial sequence of ACTH and due to the presence of a tryptophan residue in position 9 of the peptide chain, fluorescence techniques could be used to investigate the conformational properties of the hormones in different environments and the mechanisms of interaction with biomimetic systems like sodium dodecyl sulphate (SDS) micelles, sodium dodecyl sulphate-poly(ethylene oxide) (SDS-PEO) aggregates and neutral polymeric micelles. In buffer solution, fluorescence parameters were typical of peptides containing tryptophan exposed to the aqueous medium and upon addition of surfactant and polymer molecules, the gradual change of those parameters demonstrated the interaction of the peptides with the microheterogeneous systems. From time-resolved experiments it was shown that the interaction proceeded with conformational changes in both peptides, and further information was obtained from quenching of Trp fluorescence by a family of N-alkylpyridinium ions, which possess affinity to the microheterogeneous systems dependent on the length of the alkyl chain. The quenching of Trp fluorescence was enhanced in the presence of charged micelles, compared to the buffer solution and the accessibility of the fluorophore to the quencher was dependent on the peptide and the alkylpyridinium: in ACTH(1–21) highest collisional constants were obtained using ethylpyridinium as quencher, indicating a location of the residue in the surface of the micelle, while in α-MSH the best quencher was hexylpyridinium, indicating insertion of the residue into the non-polar region of the micelles. The results had shown that the interaction between the peptides and the biomimetic systems where driven by combined electrostatic and hydrophobic effects: in ACTH(1–24) the electrostatic interaction between highly positively charged C-terminal and negatively charged surface of micelles and aggregates predominates over hydrophobic interactions involving residues in the central region of the peptide; in α-MSH, which presents one residual positive charge, the hydrophobic interactions are relevant to position the Trp residue in the non-polar region of the microheterogeneous systems.

Mutational analysis of evolutionarily conserved ACTH residues

α-Melanocyte stimulating hormone (MSH) and adrenocorticotropin (ACTH)1–24, the minimal ACTH sequence required for full activity, differ only by the 10 C-terminal amino acids of ACTH1–24. Interestingly, these ten C-terminal residues have been highly conserved throughout vertebrate evolution. To understand the functional constraints of these 10 amino acids we analyzed the effects of mutating these residues on steroidogenic activity in vivo and in vitro. Alanine substitutions of some of the first four amino acid residues (the basic core residues KKRR, 15–18) greatly reduces ACTH activity in vitro and in vivo; replacement of mutant alanines at residues 15 and 17 with glutamine residues partially restores ACTH activity. Thus, for ACTH receptor binding and activation, the amino acid residues 15–18 are important for their side chains. Surprisingly, conversion of the five C-terminal residues (20–24) to alanines increases ACTH activity in vivo over that of native ACTH. With respect to receptor binding and activity, the last five amino acid residues are important only for the peptide length they contribute; however, with respect to serum stability, their side chains are significant.

α-Melanocyte Stimulating Hormone in the Modulation of Host Reactions*

I. Introduction α-MELANOCYTE-stimulating hormone (αMSH), a basic tridecapeptide, is derived from the precursor molecule POMC(l). The amino acid sequence of αMSH is identical to the 1–13 (N-terminal) amino acid sequence of ACTH although αMSH has little direct influence on glucocorticoid release from the adrenal gland. αMSH is found mainly in the pituitary, but it also occurs in lower concentrations in the central nervous system (CNS), the skin, and other sites (1). This peptide was named for its effect on pigmentation in amphibian skin, although its distribution in tissues of higher organisms suggests that it is important to other functions. Phylogenetically, αMSH is an “ancient” molecule, little changed over the last few hundred million years, and there is remarkable conservation of its amino acid sequence across species. Evidence that this endogenous neuropeptide is important to limitation of fever and inflammation in mammals, including man, suggests that αMSH has retained a role as a modulator of host r...

Corticostatic peptides

In the last four years corticostatic (anti-ACTH) peptides have been isolated from human, rabbit, guinea pig and rat tissues. These peptides do not act via the cAMP cell signalling system but rather via the inhibition of the binding of ACTH to its receptor most probably through direct competition with the 14–18 sequence of ACTH for receptor binding. ACTH has specific high affinity receptors on adrenal cells but rabbit corticostatin I (CSI) has high capacity, low affinity receptors which are competed for by unlabelled excess CSI but not by excess ACTH. This indicates the presence of specific CSI adrenal cell receptors. The rabbit pituitary, hypothalamus, thalamus, adrenals, lungs and placenta contain sizeable amounts of immunoassayable CSI. Immunochemical localization of CSI indicates that it is present in the large macrophages and in neutrophils in rabbit lung, in macrophages and “supporting” endothelial cells in the spleen and in the adrenals in the cells of the zona reticularis. We have also isolated and identified new peptides which contain 12 cysteines from immune cells of humans, rats and a teleost, the carp. The functions of these peptides are now being determined. This large family of peptides may have many other, yet unidentified functions but at present we can only describe a small number of these.

Isolation and full structural characterisation of six adrenocorticotropin‐like peptides from porcine pituitary gland

A partially purified fraction of extracted porcine pituitary glands which possesses lipolytic and adrenocorticotropic activity has been characterised. It consists of six adrenocorticotropin(ACTH)-like peptides (five of which have not been previously described) which were each purified by sequential reverse-phase (rp) HPLC. Their complete primary structures were determined following amino acid compositional analysis, extensive peptide mapping and partial sequencing. Four of the fragments represent the following ACTH fragments; ACTH(1-31), ACTH(7-34), ACTH(7-36) and ACTH(7-38). By combined analytical rpHPLC and an ACTH radioimmunoassay (with an antiserum exhibiting full cross-reaction with all six ACTH variants isolated here), evidence was obtained from analysis of extracts of whole pituitary that these fragments of ACTH exist in significant amounts relative to intact ACTH(1-39). This suggests that ACTH can undergo more extensive differential proteolytic processing than previously thought. These peptides were found to possess reduced or a complete absence of ACTH-like biological activity. Therefore the biological significance of this processing needs to be resolved. The other two fragments also resembled fragments of ACTH but each possessed the same, single amino acid substitution: a threonine replacing the arginine at the position corresponding to position 8 in the ACTH sequence and had the structures [Thr8]ACTH(1-31) and [Thr8]ACTH(7-31). They possess little ACTH-like biological activity. If these variants are derived from a variant ACTH, this would be a significant finding in view of the site of the amino acid substitution and the highly conserved nature of the ACTH primary structure. The possible physiological and genetic implications are briefly discussed. In this study attempts were also made to identify the DNA coding for the mutant ACTH sequence.

CUSHING'S SYNDROME, IDENTIFICATION OF ACTH SOURCE BY SELECTIVE CATHETERIZATION AND ACTH AND β-LPH ASSAYS

The differential diagnosis of ACTH dependent Cushing's syndrome (85% of patients) can be very difficult. Localization of an ACTH source is not always possible with CAT-scan and MRI imaging. A bilateral catheterization of the inferior petrosal sinus with blood sampling for ACTH/LPH during a CRH test has been used for localizing a pituitary adenoma. ACTH was measured with a solid-phase antiserum against the 11-24 sequence of ACTH (sensitivity 16-32 pg/ml). For β-LPH, the reagents of NIAMDD have been used. Samples were obtained before and at 5-min. intervals for up to 30 min. after injection of CRH, 100 ug/1.73 m2. In 18 of 21 patients, a pituitary ACTH source could be proven and in 16 an adenoma localized. In 12 of 18, peripheral ACTH levels were <100 pg/ml (6 <40 pg/ ml). Mean basal/peak ACTH (LPH) pg/ml: Periphery 87/256 (551/845), tumorside 899/1870 (3424/11572), contralateral 427/647 (1467/ 2034). In 6 of 18 patients, only CRH stimulated levels pointed to the correct side. The mean Δ ACTH (LPH)(ipsilateral to contralateral) increased from 172 (1957) before to 1223 (9529) after CRH. In conclusion, selective catheterization and ACTH/LPH assay after CRH is valuable for detecting the ACTH source and thus allows a selective surgery, preserving the pituitary tissue, which is of outmost importance, especially in the pediatric age group.

Use of the silylation reaction in synthesis of fragment 1?4 of the ACTH sequence

A new effective method of synthesizing fragment 1–4 of the ACTH sequence ensuring a high overall yield of the desired product is proposed. This result is achieved thanks to the wide use of the silylation reaction in the synthesis, which has permitted a considerable simplification of the process and the avoidance of the formation of by-products. The peptides synthesized have been characterized by their angles of optical rotation, chromatographic mobilities, and melting points. A table of chemical shifts in the13C NMR spectra of the final and intermediate compounds is given.

Evidence for local stimulation of ACTH secretion by corticotropin-releasing factor in human placenta

The hypothalamic-pituitary-adrenocortical axis is activated in pregnancy and parturition. Levels of immunoreactive corticotrophin releasing factor (irCRF), immunoreactive adrenocorticotropic hormone (irACTH) and cortisol concentrations in maternal plasma are elevated throughout gestation, increase further during labour and fall precipitously after parturition. The placenta contains biologically active CRF and ACTH and it has been suggested that the placenta produces these peptides during pregnancy. Here we show that irCRF is located in the cytotrophoblast cells of placenta collected at term. Using a monolayer primary culture of human placental cells we have found that CRF stimulates secretion of peptides containing the ACTH sequence in the placenta in a dose-dependent manner, as it does in the pituitary. This effect is reversed by a CRF antagonist and is mimicked by dibutyryl cyclic AMP and forskolin. Glucocorticoids, which suppress the secretion of pituitary ACTH, were found to have no influence on release of irACTH by the placenta. Oxytocin and prostaglandins stimulate irACTH and irCRF secretion from cultured placental cells and the irACTH-releasing activity of two prostaglandins is partially reversed by a CRF antagonist. Thus CRF may be involved in the paracrine regulation of placental irACTH secretion.

Neurotensin-like immunoreactivity in the pituitary and hypothalamus of bony fishes.

Using an antiserum raised against synthetic neurotensin (NT), the distribution of immunoreactivity in the pituitary and hypothalamus has been examined by immunocytochemistry at light and electron microscope level in a number of species of bony fishes. In most species immunoreactive perikarya were found in the preoptic region of the hypothalamus, with fibres throughout the tuberal hypothalamus and neurohypophysis (neural lobe and median eminence). In the neurohypophysis of teleosts NT-like immunoreactivity was seen in a dense band of fibres bordering the ACTH cells of the rostral pars distalis: absorption controls showed that this was due to the presence of an NT(8-13)-like or xenopsin-like sequence, which, according to electron microscopic observations, was contained in small dense cored vesicles. The antiserum also stained the pituitary ACTH cells of some species, apparently due to cross-reaction with the 17-19 sequence of ACTH. These results suggest that an NT-like peptide may have a role in control of the adenohypophysis in fishes.

Selection and optimisation of monoclonal antibodies for a two-site immunoradiometric assay for ACTH

Four monoclonal antibodies with predominant specificities differences within the ACTH molecule were investigated in a 2-site immunoradiometric assay (IRMA) for human ACTH. Antibody 3H9 recognises the extreme N-terminal sequence, antibodies 1A12 and 1D1 are specific for the mid N-terminal sequence but differ in that the former cross-reacts with αMSH whereas the latter does not, and antibody 2A3 recognises the C-terminal sequence. Combination of iodinated antibodies with antibodies covalently linked to Sephacryl S300 were tested for their compatibility and potential for a sensitive assay. Two antibody combinations (1D1 plus 3H9 or 1A12)_gave no dose-response curve indicating severe steric inhibition, whereas other combinations yielded assays with widely different detection limits (2–2400 ng ACTH/1). The combination of labelled 1D1 and solid-phase 2A3 gave the most sensitive assay and when optimised for antibody concentrations and incubation times the working range was 10−5 × 10 4 ng/l (CV < 20%). The optimised sequential 2-step IRMA involves incubation of standard or test sample with labelled 1D1 for 18 h at 4°C followed by incubation with solid-phase 2A3 for 2 h at room temperature, after which the labelled complex is separated by the sucrose layering technique. The detection limit of this IRMA was several 100-fold lower than by RIA using the same antibodies. The IRMA detected large molecular weight precursors containing the full ACTH sequence (22 000, 31 000 and 34 000) but not ACTH fragments (1–18, 1–24, 19–39). It is concluded that selected monoclonal antibodies provide a sensitive and rapid 2-site IRMA for intact ACTH and its precursors.

Demonstration of in vivo synthesis of pro-opiomelanocortin-, beta- endorphin-, and alpha-melanotropin-like species in the adult rat brain

An in vivo labeling technique was utilized to demonstrate the in situ biosynthesis of pro-opiomelanocortin (POMC)-, beta-endorphin- and alpha-melanotropin (MSH)-like molecular species in rat brain. Unrestrained adult female rats were bilaterally cannulated in the hypothalamic arcuate nuclear region; [35S]methionine was infused either over a 15-min period with sacrifice 2 hr subsequently, or at a constant rate for 6 hr prior to sacrifice. Sequential immune-affinity chromatography and several chromatographic and electrophoretic techniques were employed to detect and characterize POMC-related material in the hypothalamic arcuate nuclear region, preoptic area, and median eminence. Four molecular species containing both corticotropin (ACTH) and beta-endorphin antigenic determinants within the same molecules were detected in the arcuate nuclear region and preoptic area. Two forms were similar to rat pituitary POMC with respect to apparent molecular weight (35,000 and 31,500) and [35S]methionine-containing tryptic fragments (one methionine each in N-terminal glycopeptide, ACTH, and beta-endorphin sequences of rat POMC). The other two forms (apparent Mr of 21,000 and 19,000) contained only the labeled tryptic fragments characteristic of ACTH and beta-endorphin. The detection of the latter forms suggests that POMC in brain, unlike its post-translational processing in rat pituitary, undergoes primary cleavage between the N-terminal peptide and the ACTH sequence. Peptides physicochemically indistinguishable from authentic beta-endorphin and des-acetyl alpha-MSH were detected in approximately equimolar amounts in all three brain regions. The ratio of POMC-like material to the alpha-MSH- and beta-endorphin-sized peptides was highest in the arcuate nuclear region, suggesting that POMC-like proteins are synthesized in the arcuate nuclear region and are processed into the smaller molecular species during axonal transport.

Alpha-melanocyte stimulating hormone increases plasma levels of glucagon and insulin in rabbits

Intravenous injections of 25 and 2.5 μg alpha-melanocyte stimulating hormone (α-MSH) increased plasma levels of glucagon, insulin and free fatty acids in fasted and fed rabbits. 45 μg beta-melanocyte stimulating hormone (β-MSH) had similar effects, whereas 22 μg gamma-2-melanocyte stimulating hormone (γ-MSH) was inactive. The α-MSH-induced increases in the plasma levels of glucagon, insulin and free fatty acids were not inhibited by alpha- or beta-adrenergic blocking drugs. The α-MSH-induced increases in the plasma levels of insulin were, however, augmented by phentolamine (an alpha-adrenergic receptor blocking drug). The plasma levels of glucose were increased by 25 μg α-MSH in fed rabbits, only, and were decreased by α-MSH during alpha-receptor blockade. The acute in vivo effects of α-MSH and β-MSH on the plasma levels of glucagon, insulin and free fatty acids were rather similar to those previously reported for corticotropin (ACTH). It is possible that the 4–10 ACTH sequence, present in α-MSH, β-MSH and ACTH, but not in γ-MSH, is a message sequence for the observed effects. However, ORG 2766, a 4–9 ACTH analogue, was inactive. The mechanism by which α-MSH increased the plasma levels of glucagon and insulin in rabbits remains to be determined. It is possible, that the effects were mediated by both a central nervous action and a direct action on the endocrine pancreas.

Co-occurrence of immunoreactive corticotrop in-like and α-melanotropin-like material in pituitary cells: Differences between young and adult rats

We examined to which extent adrenocorticotropin (ACTH)-like substances are cleavable into α-melanotropin (MSH)-like material by application of exogenous enzymes on pituitary sections prior to immunocytochemical stainings. In pro-opiomelanocortin (POMC) cells of the anterior lobe from developing rats (1 to 21 postnatal days) at least one of two cells contained α-MSH-like immunoreactivity besides ACTH-like immunostaining. Pretreatment of these tissue sections with tryptic enzymes enhanced remarkably α-MSH-like immunostaining. In contrast, α-MSH-lik immunoreactivity was almost not detectable in anterior lobes from adult rats, also after treatment with enzymes. After adrenalectomy (ax) POMC cells in anterior lobes behaved similarly: a few days after the operation almost all ACTH cells contained also α-MSH immunostaining, whereas after more than two weeks α-MSH immunostaining was nearly absent, also after application of enzymes. In contrast to rats, in young and adult guinea pig pituitaries all ACTH-like immunoreactivity was prevented by treatment with tryptic enzymes. These observations are consistent with the assumption that occurrence of ACTH- and α-MSH-like substances in rat pituitaries is regulated by a protection mechanism against proteolytic cleavage in the ACTH sequence which appears with delay in differentiating and maturing cells.

Use of 2-alkoxy-1-alkoxycarbonyl-1,2-dihydroquinolines as condensing agents in the synthesis of intermediate fragments of the 1?4 sequence of ACTH

The use of 2-alkoxy-l-alkoxycarbonyl-1,2-dihydroquinolines as a condensing agent in a number of stages in the synthesis of fragments of the 1–4 sequence of ACTH is considered. In a number of cases, these compounds have successfully replaced dicyclohexylcarbodiimide, which is a strong allergen. All the compounds described were obtained in satisfactory yields and had fairly high purities. They were characterized from their angles of optical rotation, chromatographic mobilities, and melting points. The conditions for performing the reaction and some physicochemical characteristics of the compounds synthesized are presented.

Use of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline as condensing agent in the synthesis of fragment 20?24 of the sequence of ACTH

Using 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline as condensing agent, synthesis of intermediate compounds and of the whole fragment 20–24 of the sequence of ACTH has been successfully performed. The products synthesized were obtained with good yields and in fairly high purity. They were characterized by their chromatographic mobilities, angles of optical rotation, melting points, and13C NMR spectra. The angles of optical rotation, melting points, and chromatographic mobilities of the fragments synthesized are given.

Identification of N- and C-terminal corticotropin peptides in the Mr 80 000 form of neurophysin

The 125I-labeled M r 80 000 form of neurophysin has been purified from bovine neurohypophysi. Tryptic digests of this species were analyzed, prior to or after treatment with carboxypeptidase B, by high-pressure liquid chromatography followed by isoelectric focusing and the fragments compared with those generated by a similar treatment of reference bovine 1–39 adrenocorticotropin. The ACTH peptides 22–39 and 1–8, as well as the 1–7 derivative of the latter were identified by those two independent criteria. This provides chemical evidence supporting the hypothesis [8] that high M r neurophysin may contain the sequence of ACTH.

Use of pentafluorophenyl esters in the synthesis of intermediate fragments of ACTH

The results are given of the synthesis of the heptapeptide 13–19 of the natural sequence of ACTH using, in a number of stages, the pentafluorophyl esters of amino acids and peptides in combination with the temporary silyl protection of the carboxy group of the amino components. The intermediate compounds were obtained with good yields (70–90%) in chromatographically homogeneous form and their physicochemical characteristics did not differ from those of similar products obtained by other methods and described in the literature. The identification and checking of the purity of the compounds synthesized was carried out not only by traditional methods but also by the13C NMR method and by high-pressure liquid chromatography. Some physicochemical characteristics of the compounds synthesized are given.