Ecdysone receptor

Bombyx and Manduca prothoracicotropic hormone

The prothoracicotropic hormone (PTTH) stimulates the prothoracic glands to synthesize and release ecdysone, and is therefore a key hormone for the regulation of insect moulting and metamorphosis. Bombyx PTTH is a 30 kDa homodimeric glycoprotein, whose carbohydrate moiety is not essential for the biological function. The Bombyx genome contains a single copy of the PTTH gene. PTTH is produced by four dorsolateral neurosecretory cells of brain. Another Bombyx brain peptide exerting prothoracicotropic activity to a heterologous moth Samia cynthia ricini but no activity to Bombyx has been identified and termed bombyxin. Bombyxin is a 5 kDa heterodimeric peptide that shows a high similarity to insulin in the amino acid sequence. The bombyxin gene structure also shows a high similarity with the insulin gene structure. The Bombyx genome contains more than 30 copies of the bombyxin gene. Bombyxin is synthesized by eight dorsomedial neurosecretory cells of brain (Ishizaki, 1994).

Two allelic variants were cloned of the gene for the Bombyx mori prothoracicotropic hormone, a homodimeric 30-kDa brain secretory protein. These PTTH genes contain five exons that encode a precursor protein consisting of 224 amino acid residues whose C-terminal 109 residues represent the PTTH subunit. The Bombyx haploid genome contains a single copy of the PTTH gene. The major site of PTTH expression is the brain but expression at a very low level occurs in the gut. One Bombyx brain at day 0 of the fifth larval instar contained 2.4-2.8 pg PTTH mRNA, and this amount did not change markedly during larval-pupal development (Adachi-Yamada, 1994).

Bombyxin G1 gene, a novel insulin-related peptide gene of the silkmoth Bombyx mori, has been identified. The G1 gene encodes a precursor peptide that shows 41%-56% and 28% sequence identities with preprobombyxins previously characterized and human preproinsulin, respectively. The G1 gene forms a pair with bombyxin C2 gene with opposite transcriptional orientation in a bombyxin gene cluster. The bombyxin G1 mRNA in Bombyx brain has been shown to locate in four pairs of medial neurosecretory cells (Yoshida, 1998).

The 28-kDa size variant of prothoracicotropic hormone (big PTTH) stimulates ecdysteroidogenesis by prothoracic glands of Manduca sexta. Big PTTH stimulates in vitro incorporation of [35S]methionine into proteins of prothoracic glands from Day 7 last instar larvae. In 2-hr incubations, big PTTH elicited an approximately 2-fold increase in total protein-specific activity. The effect appeared to be tissue specific, as big PTTH had no effect on incorporation of label into proteins of control tissue (fat body). Electrophoretic separation of tissue homogenates, followed by autoradiography and densitometric analysis, reveals increased incorporation of radiolabel into numerous glandular proteins. The result suggest that the effect of big PTTH was a general stimulation of protein synthesis, not specific stimulation of a subset of glandular proteins. Big PTTH-stimulated ecdysteroidogenesis was inhibited by cycloheximide, indicating that the increase in protein synthesis is a requisite for enhanced hormone production. Analysis of gland incubation media revealed numerous radiolabeled proteins. The effect of big PTTH on incorporation of [35S]methionine into media proteins was considerably more variable than the effect of big PTTH on tissue incorporation. The result is consistent with the hypothesis that prothoracic glands may release proteins in addition to ecdysteroids (Kulesza, 1994).

Prothoracicotropic hormone (PTTH) is a brain neurosecretory protein that controls insect development. PTTH of the silkmoth Bombyx mori is a homodimeric protein, the subunit of which consists of 109 amino acids. Clear-cut sequence similarity to any other proteins has not been observed. By disulfide-bond pattern analysis and modeling of the PTTH structure based on the known three-dimensional (3D) structures of growth factor family with cystine-knot motif, it is proposed that the PTTH protomer adopts the fold unique to the structural superfamily of the growth factors, beta-nerve growth factor (beta-NGF), transforming growth factor-beta 2 (TGF-beta 2), and platelet-derived growth factor-BB (PDGF-BB). The insect neurohormone PTTH appears to be a member of the growth factor superfamily, sharing a common ancestral gene with the three vertebrate growth factors, beta-NGF, TGF-beta 2 and PDGF-BB (Noguti, 1995).

PTTH also stimulates the specific synthesis of three proteins in the prothoracic glands of the tobacco hornworm Manduca sexta. One of these proteins, p50 is identified as a beta tubulin. The ability of PTTH to stimulate beta tubulin synthesis increased dramatically late on Day 3 of the 10-day fifth larval instar. At this time and later, beta tubulin synthesis in response to PTTH in vitro could be detected in some prothoracic glands 5-10 min after the onset of stimulation, and newly synthesized beta tubulin entered the microtubule pool within 12 min. Levels of beta tubulin in the glands of fifth instar larvae, measured by immunoblot, changed in a tissue-specific manner that paralleled or presaged circulating ecdysteroid levels. The accumulation of beta tubulin in PTTH-stimulated prothoracic glands resulted from increased transcription and translation and not from a decreased protein turnover rate. Pulse-chase experiments indicate that the newly synthesized beta tubulin had a very short half-life in vitro (approximately 0.5 hr). Studies with cycloheximide and actinomycin D indicated that beta tubulin synthesis and ecdysteroid synthesis are coregulated and that beta tubulin synthesis is regulated in a unique manner relative to most other prothoracic gland proteins. Beta tubulin levels may play an important role in ecdysteroidogenesis, perhaps by influencing the dynamics of microtubule-dependent secretion or interorganelle movement of ecdysteroid precursors (Rybczynski, 1995).

Secretion of ecdysteroid molting hormones by insect prothoracic glands is stimulated by neuropeptide prothoracicotropic hormones (PTTH). Studies reported here were conducted to assess the effects of microfilament and microtubule inhibitors on in vitro ecdysteroidogenesis by prothoracic glands of Manduca sexta. Microfilament inhibitors (cytochalasins B and D) have no effect on basal or big PTTH-stimulated ecdysteroidogenesis. Microtubule inhibitors (colchicine, podophyllotoxin, nocodazole) have no effect on basal ecdysteroid secretion, but suppress PTTH-stimulated secretion in a concentration-dependent manner. The effect of nocodazole is partially reversible, suggesting it is not due to nonspecific toxicity. Colchicine has no effect on glandular ecdysteroid levels, indicating that inhibition is not due solely to blockage of secretion. The combined results are consistent with the hypothesis that microtubule-mediated transport of ecdysteroid precursors plays a critical role in stimulation of ecdysteroidogenesis by PTTH (Watson, 1996).

The insect prothoracic glands are the source of steroidal molting hormone precursors and the glands are stimulated by a brain neuropeptide, prothoracicotropic hormone (PTTH). PTTH acts via a cascade including Ca2+/calmodulin activation of adenylate cyclase, protein kinase A, and the subsequent phosphorylation of a 34 kDa protein (p34) hypothesized, but not proven, to be the S6 protein of the 40S ribosomal subunit. The immunosuppressive macrolide, rapamycin, is a potent inhibitor of cell proliferation, a signal transduction blocker, and also prevents ribosomal S6 phosphorylation in mammalian systems. Rapamycin inhibits PTTH-stimulated ecdysteroidogenesis in vitro by the prothoracic glands of the tobacco hornworm, Manduca sexta, with half-maximal inhibition at a concentration of about 5 nM. At concentrations above 5 nM, there is a 75% inhibition of ecdysteroid biosynthesis. Similar results are observed with the calcium ionophore (A23187), a known stimulator of ecdysteroidogenesis. Most importantly, the inhibition of ecdysteroid biosynthesis is accompanied by the specific inhibition of the phosphorylation of p34, indicating that p34 indeed is ribosomal protein S6. In vivo assays reveal that injection of rapamycin into day 6 fifth instar larvae results in a decreased hemolymph ecdysteroid titer and a dose-dependent delay in molting and metamorphosis. When S6 kinase (S6K: see Drosophila RPS6-p70-protein kinase) activity is examined using rapamycin-treated prothoracic glands as the enzyme source and a synthetic peptide (S6-21) or a 40S ribosomal subunit fraction from Manduca tissues as substrate, the data reveal that rapamycin inhibits S6K activity. It is concluded that S6 kinase plays a role in prothoracicotropic hormone stimulation of insect prothoracic glands by targeting ribosomal protein S6 (Song, 1994).

Phosphorylation of ribosomal protein S6 is requisite for prothoracicotropic hormone (PTTH)-stimulated specific protein synthesis and subsequent ecdysteroidogenesis in the prothoracic glands of the tobacco hornworm, Manduca sexta. To better understand the role of S6 in regulating ecdysteroidogenesis, S6 cDNA was isolated from a Manduca prothoracic gland cDNA library and sequenced. The deduced protein is comprised of 253 amino acids, has a molecular weight of 29,038, and contains four copies of a 10-amino acid motif defining potential DNA-binding sites. This Manduca S6 possesses a consensus recognition sequence for the p70(s6k) binding domain as well as six seryl residues at the carboxyl-terminal sequence of 17 amino acids. Phosphoamino acid analysis reveals that the phosphorylation of Manduca prothoracic gland S6 is limited exclusively to serine residues: although alterations in the quantity of S6 mRNA throughout the last larval instar and early pupal-adult development are not well correlated with the hemolymph ecdysteroid titer, developmental expression and phosphorylation of S6 are temporally correlated with PTTH release and the hemolymph ecdysteroid titer. These data provide additional evidence that S6 phosphorylation is a critical element in the transduction pathway leading to PTTH-stimulated ecdysteroidogenesis (Song, 1997).

Development of the corpora allata

The prothoracicotropic hormone (PTTH) is an insect cerebral peptide that stimulates the prothoracic glands to produce ecdysteroids, which initiate moulting and metamorphosis. During the last larval instar of holometabolous insects, a reduction in the hemolymph juvenile hormone levels is a necessary step in initiating larval-pupal transformation. Very low ecdysteroid levels in the early last larval instar of Bombyx mori initiate the complete inactivation of corpora allata. PTTH signal transduction pathways undergo specific developmental changes, with a deficiency in transduction in prothoracic gland cells occurring during the early last instar. Glands from the early last instar show no increase in either cAMP levels or steroidogenesis as a result of PTTH stimulation, indicating the absence of the PTTH receptors in gland cells. It is proposed that this absence of PTTH receptors plays a critical role in directing larval-pupal transformation (Gu, 1996).

A deficiency in prothoracicotropic hormone (PTTH) transduction during the early last larval instar of Bombyx mori has been found to play a role leading to very low ecdysteroid levels in the hemolymph, inactivation of corpora allata, as well as larval-pupal transformation. In the present study, the role of juvenile hormone (JH) in the regulation of PTTH transduction has been clarified. When JH analog (hydroprene) is applied to early last instar larvae, the development of larvae is greatly inhibited. It is not PTTH release, but rather prothoracic gland competency in both cAMP generation and ecdysteroidogenesis that is developmentally inhibited by hydroprene application, as a result of PTTH stimulation. Glands in hydroprene-treated larvae show no response in ecdysteroidogenesis to either PTTH or 1-methyl-3-isobutylxanthine (MIX) until day 7, 4 days later than those of control larvae. JH-I application shows the same effects as those of hydroprene. By contrast, allatectomy on day 0 of the last instar accelerates development, and glands show the activation response to either PTTH or MIX in both cAMP generation and ecdysteroidogenesis 1 day after allatectomy. From these results, it is concluded that the absence of JH is a prerequisite for successful PTTH transduction and for acquisition of the cAMP generating system of gland cells (Gu, 1998).

Ca2+ signaling and PTTH action

Ecdysteroidogenesis in the prothoracic glands of the tobacco hornworm Manduca sexta is stimulated by the cerebral neuropeptide prothoracicotropic hormone (PTTH). PTTH-stimulated cAMP synthesis and ecdysone secretion are dependent on the presence of extracellular calcium, suggesting that PTTH enhances calcium entry into the cytosol. Such entry into the cytosol might involve the opening of a plasma membrane calcium channel, or a mechanism dependent on prior inositol triphosphate (IP3)-mediated release of intracellularly stored calcium. In pupal prothoracic glands, PTTH does not increase IP3 or other inositol phosphates over the course of times ranging from seconds up to 30 min, even in the presence of lithium. However, the L-type calcium channel antagonist nitrendipine completely prevents PTTH-stimulated ecdysone synthesis. A 41 kDa G-protein in prothoracic glands is ADP-ribosylated by pertussis toxin. However, PTTH-stimulated ecdysone synthesis is unaffected by prior exposure to pertussis toxin, indicating that the 41 kDa protein is not involved in the acute stimulation of steroidogenesis. By contrast, cholera toxin has a stimulatory effect on ecdysone secretion, suggesting the involvement of a Gs-like protein (see Drosophila G protein salpha 60A). Based on the absence of PTTH-stimulated inositol phosphate formation in pupal prothoracic glands, it is suggested that calcium mobilization may occur through the opening of a calcium channel, possibly regulated by Gs (Girgenrath, 1996).

Prothoracicotropic hormone (PTTH), a peptide produced by the insect brain, stimulates the prothoracic glands to secrete ecdysteroids. The big form of this peptide (25.5 kDa) has been postulated to act through cyclic AMP in larval Manduca sexta, but the role of the cyclic nucleotide in the action of PTTH in pupal glands has been less clear. PTTH-stimulated ecdysteroid secretion and protein phosphorylation by glands removed from pupal Manduca sexta are blocked by two inhibitors of cAMP-dependent protein kinase: Rp-cAMPS (see Drosophila cAMP-dependent protein kinase 1), an antagonist of cAMP binding to the regulatory subunit of the kinase, and H-89, an inhibitor of the catalytic subunit of the kinase. Further, PTTH stimulates significant accumulation of cAMP in pupal glands, although less than that previously seen in PTTH-stimulated larval glands. Cyclic AMP-dependent protein kinase is found in cytoplasmic and membrane-associated glandular subfractions, as measured by incorporation of radioactively labeled cAMP into the regulatory subunit of the kinase. PTTH enhances cytoplasmic cAMP content and appears to increase the amount of cAMP bound to a cytoplasmic type II regulatory subunit of cAMP-dependent protein kinase. The results indicate that cAMP plays a requisite role in PTTH action in pupal glands, thus arguing in favor of a uniform mechanism of action for the peptide during Manduca development (Smith, 1996).

In Manduca sexta, levels of basal and PTTH-stimulated secretion of ecdysteroids by prothoracic glands in vitro increase with time from day 1 to day 4 of the fifth larval stage. Glandular content of cAMP-dependent protein kinase was analyzed to determine if the enzyme changes in concert with increased secretory response. Photoaffinity labeling with radioactively labeled cAMP reveals a 55-kDa cAMP-binding protein characteristic of the regulatory subunit of type-II cAMP-dependent protein kinase (RII). It appears that RII is one of a limited number of cellular proteins that is phosphorylated in the presence of [gamma-35S]ATP: the thiophosphorylated protein and the photoaffinity-labeled regulatory subunit possess the same M(r) and pI, and thiophosphorylation is blocked by mammalian cAMP-dependent protein kinase inhibitor. From day 1 to day 4 of the fifth instar, glandular content of RII increases in conjunction with increased ecdysteroid secretory capacity. Application of JH analog on day 1 significantly inhibits the observed increase in RII. Catalytic subunit activity does not change from days 1 to 4 of the fifth instar, nor does cellular content of a 34-kDa protein previously shown to be phosphorylated in response to PTTH. While it is unlikely that increased content of RII is solely responsible for enhanced ecdysteroid secretion by the prothoracic glands, it may serve as a convenient marker for investigating the mechanism by which steroidogenic capacity is regulated (Smith, 1993).

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