A nuclear juvenile hormone-binding protein from larvae of Manduca sexta: a putative receptor for the metamorphic action of juvenile hormone.

Abstract

A 29-kDa nuclear juvenile hormone (JH)-binding protein from the epidermis of Manduca sexta larvae was purified by using the photoaflinity analog for JH II ([3H] Juvenile hormone (JH), a sesquiterpenoid, regulates both metamorphosis and reproduction in insects (1). In the larva, JH is present throughout the growth phase, and its presence at the time of the ecdysteroid-induced molt is critical to prevent metamorphosis. The primary morphogenetic effect of JH is to guide ecdysteroid action so as to prevent its activation of new programs resulting in a change of form. In the adult, by contrast, JH apparently acts alone to stimulate vitellogenin synthesis in the fat body or vitellogenin uptake into the oocyte. The fat body has high-affinity cytosolic and nuclear-binding proteins for JH, whereas in facilitating yolk uptake, JH apparently acts via a membrane receptor on the follicle cells (for review, see refs. 1 and 2).In the epidermis of tobacco hornworm (Manduca sexta) larvae, one-third of the JH that enters the cell is retained in the nucleus by two proteins (Kd = 7 and 88 nM) (3). Palli et al. (4), using tritiated photoaffinity analogs of JH, identified a 29-kDa nuclear protein and a 38-kDa cytosolic protein that specifically bound JH. The 29-kDa protein was present in the epidermis during the fourth-and fifth-instar growth phases but declined during the molt and disappeared at the time of pupal commitment of the epidermis (5). Its reappearance in both the larval and pupal epidermis after ecdysis depended on the presence of JH during the molt. Our paper reports the purification and subsequent cloning of the cDNA for this

Comparative genomics of insect juvenile hormone biosynthesis☆

“…Takeout is a circadian clock-regulated output gene and has been proposed as a direct molecular link between the circadian clock and the feeding/starvation response in Drosophila (SarovBlat et al, 2000). There are 20 genes of the takeout family in the fruit fly (Dauwalder et al, 2002) and proteins of this family are related to hemolymph JHBPs of Lepidoptera and to the JP29 protein initially thought to represent a JH receptor in M. sexta (Palli et al, 1994). It has been proposed that Takeout participates in a circadian output pathway that conveys temporal and food status information to feeding-relevant metabolism and activities (Sarov-Blat et al, 2000).…”

Hormonal regulation of JP29 in the epidermis during larval development and metamorphosis in the tobacco hornworm,Manduca sexta

“…Yet this rJP29 bound epoxybishomofarnesyldiazoacetate (EBDA), the photoaffinity analog of JH I, and this binding could be prevented by excess JH I or other similar hydrophobic molecules. Immunocytochemistry using a polyclonal antibody prepared against bacterially produced JP29 showed that this protein was located in the nuclei (Palli et al, 1994); however, studies reported below show that it is also present in the insecticyanin granules.…”

“…Immunodetection of JP29 was either with a monoclonal antibody (1:3000) (Charles et al, 1996) or with an affinity-purified polyclonal antiserum (Palli et al, 1994) (1:1000) followed by detection with O-dianisidine (Minnick and Spence, 1988) or the more sensitive Renaissance immunoblot chemiluminescence reagent (DuPont NEN). Immunodetection of insecticyanin was with a polyclonal antibody (1:1000) and O-dianisidine.…”

“…Two to 5 µg total RNA were dotted onto a Duralon membrane (Stratagene), and the membrane was processed as described by Hiruma and Riddiford (1990). The 870 bp BglII-EcoRI fragment was excised from JP29 cDNA (Palli et al, 1994), and labeled using 32 P-dATP (>3000 Ci/mmole, Amersham) and PrimeIT Kit II (Stratagene). The 4.6-kb BglII/BamHI genomic fragment of LCP14 (Rebers and Riddiford, 1988), which contains exons 2, 3, and 4, was labeled similarly.…”

“…Using both MDK and the photoaffinity analog of JH II, epoxyhomofarnesyldiazoacetate (EHDA), Palli et al (1994) isolated the 29 kDa JH binding protein (JP29) and then obtained its cDNA. Partially purified recombinant JP29 (rJP29) produced by baculovirus specifically bound EHDA and appeared to bind JH I with high affinity.…”