Presence of membrane ecdysone receptor in the anterior silk gland of the silkworm Bombyx mori

Abstract

Nongenomic action of an insect steroid hormone, 20‐hydroxyecdysone (20E), has been implicated in several 20E‐dependent events including the programmed cell death of Bombyx anterior silk glands (ASGs), but no information is available for the mode of the action. We provide evidence for a putative membrane receptor located in the plasma membrane of the ASGs. Membrane fractions prepared from the ASGs exhibit high binding activity to [3H]ponasterone A (PonA). The membrane fractions did not contain conventional ecdysone receptor as revealed by Western blot analysis using antibody raised against Bombyx ecdysone receptor A (EcR‐A). The binding activity was not solubilized with 1 m NaCl or 0.05% (w/v) MEGA‐8, indicating that the binding sites were localized in the membrane. Differential solubilization and temperature‐induced phase separation in Triton X‐114 showed that the binding sites might be integrated membrane proteins. These results indicated that the binding sites are located in plasma membrane proteins, which we putatively referred to as membrane ecdysone receptor (mEcR). The mEcR exhibited saturable binding for [3H]PonA (Kd = 17.3 nm, Bmax = 0.82 pmol·mg−1 protein). Association and dissociation kinetics revealed that [3H]PonA associated with and dissociated from mEcR within minutes. The combined results support the existence of a plasmalemmal ecdysteroid receptor, which may act in concert with the conventional EcR in various 20E‐dependent developmental events.

The Steroid Hormone 20-Hydroxyecdysone Enhances Gene Transcription through the cAMP Response Element-binding Protein (CREB) Signaling Pathway

“…Diethylstilbestrol induces the expression and activation of melaninrelated enzymes by activating cAMP/PKA signaling (73). 20E also activates the PKC pathway in B. mori (22,23) and H. armigera (18,24) and the PKA pathway in D. melanogaster (27). The PKC pathway regulates USP phosphorylation, formation of the EcR-USP transcription complex (25,26), and acetylation of USP1 (2) for 20E pathway gene expression.…”

“…The 20E-induced nongenomic pathway has been reported in the Bombyx mori anterior silk gland; this pathway includes the activation of G-protein-coupled receptors (GPCRs), a rapid increase in calcium, and protein kinase C (PKC) activation (22,23). Two ecdysone-responsive GPCRs, ErGPCR1 and ErG-PCR2, transmit the 20E signal in the cell membrane in the lepidopteran insect cotton bollworm H. armigera (18,24).…”

Solubilization of the Ecdysone Binding Protein from Anterior Silk Gland Cell Membranes of the Silkworm, Bombyx mori

“…ASGs were dissected on the day of gut purge (Terashima et al, 2000) and cultured separately in 0.3 ml Grace's insect culture medium (Gibco BRL, Rockville, MD) at 25°C for 18 h with 1 μM 20E, followed by culture in a hormone-free medium for further 12 h. Because the binding activity in membrane fractions prepared from cultured ASGs is higher than that in freshly dissected ASGs (Elmogy et al, 2004), we mainly used cultured ASGs.…”

“…The biologically active form of ecdysone, 20-hydroxyecdysone (20E), binds to a functional nuclear ecdysone receptor consisting of an ecdysone receptor (EcR) and its heterodimeric partner, Ultraspiracle (USP), and thereby controls the transcriptional activity of target genes (Riddiford et al, 2000). We previously provided preliminary evidence for the presence of a putative membrane ecdysone receptor (mEcR) located in the plasma membranes of B. mori ASGs, which may act in concert with the conventional nuclear EcR (Elmogy et al, 2004(Elmogy et al, , 2006. Thus, to further characterize mEcR, we tried to solubilize from ASGs a functional mEcR retaining its binding activity to ecdysteroids.…”

The variety of complexes formed by EcR and Usp nuclear receptors in the nuclei of living cells

“…Consequently, a transcriptionally active EcR/Usp heterocomplex is believed to be the only functional form of the 20E receptor. This paradigm is not consistent with gene repression and activation by apo-and holo-EcR (Hu et al, 2003), dimerization of EcR and Usp with alternative partners, and very recent data suggesting that membraneassociated EcR may be involved in the transmission of non-genomic signals elicited by ecdysteroids (Elmogy et al, 2004). Thus, in contrast to the traditional static view, EcR and Usp appear to be key components of a dynamic network that triggers genomic and nongenomic action in different compartments of a cell (Schlattner et al, 2006).…”