DNA binding properties of the ecdysteroid receptor in the salivary gland of the female ixodid tick, Amblyomma hebraeum.
Salivary gland degeneration in the female tick, Amblyomma hebraeum Koch (Acari: Ixodidae) is controlled by an ecdysteroid hormone. In an earlier study (Mao, H., McBlain, W.A., Kaufman, W.R., 1995. Some properties of the ecdysteroid receptor in the salivary gland of the ixodid tick, Amblyomma hebraeum. Gen. Comp. Endocrinol. 99, 340-348), we demonstrated that a protein component of a salivary gland extract binds to ponasterone A (Pon A) with high affinity (Kd-1 nM), suggesting a tick ecdysteroid receptor (EcR). In this study, the Pon A binding protein bound to calf thymus DNA; this binding could be dissociated by Drosophila hsp27 EcRE. The binding protein shifted the [32P]hsp27 EcRE band on a gel mobility shift assay; formation of the complex with hsp27 EcRE required KCl (optimal concentration was approximately 75 mM). A number of physiologically effective ecdysteroids enhanced the binding with the following order of potency: Pon A > Mur A > Mak A > 20E > ecdysone, whereas vertebrate steroids (estradiol, cholesterol, corticosterone, progesterone, testosterone) had no such effect. Using monoclonal antibodies against Drosophila EcR and USP, we found that AG 10.2 recognized three bands (90.5, 87.3 and 84 kDa for EcR) and AB11 recognized at least two major bands (50.3 and 47.1 kDa for USP) in the salivary gland extract by western blot analysis. In addition, AB11 supershifted the tick EcR-hsp27 EcRE band on a gel mobility shift assay, indicating that the tick EcR heterodimerized with a USP-like protein for DNA binding. Furthermore, selective mutations to the 15-basepair palindrome of hsp27 EcRE at positions-5, + 2, or adding a base to the spacer, resulted in considerably reduced affinity to the tick EcR/USP. We thus propose a sequence similarity of EcREs between A. hebraeum and its insect counterpart.
Mao
H
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Department of Biological Sciences, University of Alberta, Edmonton, Canada.
Kaufman
W R
WR
D000818:Animals; D002417:Cattle; D002482:Cellulose; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005260:Female; D047908:Intracellular Signaling Peptides and Proteins; D007447:Invertebrate Hormones / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D011987:Receptors, Steroid / Q000378:metabolism*; D020218:Response Elements; D012469:Salivary Glands / Q000378:metabolism; D013987:Ticks / Q000378:metabolism*; D000099190:MAP-Kinase-Activated Kinase 2
D002482:Cellulose; D004247:DNA; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D007447:Invertebrate Hormones; D017346:Protein Serine-Threonine Kinases; D011987:Receptors, Steroid; D000099190:MAP-Kinase-Activated Kinase 2; C034808:ecdysteroid receptor