Juvenile hormone receptors in insect larval epidermis - Europe PMC

Proc. Nati. Acad. Sci. USA Vol. 87, pp. 796-800, January 1990 Biochemistry

Juvenile hormone receptors in insect larval epidermis: Identification by photoaffinity labeling (epoxybishomofarnesyl diazoacetate/epoxyhomofarnesyl diazoacetate/methoprene diazoketone/DNA binding protein/Manduca sexta)

SUBBA R. PALLI*, ELLIE 0. OSIR*t, WAI-SI ENGt§, MARCUS F. BOEHMt¶, MARTEN EDWARDS*, PETER KULCSARt I, ISTVAN UJVARYt**, KIYOSHI HIRUMA*, GLENN D. PRESTWICHttt, AND LYNN M. RIDDIFORD*tt *Department of Zoology, University of Washington, Seattle, WA 98185; and tDepartment of Chemistry, State University of New York, Stony Brook, NY 11794-3400

Communicated by Fotis C. Kafatos, October 26, 1989 (receivedfor review March 20, 1989)

ABSTRACT Tritiated photoaffinity analogs of the natural lepidopteran juvenile hormones, JH I and H {epoxy[3H]bishomofarnesyl diazoacetate ([3HJEBDA) and epoxy[3H]homofarnesyl diazoacetate ([3H]EHDA)}, and of the JH analog methoprene {[3H]methoprene diazoketone ([3H]MDK)} were synthesized and used to identify specific JH binding proteins in the larval epidermis of the tobacco hornworm (Manduca sexta). EBDA and EHDA specifically photolabeled a 29-kDa nuclear protein (pI 5.8). This protein and a second 29-kDa protein (pI 6.0) were labeled by MDK, but excess unlabeled methoprene or MDK only prevented binding to the latter. These 29-kDa proteins are also present in larval fat body but not in epidermis from either wandering stage or allatectomized larvae, which lack high-affinity JH binding sites. A 29-kDa nuclear protein with the same developmental specificity as this JH binder bound the DNA of two larval endocuticle genes. A 38-kDa cytosolic protein was also specifically photolabeled by these photoaffinity analogs. The 29-kDa nuclear protein is likely the high-affiity receptor for JH that mediates its genomic action, whereas the 38-kDa cytosolic protein may serve as an intracellular carrier for these highly lipophilic hormones and hormone analogs.

finity analog of JH I, was first synthesized from the vinyl oxirane (R = C2H5) (Fig. 1 Middle) (8). Selective reduction of the vinyl group with carrier-free 3H gas was mediated by tris(triphenylphosphine)chlororhodium in benzene solution to give a 40o radiochemical yield of the chiral, ditritioethyl oxirane compound with a specific activity of 55-58 Ci/mmol (1 Ci = 37 GBq) (9). The hydrolysis of the acetate and introduction of the photolabile diazoacetate group were performed as described for the synthesis of racemic 10,11epoxy[3H]farnesyl diazoacetate ([3H]EFDA) (10). [3H]EHDA, the corresponding photoaffinity analog of JH II, was synthesized from a homologous vinyl oxirane, where R = CH3 (Fig. 1 Middle). More recently, [3H]EBDA and [3H]EHDA were prepared directly from [3H]JH I and [3H]JH II, respectively, by selective reduction and diazoacetylation (11). Unlabeled MDK was prepared from (7S)-methoprene (Zoecon, Palo Alto, CA) (Fig. 1 Bottom). A 500-mg sample (1.86 mmol) of methoprene acid, prepared by basic hydrolysis of methoprene with methanolic KOH, was dissolved in 2 ml of dry benzene at 0°C and treated with 470 mg (3.73 mmol) of distilled oxalyl chloride. The solution was warmed to room temperature for 1 hr; the benzene and excess oxalyl chloride were then removed in vacuo. The reaction flask containing the crude acid chloride was cooled to 0°C, and a cold, anhydrous solution of diazomethane in ether was added rapidly. The mixture was stirred for 20 min and brought to ambient temperature. The excess diazomethane was decomposed with a dilute acetic acid solution and the solvent was removed. Purification (10% ethyl acetate/hexane, silica gel) gave 350 mg (1.2 mmol, 65% yield) of MDK. TLC:Rf (10% ethyl acetate/hexane) = 0.29; 1H NMR (CDC13, 300 MHz) 8 0.85 (d, 6.6 Hz, C-8 CH3), 1.14 (s, C-12 CH3 + H-13), 2.28 (d, J = 1.2 Hz, C-4 CH3), 3.15 (s, OCH3), 5.16 (s, H-1), 5.68 (s, H-3), 6.02 (m, H-5 + H-6). 13C NMR (CDC13, 75 MHz) 14.4 (C-4 CH3), 19.7 (C-8 CH3), 2.13 (C-7), 25.0 (C-13 + C-12 CH3), 33.1 (C-8), 37.2 (C-10), 40.0 (C-11), 50.8 (C-9), 49.1 (OMe), 57.2 (C-1), 74.5 (C-12), 123.1 (C-3), 135.0 (C-5), 137.4 (C-6), 150.0 (C-4), 186.0 (C-2).

The juvenile hormones (JHs) of insects are sesquiterpenoid molecules that prevent metamorphosis of larvae and in most insects also regulate reproductive maturation in the adult (1, 2). The action of JH in the larva is manifest only in the presence of the molting hormone ecdysone (1). The presence of JH allows the ecdysteroids to modulate ongoing gene expression but prevents activation of new genes and thus metamorphosis. In the larval epidermis of the tobacco hornworm, Manduca sexta, JH I, the naturally occurring hormone in lepidopteran larvae (3), and iodovinylmethoprenol, an iodinated derivative of methoprene (4) and biologically active JH analog (5), are taken up by the epidermis. Approximately 33% is bound in the nucleus by two binding components, one that shows high affinity and about 10,000 sites per nucleus (6). These studies further suggested that the binding sites were different for the natural JH homologs and the dodecadienoate analogs since neither competed with the other for binding. We now show, by using photoafflinity analogs (7) of these compounds, that the two types of compounds specifically bind to two different 29-kDa nuclear proteins. This nuclear protein complex is only present in epidermis that shows specific binding of JH and appears to bind to two Manduca larval cuticle genes.

Abbreviations: JH, juvenile hormone; EBDA, epoxybishomofarnesyl diazoacetate; EFDA, epoxyfarnesyl diazoacetate; EHDA, epoxyhomofarnesyl diazoacetate; MDK, methoprene diazoketone. tPresent address: International Center for Insect Physiology and Ecology, P.O. Box 30772, Nairobi, Kenya. §Present address: Research Laboratories, Merck Sharpe & Dohme, West Point, PA 19486. Present address: Department of Chemistry, Columbia University, New York, NY 10027. Present address: Research Institute for Plant Protection, P.O. Box 102, H-1525 Budapest, Hungary. **Present address: Department of Entomology, University of California, Berkeley, CA 94720. ttTo whom reprint requests should be addressed.

MATERIALS AND METHODS Synthesis of Photoaffinity Analogs. Labeled 10R,llS-epoxybishomofarnesyl diazoacetate ([3H]EBDA), the photoafThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

796

Proc. Natl. Acad. Sci. USA 87 (1990)

Biochemistry: Palli et al. 0

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FIG. 1. (Top) Structure of the two natural lepidopteran larval JHs, JH I and JH II. (Middle) Preparation of 3H-labeled photoaffinity analogs of JH I and JH II, EBDA and epoxyhomofamesyl diazoacetate (EHDA), respectively, from a chiral vinyl oxirane precursor. (Bottom) Synthesis of [3H]methoprene diazoketone ([3H]MDK) from (7S)-methoprene.

To prepare [3H]MDK, a 250-mCi portion (0.91 mg, 0.0029 mmol) of [3H]-(7S)-methoprene (84 Ci/mmol) (12) was dissolved in 1 ml of methanol containing 0.2 ml of 2 M KOH in methanol and heated for 1 hr at 60°C. The reaction mixture was cooled and then concentrated in vacuo, 1 ml 1 M HCI was added, and the residue was extracted with ether (three times 2 ml). The extracts were dried (MgSO4), concentrated in vacuo, and purified by flash chromatography on silica gel with 5% ethyl acetate/hexane elution to give 150 mCi (0.47 mg, 0.0018 mmol) of thin-layer chromatography (TLC)homogeneous [3H]methoprene acid. This entire sample was dissolved in anhydrous benzene and treated with 0.2 ml (100-fold excess) of oxalyl chloride. The reaction was stirred for 1 hr and solvent and excess reagent were removed in vacuo. After addition of a cold, anhydrous solution of excess diazomethane in ether (dried over MgSO4), solvents were removed under a stream of nitrogen. Flash chromatography of the residue on silica gel with 5% ethyl acetate/hexane afforded 50 mCi (20% overall radiochemical yield) of homogeneous [3H]MDK that coeluted on TLC with the unlabeled MDK produced above. Unlabeled and labeled hormones were checked periodically by TLC or radio-TLC for purity; radioligands were repurified, when necessary, by silica gel chromatography or HPLC. Animals, Bioassay, and Tissue Preparation. M. sexta larvae were reared on artificial diet at 26°C in a 12:12 light:dark photoperiod as described (5). The photoaffinity analogs were assayed for JH activity on the black larval mutant of Manduca (13). The epidermis of day 1 fifth instar larvae (3.0-4.5 g) was dissected and homogenized in cold buffer A [20 mM Tris, pH 7.9, containing 50 mM KCI, 300 mM sucrose, and 1 mM (each) EDTA, dithiothreitol, phenylmethylsulfonyl fluoride, and diisopropyl phosphofluoridate] (6). The crude nuclear pellet (1000 x g, 30 min) was washed three times with 1 ml of buffer A and then washed three times with 1 ml of buffer C (6) [20 mM Tris, pH 7.9, containing 5 mM magnesium acetate and 1 mM (each) above inhibitors]. This pellet was highly enriched for nuclei as judged by phase-contrast mi-

croscopy. Nuclei were extracted with 0.5 M KCI in buffer C (100-200 Ag of DNA per ml) for 2 hr at 00C with occasional mixing. After centrifugation (12,000 x g, 15 min, 40C), the supernatant was dialyzed against buffer C (12 hr, three changes) to remove the salt. The 1000 x g epidermal supernatant was subjected to further centrifugation at 100,000 x g for 1 hr to obtain the

cytosolic fraction. Protein concentrations were determined colorimetrically (14). DNA concentrations were determined fluorimetrically using Hoechst dye 33258 (15). Photoafflinity Labeling. All glassware was precoated with 1% (wt/vol) polyethylene glycol (Mr 20,000; Sigma). Solutions of the labeled photoaffinity analogs and of the unlabeled competitors [JH I and JH II (Sigma) and (7S)-methoprene (Zoecon)] were dissolved in buffer C by sonication (Branson sonicator bath) for 10 min at 0C followed by a 2-hr period at room temperature. The concentration of the labeled photoaffinity analogs was determined by liquid scintillation counting and that of the unlabeled hormones and analogs was determined spectrophotometrically using the following extinction coefficients: JH I and JH II, -217nm = 14,800 in methanol; methoprene, C261nm, 28,000 in hexane. For photoaffinity labeling, an aliquot of the nuclear (40 ,tg) or cytosolic (20 Iug) protein or an aliquot of intact nuclei containing 50-100 1Lg of DNA was added to buffer C (total volume, 400 p.l) containing the labeled photoaffinity analog with or without an excess of unlabeled hormone or analog in a 0.5-ml quartz tube and incubated either 2 hr at 21'C or 10-12 hr at 40C. One milligram of gamma globulin per milliliter was added to the cytosolic fraction to reduce nonspecific binding. Then the samples were irradiated 30 sec at 254 nm (Rayonet reactor). The same pattern of labeled proteins was seen for photolysis times between 30 sec and 2 min. Efficiency of labeling was found to be 12-14% from 10-100 nM solutions of the various analogs. After irradiation, the 0.5 M KCI nuclear extracts and the cytosolic extracts were lyophilized. Whole nuclei were sonicated, and the resultant 15,000 X g supernatant (15 min, 4°C) was lyophilized.

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Biochemistry: Palli et al.

Electrophoresis. Proteins were electrophoresed on 10% SDS/polyacrylamide gels or on nonequilibrium pH gradient gels, followed by 10% SDS gels (16) followed by fluorography using EN3HANCE (New England Nuclear). DNA Binding Studies. Electrophoretically separated proteins from the 0.5 M KCl nuclear extract were electroblotted to nitrocellulose (17); then the filter was preincubated overnight in 10 mM Tris (pH 7.5), 1 mM EDTA containing 0.02% (each) of bovine serum albumin, Ficoll, and polyvinylpyrrolidone (18). It was then exposed to 10 ng of [32P]dATPoligo-labeled (19) DNA (106 cpm/ml) for 4 hr at 21'C in the same buffer containing 250 .ug of herring sperm DNA per ml, followed by three washes with buffer containing 50 mM NaCl (18) and autoradiography on X-Omat AR film. The Manduca genomic clones used were as follows: (i) 26-10, an 8-kilobase (kb) fragment containing the entire gene coding for a 14-kDa cuticular protein (LCP14) and 2 and 3 kb of the 3' and 5' flanking regions, respectively (20); (ii) pGFH-17, a 2.9-kb fragment containing the entire larval cuticle protein LCP16/ 17 gene II and 0.24 and 0.6 kb of the 3' and 5' flanking regions, respectively (21).

RESULTS Activity of Photoaffinity JH Analogs. In the Manduca black larval assay for JH, EBDA had an ED50 of 1.4 pmol (n = 15 for each of four doses), as compared to 1.5 pmol for JH I (5), and EHDA had an ED50 of 17 pmol (n = 20 per dose), as compared to 1.8 pmol for JH II (10R,11S) (5). MDK (ED50 = 4.5 pmol) was half as active as (7S)-methoprene (ED50 = 2.0 pmol) (5). Photoaffinity Labeling of Nuclear and Cytosolic Proteins. When either [3H]EBDA or [3H]EHDA was incubated with isolated epidermal nuclei or with nuclear proteins extractable with 0.5 M KCl and then the mixture was irradiated at 254 nm, only a 29-kDa protein was covalently modified by the photoaffinity analog (Fig. 2 Upper). This binding was reduced 5-fold by 100-fold excess and was prevented by 250-fold excess of either unlabeled JH I or JH II but not by excess methoprene (Fig. 2 Upper). These analogs also bound specifically to the 32-kDa hemolymph JH binding protein (22). The 29-kDa protein in the nuclear extract was also covalently modified by MDK (Fig. 2 Upper). In this case, binding was only partially prevented by 250-fold excess of MDK or methoprene. No reduction of binding was seen with excess JH I. Even 1-hr preincubation with 1000-fold excess of methoprene or MDK failed to eliminate the MDK binding (data not shown). Several other nuclear proteins were also labeled by MDK, but these were also labeled to a variable extent without photolysis (Fig. 2 Upper), indicating some nonspecific chemical modification by this diazoketone. When the photoaffinity-labeled nuclear proteins were separated on a two-dimensional gel (Fig. 2 Lower), only one 29-kDa protein (pI 5.8) was labeled by [3H]EHDA; 250-fold excess of JH II prevented this labeling. By contrast, [3H]MDK labeled two 29-kDa proteins (pI 5.8 and 6.0); excess methoprene only prevented binding to the pI 6.0 protein (Fig. 2 Lower). JH I had no effect on MDK binding to either protein (data not shown). Thus, there appear to be two specific 29-kDa binding proteins, one for the natural JH homologs and one for the dodecadienoate analog. Since MDK also binds nonspecifically to the pI 5.8 protein, its specific binding to the second protein is obscured on onedimensional gels (Fig. 2 Upper). Since [3H]MDK has higher specific activity, we took advantage of its high affinity for the natural JH binding protein for studies of this protein's temporal distribution (see below). The cytosolic fraction contained 29-kDa and 38-kDa polypeptides that were photolabeled by all three photoaffinity analogs and a 55-kDa protein photolabeled by MDK (Fig. 3).

Proc. Natl. Acad. Sci. USA 87 (1990) Nuclei

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M. sexta larvae. (Upper) Photolabeling of hemolymph (Hemo) (5 ,ug) or of nuclear proteins (40 ,.g) by 10 nM [3H]EBDA, [3H]EHDA, or

[3H]MDK in the absence (0) or presence of 250-fold excess of unlabeled hormone [JH I, JH II, methoprene (MET), or MDK] or in the absence of photolysis (NP). The arrow indicates the 29-kDa nuclear protein covalently modified by photoaffinity analogs. (Lower) Two-dimensional gels of photolabeled nuclear proteins (40 jug) in the absence (0) or presence of 250-fold excess of unlabeled hormone as in Upper. The EHDA used for lanes marked "0" and "MET" in Upper and for the two gels in Lower was from a second preparation of [3H]EHDA. Molecular masses are given in kDa. Addition of 250-fold excess of the appropriate unlabeled hormone completely prevented binding to the 38-kDa protein and reduced binding to the 29-kDa and 55-kDa bands. Although excess JH I had little effect on binding of MDK to the 29-kDa and 55-kDa proteins, it completely prevented binding to the 38-kDa protein (Fig. 3). Temporal and Tissue Distribution of the 29-kDa JH Binding Protein. Previous biochemical analysis of JH analog binding in Manduca epidermis had indicated the absence of specific nuclear binding sites in epidermis of day 1 fifth instar larvae that had had their corpora allata (the source of JH) removed during the molt to that stage (E.O.O., K.H., and L.M.R., unpublished data) and in pupally committed epidermis from wandering stage larvae (5). By contrast, specific binding sites 6 6m'4 311.

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[3H]EBDA, [3H]EHDA, or [3H]MDK in the absence (0) or presence of 250-fold excess of unlabeled hormone [JH I, JH II, or methoprene (MET)]. Molecular masses are given in kDa. The arrow indicates the 38-kDa cytosolic protein specifically modified by all three photoaffinity analogs.


Proc. Natl. Acad. Sci. USA 87 (1990)

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1000-fold excess of herring sperm DNA (data not shown). Only trace binding was seen with pUC18 (Fig. 5). Importantly, when nuclear proteins from either allatectomized or wandering stage larval epidermis were probed with either of the two cuticle genes (LCP14 shown in Fig. 5; LCP16/17, data not shown), little or no binding to a 29-kDa protein was seen. Nuclei from pupal epidermis contained 29-kDa and 35-kDa proteins that bound LCP14 (Fig. 5). This developmental correlation suggests that this 29-kDa nuclear protein that binds specifically to these two cuticular genes may be the same as the putative JH receptor identified above.

0 JHI 0 JHII 0 MET EBDA EHDA MDK

FIG. 4. Temporal and tissue distribution of photolabeled proteins. (Upper) Epidermal nuclear proteins (40 ,g) (left) and cytosolic proteins (20 u±g) (right) labeled by MDK from normal (dl) and allatectomized (-CA) day 1 fifth instar larvae, wandering stage larvae (W), and day 1 pupae (P). Arrows indicate the 29-kDa and 38-kDa proteins. (Lower) Day 1 fifth instar larval fat body nuclear proteins labeled with 10 nM [3H]EBDA, [3H]EHDA, or [3H]MDK in the absence (0) or presence of 250-fold excess of unlabeled hormone [JH I, JH II, or methoprene (MET) respectively]. Molecular masses are given in kDa. The arrow indicates the 29-kDa nuclear protein. present in the pupal abdominal epidermis after pupal ecdysis (5). Fig. 4 Upper shows that 0.5 M KCI extracts of were

epidermal nuclei from pupal abdomens contained a 29-kDa protein and three proteins between 35 and 40 kDa that bind MDK, whereas those from either wandering or allatectomized larvae showed no binding. Moreover, the cytosolic fractions from these same larvae only contained the 38-kDa binding protein when the 29-kDa nuclear protein was present (Fig. 4 Upper). The 55-kDa binding protein was not stagespecific. Thus, the presence of the 29-kDa nuclear and the 38-kDa cytosolic binding proteins is correlated with the presence of high-affinity nuclear binding sites. Nuclei isolated from day 1 fifth instar fat body also have a 29-kDa protein that is covalently bound by all three photoaffinity analogs (Fig. 4 Lower). As in the epidermis, the EBDA and EHDA binding is prevented by 250-fold excess of JH I and JH II respectively, whereas MDK binding is not prevented by excess methoprene. When separated on twodimensional gels, the same two proteins as in the epidermis are labeled by MDK (data not shown). DNA Binding. Preliminary studies showed that the 29-kDa nuclear JH binding protein from the epidermis binds to DNA cellulose. Exposure of electrophoretically separated and blotted nuclear proteins from day 1 epidermis to two different Manduca endocuticle genes (20, 21) showed that each gene bound strongly to a 29-kDa protein and weakly to a 21.5-kDa protein (Fig. 5). This binding was eliminated by 100-fold excess of homologous unlabeled DNA (Fig. 5) but not by

DISCUSSION This study clearly shows that the 0.5 M KCI fraction of the larval epidermal nuclei contains a major specific JH I and JH II binding protein that is different from the hemolymph JH binding protein and a cytosolic binding protein. This 29-kDa protein is absent from epidermal nuclei at times that no highaffinity JH binding sites (5) are detected, such as in wandering or allatectomized larvae. Also, the same 29-kDa binding protein is found in nuclei of larval fat body, a tissue that is also responsive to JH (23). The photoaffinity analog of methoprene, MDK, on photolysis covalently binds to the same 29-kDa, pI 5.8 nuclear protein as EBDA and EHDA. This binding however is not displaceable by excess methoprene, MDK, or JH II, indicating modification at a site other than the ligand binding site. In addition, MDK specifically binds to a second 29-kDa protein (pI 6.0), which may be the true methoprene receptor in the nucleus and thus accounts for the previously observed lack of competition of iodovinylmethoprenol by the natural JH homologs (6). Determination of whether these two binders are different proteins or are differently charged forms of one protein awaits their isolation and purification. Intriguingly, a 29-kDa nuclear protein having the same temporal specificity as the 29-kDa JH binding protein specifically binds to two larval endocuticle genes, both of which are regulated by JH. LCP14 is expressed throughout larval life except during the molts when it is transiently suppressed by high ecdysteroid acting in the presence of JH and then is permanently repressed by ecdysteroid in the absence of JH at the onset of metamorphosis (20). By contrast, LCP16/17

800


is only induced late in larval life by low ecdysteroid acting in the absence of JH (21). Although final confirmation awaits the purification of the putative 29-kDa JH receptor, these data suggest that this receptor may bind directly to these two genes. In contrast to nuclear binding that is specific for either the natural JHs or the dodecadienoate analogs, binding of the 38-kDa cytosolic protein to any of the photoaffinity analogs can be prevented by excess hormone ofeither type. Since this protein is not found in the nucleus, it likely is a general intracellular carrier for these lipophilic molecules as they traverse the cytoplasm to the nucleus, analogous to the

cellular retinoic acid binding protein (24). These studies and those of Wang et al. (25) with JH III cytosolic binding protein from Drosophila Kc cells have shown the utility of photoaffinity labels for the isolation of these elusive intracellular binding proteins that are presumably critical for the action of JH. Further purification of these cellular JH binding proteins followed by a detailed study of their interaction with JH-responsive genes is necessary for understanding JH action at the molecular level. We thank Jan Green for the black larval assays, Dr. Gerardus Staal of Zoecon for the gift of the (7S)-methoprene, and Dr. James Truman and Ms. Catherine Fittinghoff for critical comments on the manuscript. The work was supported by National Science Foundation Grants DCB85-09629 and DCB88-12322 (to G.D.P.) and Grants DCB85-18696 and DCB88-18876 (to L.M.R.). G.D.P. also thanks the Alfred P. Sloan Foundation, the Camille and Henry Dreyfus Foundation, and the Rohm and Haas Company for unrestricted funds. 3H labeling was performed by G.D.P., I.U., and W.-s.E. in conjunction with Dr. H. Morimoto at the National Tritium Labeling Facility (Berkeley, CA). 1. Riddiford, L. M. (1985) in Comprehensive Insect Biochemistry, Physiology, and Pharmacology, eds. Kerkut, G. A. & Gilbert, L. I. (Pergamon, Oxford, U.K.), Vol. 8, pp. 37-84. 2. Koeppe, J. K., Fuchs, M., Chen, T. T., Hunt, L. M., Kovalick, G. E. & Briers, T. (1985) in Comprehensive Insect Biochemistry, Physiology, and Pharmacology, eds. Kerkut, G. A. & Gilbert, L. I. (Pergamon, Oxford, U.K.), Vol. 8, pp. 165204.

Proc. Natl. Acad. Sci. USA 87 (1990) 3. Schooley, D. A., Baker, F. C., Tsai, L. W., Miller, C. A. & Jamieson, C. G. (1984) in Biosynthesis, Metabolism and Mode of Action of Invertebrate Hormones, eds. Hoffmann, J. & Porchet, M. (Springer, Berlin), pp. 373-383. 4. Boehm, M. F. & Prestwich, G. D. (1986) J. Org. Chem. 51, 5447-5450. 5. Riddiford, L. M., Osir, E. O., Fittinghoff, C. M. & Green, J. M. (1987) Insect Biochem. 17, 1039-1043. 6. Osir, E. 0. & Riddiford, L. M. (1988) J. Biol. Chem. 263, 13812-13818. 7. Prestwich, G. D. (1987) Science 237, 999-1006. 8. Eng, W.-s. (1987) Ph.D. Dissertation (State University of New York, Stony Brook, NY). 9. Prestwich, G. D. & Wawrzenczyk, C. (1985) Proc. Natl. Acad. Sci. USA 82, 5290-5294. 10. Eng, W.-s. & Prestwich, G. D. (1986) Bull. Soc. Chim. Belg. 95, 895-913. 11. Ujvary, I., Eng, W.-s. & Prestwich, G. (1989) J. Labelled Compd. Radiopharmacol. 27, in press. 12. Boehm, M. F. & Prestwich, G. D. (1988) J. Labelled Compd. Radiopharmacol. 25, 653-659. 13. Fain, M. J. & Riddiford, L. M. (1975) Biol. Bull. 149, 506-521. 14. Bearden, J. C. (1978) Biochim. Biophys. Acta 533, 525-529. 15. Labarca, C. & Paigen, K. (1980) Anal. Biochem. 102, 344-352. 16. Jones, P. P. (1980) in Selected Methods in Immunology, eds. Mishell, B. B. & Shiigi, S. M. (Freeman, San Francisco), pp. 398-440. 17. Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4354. 18. Bowen, B., Steinberg, J., Laemmli, U. K. & Weintraub, H. (1980) Nucleic Acids Res. 8, 1-20. 19. Feinberg, A. P. & Vogelstein, B. (1984) Anal. Biochem. 137, 266-267. 20. Rebers, J. & Riddiford, L. M. (1988)J. Mol. Biol. 203, 411-423. 21. Horodyski, F. M. & Riddiford, L. M. (1989) Dev. Biol. 132, 292-303. 22. Koeppe, J. K., Prestwich, G. D., Brown, J. J., Goodman, W. G., Kovalick, G. E., Briers, T., Pak, M. D. & Gilbert, L. I. (1984) Biochemistry 23, 6674-6679. 23. Webb, B. A. & Riddiford, L. M. (1988) Dev. Biol. 130, 682692. 24. Chytil, F. (1984) Pharmacol. Rev. 36, 93S-100S. 25. Wang, X., Chang, E. S. & O'Connor, J. D. (1989) Insect Biochem. 19, 327-335.