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

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 naturallepidopteran juvenile hormones, JH I and H {epoxy[3H]bisho-mofarnesyl diazoacetate ([3HJEBDA) and epoxy[3H]homo-farnesyl diazoacetate ([3H]EHDA)}, and of the JH analog meth-oprene {[3H]methoprene diazoketone ([3H]MDK)} were synthe-sized and used to identify specific JH binding proteins in thelarval epidermis of the tobacco hornworm (Manduca sexta).EBDA and EHDA specifically photolabeled a 29-kDa nuclearprotein (pI 5.8). This protein and a second 29-kDa protein (pI6.0) were labeled by MDK, but excess unlabeled methoprene orMDK only prevented binding to the latter. These 29-kDaproteins are also present in larval fat body but not in epidermisfrom either wandering stage or allatectomized larvae, whichlack high-affinity JH binding sites. A 29-kDa nuclear proteinwith the same developmental specificity as this JH binder boundthe DNA of two larval endocuticle genes. A 38-kDa cytosolicprotein was also specifically photolabeled by these photoaffinityanalogs. The 29-kDa nuclear protein is likely the high-affiityreceptor for JH that mediates its genomic action, whereas the38-kDa cytosolic protein may serve as an intracellular carrier forthese highly lipophilic hormones and hormone analogs.

The juvenile hormones (JHs) of insects are sesquiterpenoidmolecules that prevent metamorphosis of larvae and in mostinsects also regulate reproductive maturation in the adult (1,2). The action ofJH in the larva is manifest only in the presenceof the molting hormone ecdysone (1). The presence of JHallows the ecdysteroids to modulate ongoing gene expressionbut prevents activation ofnew genes and thus metamorphosis.

In the larval epidermis ofthe tobacco hornworm, Manducasexta, JH I, the naturally occurring hormone in lepidopteranlarvae (3), and iodovinylmethoprenol, an iodinated derivativeof methoprene (4) and biologically active JH analog (5), aretaken up by the epidermis. Approximately 33% is bound inthe nucleus by two binding components, one that shows highaffinity and about 10,000 sites per nucleus (6). These studiesfurther suggested that the binding sites were different for thenatural JH homologs and the dodecadienoate analogs sinceneither competed with the other for binding. We now show,by using photoafflinity analogs (7) of these compounds, thatthe two types of compounds specifically bind to two different29-kDa nuclear proteins. This nuclear protein complex is onlypresent in epidermis that shows specific binding of JH andappears to bind to two Manduca larval cuticle genes.

MATERIALS AND METHODSSynthesis of Photoaffinity Analogs. Labeled 10R,llS-ep-

oxybishomofarnesyl diazoacetate ([3H]EBDA), the photoaf-

finity analog of JH I, was first synthesized from the vinyloxirane (R = C2H5) (Fig. 1 Middle) (8). Selective reduction ofthe vinyl group with carrier-free 3H gas was mediated bytris(triphenylphosphine)chlororhodium in benzene solutionto give a 40o radiochemical yield of the chiral, ditritioethyloxirane compound with a specific activity of 55-58 Ci/mmol(1 Ci = 37 GBq) (9). The hydrolysis of the acetate andintroduction of the photolabile diazoacetate group were per-formed as described for the synthesis of racemic 10,11-epoxy[3H]farnesyl diazoacetate ([3H]EFDA) (10). [3H]-EHDA, the corresponding photoaffinity analog ofJH II, wassynthesized 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 (Zo-econ, Palo Alto, CA) (Fig. 1 Bottom). A 500-mg sample (1.86mmol) of methoprene acid, prepared by basic hydrolysis ofmethoprene with methanolic KOH, was dissolved in 2 ml ofdry benzene at 0°C and treated with 470 mg (3.73 mmol) ofdistilled oxalyl chloride. The solution was warmed to roomtemperature for 1 hr; the benzene and excess oxalyl chloridewere then removed in vacuo. The reaction flask containingthe crude acid chloride was cooled to 0°C, and a cold,anhydrous solution of diazomethane in ether was addedrapidly. The mixture was stirred for 20 min and brought toambient temperature. The excess diazomethane was decom-posed with a dilute acetic acid solution and the solvent wasremoved. 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) 80.85 (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-12CH3), 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).

Abbreviations: JH, juvenile hormone; EBDA, epoxybishomofarne-syl diazoacetate; EFDA, epoxyfarnesyl diazoacetate; EHDA, ep-oxyhomofarnesyl diazoacetate; MDK, methoprene diazoketone.tPresent address: International Center for Insect Physiology andEcology, 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. Box102, H-1525 Budapest, Hungary.

**Present address: Department of Entomology, University of Cali-fornia, Berkeley, CA 94720.

ttTo whom reprint requests should be addressed.

796

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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R 0

oll""">~ OMe

HR = C2H, (1OR, 11S) - JH IR= CH3, (1OR, 11S) - JH II

R

'-e-OQAcH

vinyl oxirane precursor

3H

1. 3H2, Rh(Ph3P)3CI H RN+

2. K2CO3 H3. ClCOCH=NNHTs, base R = C2H5, [3H] - EBDA

R = CH3, [3H] - EHDA

MeO 3H 1 0 1. KOH; then H30O

3H 2. (COCl)2[3H] - (7S) - methoprene 3. CH2N2

MeO 3H N0

3H[3H] - MDK

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

To prepare [3H]MDK, a 250-mCi portion (0.91 mg, 0.0029mmol) of [3H]-(7S)-methoprene (84 Ci/mmol) (12) was dis-solved in 1 ml of methanol containing 0.2 ml of 2 M KOH inmethanol and heated for 1 hr at 60°C. The reaction mixturewas cooled and then concentrated in vacuo, 1 ml 1 M HCI wasadded, and the residue was extracted with ether (three times2 ml). The extracts were dried (MgSO4), concentrated invacuo, and purified by flash chromatography on silica gelwith 5% ethyl acetate/hexane elution to give 150 mCi (0.47mg, 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 stirredfor 1 hr and solvent and excess reagent were removed invacuo. After addition of a cold, anhydrous solution of excessdiazomethane in ether (dried over MgSO4), solvents were

removed under a stream of nitrogen. Flash chromatographyof the residue on silica gel with 5% ethyl acetate/hexaneafforded 50 mCi (20% overall radiochemical yield) of homo-geneous [3H]MDK that coeluted on TLC with the unlabeledMDK produced above.

Unlabeled and labeled hormones were checked periodi-cally by TLC or radio-TLC for purity; radioligands wererepurified, 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:darkphotoperiod as described (5). The photoaffinity analogs were

assayed for JH activity on the black larval mutant of Man-duca (13).The epidermis of day 1 fifth instar larvae (3.0-4.5 g) was

dissected and homogenized in cold buffer A [20mM Tris, pH7.9, containing 50 mM KCI, 300 mM sucrose, and 1 mM(each) EDTA, dithiothreitol, phenylmethylsulfonyl fluoride,and diisopropyl phosphofluoridate] (6). The crude nuclearpellet (1000 x g, 30 min) was washed three times with 1 mlof buffer A and then washed three times with 1 ml of bufferC (6) [20 mM Tris, pH 7.9, containing 5 mM magnesiumacetate 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 occasionalmixing. After centrifugation (12,000 x g, 15 min, 40C), thesupernatant was dialyzed against buffer C (12 hr, threechanges) 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 thecytosolic fraction.

Protein concentrations were determined colorimetrically(14). DNA concentrations were determined fluorimetricallyusing Hoechst dye 33258 (15).

Photoafflinity Labeling. All glassware was precoated with1% (wt/vol) polyethylene glycol (Mr 20,000; Sigma). Solu-tions of the labeled photoaffinity analogs and of the unlabeledcompetitors [JH I and JH II (Sigma) and (7S)-methoprene(Zoecon)] were dissolved in buffer C by sonication (Bransonsonicator bath) for 10 min at 0C followed by a 2-hr period atroom temperature. The concentration of the labeled photo-affinity analogs was determined by liquid scintillation count-ing and that of the unlabeled hormones and analogs wasdetermined spectrophotometrically using the following ex-tinction coefficients: JH I and JH II, -217nm = 14,800 inmethanol; 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 nucleicontaining 50-100 1Lg of DNA was added to buffer C (totalvolume, 400 p.l) containing the labeled photoaffinity analogwith or without an excess of unlabeled hormone or analog ina 0.5-ml quartz tube and incubated either 2 hr at 21'C or 10-12hr at 40C. One milligram ofgamma globulin per milliliter wasadded to the cytosolic fraction to reduce nonspecific binding.Then the samples were irradiated 30 sec at 254 nm (Rayonetreactor). The same pattern of labeled proteins was seen forphotolysis times between 30 sec and 2 min. Efficiency oflabeling was found to be 12-14% from 10-100 nM solutionsof the various analogs. After irradiation, the 0.5 M KCInuclear extracts and the cytosolic extracts were lyophilized.Whole nuclei were sonicated, and the resultant 15,000 X gsupernatant (15 min, 4°C) was lyophilized.

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Electrophoresis. Proteins were electrophoresed on 10%SDS/polyacrylamide gels or on nonequilibrium pH gradientgels, followed by 10% SDS gels (16) followed by fluorographyusing EN3HANCE (New England Nuclear).DNA Binding Studies. Electrophoretically separated pro-

teins from the 0.5 M KCl nuclear extract were electroblottedto nitrocellulose (17); then the filter was preincubated over-night in 10 mM Tris (pH 7.5), 1 mM EDTA containing 0.02%(each) of bovine serum albumin, Ficoll, and polyvinylpyr-rolidone (18). It was then exposed to 10 ng of [32P]dATP-oligo-labeled (19) DNA (106 cpm/ml) for 4 hr at 21'C in thesame 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 Manducagenomic clones used were as follows: (i) 26-10, an 8-kilobase(kb) fragment containing the entire gene coding for a 14-kDacuticular protein (LCP14) and 2 and 3 kb of the 3' and 5'flanking regions, respectively (20); (ii) pGFH-17, a 2.9-kbfragment 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).

Hemo Nuclei

0

:;O JHII 0 JHII 0 IIMET

EHDA

pH . 5.8MrV

5.8F

466

.4 3

a ~a .43 1

2 1

0 JIHI 0 METMDKJHI NP

EBDA MDK

5.8 6 J.

5 8 6.0'FT

291'

EHDA-O E H D A - J H MD K -- 0 M D K - M E T

RESULTSActivity of Photoaffinity JH Analogs. In the Manduca black

larval assay for JH, EBDA had an ED50 of 1.4 pmol (n = 15for 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), ascompared to 1.8 pmol for JH II (10R,11S) (5). MDK (ED50 =4.5 pmol) was half as active as (7S)-methoprene (ED50 = 2.0pmol) (5).

Photoaffinity Labeling of Nuclear and Cytosolic Proteins.When either [3H]EBDA or [3H]EHDA was incubated withisolated epidermal nuclei or with nuclear proteins extractablewith 0.5 M KCl and then the mixture was irradiated at 254 nm,only a 29-kDa protein was covalently modified by the pho-toaffinity analog (Fig. 2 Upper). This binding was reduced5-fold by 100-fold excess and was prevented by 250-foldexcess of either unlabeled JH I or JH II but not by excessmethoprene (Fig. 2 Upper). These analogs also bound spe-cifically to the 32-kDa hemolymph JH binding protein (22).The 29-kDa protein in the nuclear extract was also cova-

lently modified by MDK (Fig. 2 Upper). In this case, bindingwas only partially prevented by 250-fold excess of MDK ormethoprene. No reduction of binding was seen with excessJH I. Even 1-hr preincubation with 1000-fold excess ofmethoprene or MDK failed to eliminate the MDK binding(data not shown). Several other nuclear proteins were alsolabeled by MDK, but these were also labeled to a variableextent without photolysis (Fig. 2 Upper), indicating somenonspecific chemical modification by this diazoketone.When the photoaffinity-labeled nuclear proteins were sep-

arated on a two-dimensional gel (Fig. 2 Lower), only one29-kDa protein (pI 5.8) was labeled by [3H]EHDA; 250-foldexcess 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 6.0protein (Fig. 2 Lower). JH I had no effect on MDK bindingto either protein (data not shown). Thus, there appear to betwo specific 29-kDa binding proteins, one for the natural JHhomologs and one for the dodecadienoate analog. SinceMDK also binds nonspecifically to the pI 5.8 protein, itsspecific binding to the second protein is obscured on one-dimensional gels (Fig. 2 Upper). Since [3H]MDK has higherspecific activity, we took advantage of its high affinity for thenatural JH binding protein for studies of this protein's tem-poral distribution (see below).The cytosolic fraction contained 29-kDa and 38-kDa poly-

peptides that were photolabeled by all three photoaffinityanalogs and a 55-kDa protein photolabeled by MDK (Fig. 3).

FIG. 2. Photolabeling of 0.5 M KCI-extractable nuclear proteinsfrom the epidermis and of hemolymph proteins of day 1 fifth instarM. 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 ofunlabeled hormone [JH I, JH II, methoprene (MET), or MDK] or inthe absence of photolysis (NP). The arrow indicates the 29-kDanuclear protein covalently modified by photoaffinity analogs. (Low-er) Two-dimensional gels of photolabeled nuclear proteins (40 jug) inthe absence (0) or presence of 250-fold excess of unlabeled hormoneas in Upper. The EHDA used for lanes marked "0" and "MET" inUpper and for the two gels in Lower was from a second preparationof [3H]EHDA. Molecular masses are given in kDa.

Addition of 250-fold excess of the appropriate unlabeledhormone completely prevented binding to the 38-kDa proteinand reduced binding to the 29-kDa and 55-kDa bands. Al-though excess JH I had little effect on binding ofMDK to the29-kDa and 55-kDa proteins, it completely prevented bindingto the 38-kDa protein (Fig. 3).Temporal and Tissue Distribution of the 29-kDa JH Binding

Protein. Previous biochemical analysis of JH analog bindingin Manduca epidermis had indicated the absence of specificnuclear binding sites in epidermis of day 1 fifth instar larvaethat had had their corpora allata (the source of JH) removedduring the molt to that stage (E.O.O., K.H., and L.M.R.,unpublished data) and in pupally committed epidermis fromwandering stage larvae (5). By contrast, specific binding sites

6 6m'-

4 311.

3 1 P- __ p

2 1. 50

0 JHI 0 JHII 0 METJHIEBDA EHDA MDK

FIG. 3. Photolabeling of epidermal cytosol (20 ug) with 10 nM[3H]EBDA, [3H]EHDA, or [3H]MDK in the absence (0) or presenceof 250-fold excess of unlabeled hormone [JH I, JH II, or methoprene(MET)]. Molecular masses are given in kDa. The arrow indicates the38-kDa cytosolic protein specifically modified by all three photoaf-finity analogs.

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EpidermisNuclei Cytosol

4 3m- 660-

a

cr_I--C)

-L -

a, -4 ~ LCP14

43".

%OI- PA.

2 1.5 ._

di-CA W P di-CAW P

66".

43'-

Fat body Nuclei

2 1.5w

0 JHI 0 JHII 0 MET

EBDA EHDA MDK

FIG. 4. Temporal and tissue distribution of photolabeled pro-teins. (Upper) Epidermal nuclear proteins (40 ,g) (left) and cytosolicproteins (20 u±g) (right) labeled by MDK from normal (dl) andallatectomized (-CA) day 1 fifth instar larvae, wandering stagelarvae (W), and day 1 pupae (P). Arrows indicate the 29-kDa and38-kDa proteins. (Lower) Day 1 fifth instar larval fat body nuclearproteins labeled with 10 nM [3H]EBDA, [3H]EHDA, or [3H]MDK inthe absence (0) or presence of 250-fold excess of unlabeled hormone[JH I, JH II, or methoprene (MET) respectively]. Molecular massesare given in kDa. The arrow indicates the 29-kDa nuclear protein.

were present in the pupal abdominal epidermis after pupalecdysis (5). Fig. 4 Upper shows that 0.5 M KCI extracts ofepidermal nuclei from pupal abdomens contained a 29-kDaprotein and three proteins between 35 and 40 kDa that bindMDK, whereas those from either wandering or allatecto-mized larvae showed no binding. Moreover, the cytosolicfractions from these same larvae only contained the 38-kDabinding protein when the 29-kDa nuclear protein was present(Fig. 4 Upper). The 55-kDa binding protein was not stage-specific. Thus, the presence of the 29-kDa nuclear and the38-kDa cytosolic binding proteins is correlated with thepresence of high-affinity nuclear binding sites.

Nuclei isolated from day 1 fifth instar fat body also have a29-kDa protein that is covalently bound by all three photo-affinity analogs (Fig. 4 Lower). As in the epidermis, theEBDA and EHDA binding is prevented by 250-fold excess ofJH I and JH II respectively, whereas MDK binding is notprevented by excess methoprene. When separated on two-dimensional gels, the same two proteins as in the epidermisare labeled by MDK (data not shown).DNA Binding. Preliminary studies showed that the 29-kDa

nuclear JH binding protein from the epidermis binds to DNAcellulose. Exposure of electrophoretically separated andblotted nuclear proteins from day 1 epidermis to two differentManduca endocuticle genes (20, 21) showed that each genebound strongly to a 29-kDa protein and weakly to a 21.5-kDaprotein (Fig. 5). This binding was eliminated by 100-foldexcess of homologous unlabeled DNA (Fig. 5) but not by

x D -CAW P-LL5-d 1--

FIG. 5. Binding of 10 ng ofDNA (106 cpm/ml) from the Manducagenomic clones coding for the larval cuticular proteins LCP16/17(pGFH-17) and LCP14 (clone 26-10) or from pUC18 to 20 jug ofnuclear proteins from abdominal epidermis of normal and allatecto-mized (-CA) day 1 fifth instar larvae (L5-dl), wandering stage larvae(W), and day 1 pupae (P). xD, 100-fold excess of unlabeled LCP14DNA added. Molecular masses are given in kDa. The arrow indicatesthe 29-kDa DNA binding protein.

1000-fold excess of herring sperm DNA (data not shown).Only trace binding was seen with pUC18 (Fig. 5). Impor-tantly, when nuclear proteins from either allatectomized orwandering stage larval epidermis were probed with either ofthe two cuticle genes (LCP14 shown in Fig. 5; LCP16/17,data not shown), little or no binding to a 29-kDa protein wasseen. Nuclei from pupal epidermis contained 29-kDa and35-kDa proteins that bound LCP14 (Fig. 5). This develop-mental correlation suggests that this 29-kDa nuclear proteinthat binds specifically to these two cuticular genes may be thesame as the putative JH receptor identified above.

DISCUSSIONThis study clearly shows that the 0.5 M KCI fraction of thelarval epidermal nuclei contains a major specific JH I and JHII binding protein that is different from the hemolymph JHbinding protein and a cytosolic binding protein. This 29-kDaprotein is absent from epidermal nuclei at times that no high-affinity JH binding sites (5) are detected, such as in wanderingor allatectomized larvae. Also, the same 29-kDa bindingprotein is found in nuclei oflarval fat body, a tissue that is alsoresponsive to JH (23).The photoaffinity analog of methoprene, MDK, on pho-

tolysis covalently binds to the same 29-kDa, pI 5.8 nuclearprotein as EBDA and EHDA. This binding however is notdisplaceable by excess methoprene, MDK, or JH II, indi-cating modification at a site other than the ligand binding site.In addition, MDK specifically binds to a second 29-kDaprotein (pI 6.0), which may be the true methoprene receptorin the nucleus and thus accounts for the previously observedlack of competition of iodovinylmethoprenol by the naturalJH homologs (6). Determination ofwhether these two bindersare different proteins or are differently charged forms of oneprotein awaits their isolation and purification.

Intriguingly, a 29-kDa nuclear protein having the sametemporal specificity as the 29-kDa JH binding protein spe-cifically binds to two larval endocuticle genes, both of whichare regulated by JH. LCP14 is expressed throughout larvallife except during the molts when it is transiently suppressedby high ecdysteroid acting in the presence of JH and then ispermanently repressed by ecdysteroid in the absence of JHat the onset of metamorphosis (20). By contrast, LCP16/17

2 1.5 - Now

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is only induced late in larval life by low ecdysteroid acting inthe absence of JH (21). Although final confirmation awaitsthe purification of the putative 29-kDa JH receptor, thesedata suggest that this receptor may bind directly to these twogenes.

In contrast to nuclear binding that is specific for either thenatural JHs or the dodecadienoate analogs, binding of the38-kDa cytosolic protein to any of the photoaffinity analogscan be prevented by excess hormone ofeither type. Since thisprotein is not found in the nucleus, it likely is a generalintracellular carrier for these lipophilic molecules as theytraverse the cytoplasm to the nucleus, analogous to thecellular retinoic acid binding protein (24).These studies and those of Wang et al. (25) with JH III

cytosolic binding protein from Drosophila Kc cells haveshown the utility of photoaffinity labels for the isolation ofthese elusive intracellular binding proteins that are presum-ably critical for the action ofJH. Further purification of thesecellular JH binding proteins followed by a detailed study oftheir interaction with JH-responsive genes is necessary forunderstanding JH action at the molecular level.

We thank Jan Green for the black larval assays, Dr. Gerardus StaalofZoecon for the gift of the (7S)-methoprene, and Dr. James Trumanand Ms. Catherine Fittinghoff for critical comments on the manu-script. The work was supported by National Science FoundationGrants DCB85-09629 and DCB88-12322 (to G.D.P.) and GrantsDCB85-18696 and DCB88-18876 (to L.M.R.). G.D.P. also thanks theAlfred P. Sloan Foundation, the Camille and Henry Dreyfus Foun-dation, and the Rohm and Haas Company for unrestricted funds. 3Hlabeling was performed by G.D.P., I.U., and W.-s.E. in conjunctionwith Dr. H. Morimoto at the National Tritium Labeling Facility(Berkeley, CA).

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