6
Insect Molecular Biology (2003) 12(5), 427–432 © 2003 The Royal Entomological Society 427 Blackwell Publishing Ltd. Functional dissection of the hexamerin receptor and its ligand arylphorin in the blowfly Calliphora vicina I. A. Hansen*, V. Gutsmann†, S. R. Meyer* and K. Scheller* *Department of Cell and Developmental Biology, Biocentre of the University, Wuerzburg, Germany; Bayer CropScience AG, Monheim, Germany Abstract The process of receptor-mediated uptake of hexamerin storage proteins from insect haemolymph by fat body cells is a unique feature of the class Insecta . We iden- tified the binding domains of the hexamerin receptor and the hexamerin ligand arylphorin in the blowfly, by means of the yeast-two-hybrid-system. The receptor- binding domain of arylphorin was located within domain 3 of the arylphorin monomer. The ligand-binding domain of the hexamerin receptor was mapped to the extreme N-terminus of the receptor. The binding domains identified exhibit no similarity to any functional protein domains known to date. Additionally, we identified two previously unknown protein-interactors of the hexam- erin receptor. The results of this study provide further insights regarding the mechanism of the receptor- mediated endocytosis of storage proteins in insects. Keywords: hexamerin, hexamerin receptor, endocytosis, yeast-two-hybrid system, EGFP, Calliphora , blowfly. Introduction Endocytotic events are involved in many different physio- logical processes with various types of receptors and lig- ands. In insects, the most conversant examples are the uptake of low density lipoprotein (LDL) and the uptake of yolk by the growing oocyte (Schneider, 1996). Both processes follow a common scheme, involving receptors of the LDL receptor supergene family, which feature one membrane- spanning domain and clusters of cysteine-rich areas that constitute the ligand-binding domain. In contrast, the process of receptor-mediated uptake of hexamerin storage proteins from insect haemolymph by fat body cells is a unique feature of the class Insecta involving a receptor that does not belong to the LDL receptor supergene family or to any other receptor family known to date. As insect pupae do not feed during metamorphosis, they depend on nutrients that have been accumulated previ- ously in the larval period. In holometabolous insects, energy and amino acid building blocks for imaginal tissues are provided by proteins, lipids and carbohydrates that have been selectively taken up by the larval fat body from the haemolymph before pupation. The incorporated pro- teins belong to the family of hexamerins also referred to as arylphorins, larval serum proteins or storage proteins (Burmester & Scheller, 1999; Haunerland, 1996). The transport of hexamerins across the fat body cell membrane requires the existence of a specific receptor. Sequences of recep- tors are only known from the dipteran clade, notably from the fleshfly, Sarcophaga peregrina (Chung et al ., 1995), the blowfly, Calliphora vicina (Burmester & Scheller, 1995), and the fruitfly, Drosophila melanogaster (Burmester et al ., 1999). Surprisingly, these receptors show a significant sim- ilarity to their ligands, the hexamerins. Phylogenetic studies suggest that hexamerin receptor proteins evolved from their own ligands, the ancient hexamerins and haemocyanin- like proteins, early in insect evolution even before the divergence of winged insects (Burmester, 2002; Burmester & Scheller, 1996). A puzzling feature of the hexamerin receptor is the absence of a typical membrane-spanning domain, which has led to various speculations as to how this protein mediates endocytosis. In Calliphora the hexam- erin receptor is synthesized as a 130 kDa precursor, which is subject to an unusual processing that involves three distinct cleavage steps. The increase in steroid hormone 20-hydroxyecdysone concentration at the end of larval development triggers the process that results in the final cleavage steps of the receptor, coinciding with the onset of hexamerin endocytosis (Burmester & Scheller, 1997b). A similar processing pattern is recorded in Drosophila mela- nogaster (Burmester et al ., 1999). In this study we report the mapping and characterization of the binding domains of the hexamerin receptor and its ligand, arylphorin, in the blowfly Calliphora vicina and discuss the putative roles of some recently identified novel interactors of the hexamerin receptor (Hansen et al ., 2002). Received 13 February 2003; accepted after revision 29 April 2003. Corre- spondence: Immo A. Hansen, Department of Entomology, Watkins Drive, University of California, Riverside, CA 92521, USA. Tel.: +1 909 7872146; fax: +1 909 7872130; e-mail: [email protected]

Functional dissection of the hexamerin receptor and its ligand arylphorin in the blowfly Calliphora vicina

Embed Size (px)

Citation preview

Insect Molecular Biology (2003)

12

(5), 427–432

© 2003 The Royal Entomological Society

427

Blackwell Publishing Ltd.

Functional dissection of the hexamerin receptor and its ligand arylphorin in the blowfly

Calliphora vicina

I. A. Hansen*, V. Gutsmann†, S. R. Meyer* and K. Scheller*

*

Department of Cell and Developmental Biology, Biocentre of the University, Wuerzburg, Germany;

Bayer CropScience AG, Monheim, Germany

Abstract

The process of receptor-mediated uptake of hexamerinstorage proteins from insect haemolymph by fat bodycells is a unique feature of the class

Insecta

. We iden-tified the binding domains of the hexamerin receptorand the hexamerin ligand arylphorin in the blowfly, bymeans of the yeast-two-hybrid-system. The receptor-binding domain of arylphorin was located withindomain 3 of the arylphorin monomer. The ligand-bindingdomain of the hexamerin receptor was mapped to theextreme N-terminus of the receptor. The binding domainsidentified exhibit no similarity to any functional proteindomains known to date. Additionally, we identified twopreviously unknown protein-interactors of the hexam-erin receptor. The results of this study provide furtherinsights regarding the mechanism of the receptor-mediated endocytosis of storage proteins in insects.

Keywords: hexamerin, hexamerin receptor, endocytosis,yeast-two-hybrid system, EGFP,

Calliphora

, blowfly.

Introduction

Endocytotic events are involved in many different physio-logical processes with various types of receptors and lig-ands. In insects, the most conversant examples are theuptake of low density lipoprotein (LDL) and the uptake of yolkby the growing oocyte (Schneider, 1996). Both processesfollow a common scheme, involving receptors of the LDLreceptor supergene family, which feature one membrane-spanning domain and clusters of cysteine-rich areas thatconstitute the ligand-binding domain. In contrast, theprocess of receptor-mediated uptake of hexamerin storage

proteins from insect haemolymph by fat body cells is aunique feature of the class

Insecta

involving a receptor thatdoes not belong to the LDL receptor supergene family or toany other receptor family known to date.

As insect pupae do not feed during metamorphosis, theydepend on nutrients that have been accumulated previ-ously in the larval period. In holometabolous insects,energy and amino acid building blocks for imaginal tissuesare provided by proteins, lipids and carbohydrates thathave been selectively taken up by the larval fat body fromthe haemolymph before pupation. The incorporated pro-teins belong to the family of hexamerins also referred to asarylphorins, larval serum proteins or storage proteins(Burmester & Scheller, 1999; Haunerland, 1996). The transportof hexamerins across the fat body cell membrane requiresthe existence of a specific receptor. Sequences of recep-tors are only known from the dipteran clade, notably fromthe fleshfly,

Sarcophaga peregrina

(Chung

et al

., 1995), theblowfly,

Calliphora vicina

(Burmester & Scheller, 1995), andthe fruitfly,

Drosophila melanogaster

(Burmester

et al

.,1999). Surprisingly, these receptors show a significant sim-ilarity to their ligands, the hexamerins. Phylogenetic studiessuggest that hexamerin receptor proteins evolved fromtheir own ligands, the ancient hexamerins and haemocyanin-like proteins, early in insect evolution even before thedivergence of winged insects (Burmester, 2002; Burmester& Scheller, 1996). A puzzling feature of the hexamerinreceptor is the absence of a typical membrane-spanningdomain, which has led to various speculations as to howthis protein mediates endocytosis. In

Calliphora

the hexam-erin receptor is synthesized as a 130 kDa precursor, whichis subject to an unusual processing that involves threedistinct cleavage steps. The increase in steroid hormone20-hydroxyecdysone concentration at the end of larvaldevelopment triggers the process that results in the finalcleavage steps of the receptor, coinciding with the onset ofhexamerin endocytosis (Burmester & Scheller, 1997b). Asimilar processing pattern is recorded in

Drosophila mela-nogaster

(Burmester

et al

., 1999). In this study we reportthe mapping and characterization of the binding domains ofthe hexamerin receptor and its ligand, arylphorin, in theblowfly

Calliphora vicina

and discuss the putative roles ofsome recently identified novel interactors of the hexamerinreceptor (Hansen

et al

., 2002).

Received 13 February 2003; accepted after revision 29 April 2003. Corre-spondence: Immo A. Hansen, Department of Entomology, Watkins Drive,University of California, Riverside, CA 92521, USA. Tel.: +1 909 7872146;fax: +1 909 7872130; e-mail: [email protected]

428

I. A. Hansen

et al.

© 2003 The Royal Entomological Society,

Insect Molecular Biology

,

12

, 427–432

Results and discussion

Hexamerins bind the hexamerin receptor in the yeast-two-hybrid system

The synthesis of hexameric storage proteins by the larvalfat body and the reuptake of these proteins by the fat bodyshortly before pupation seems to be a common process in

the life cycle of all holometabolous insects (Burmester &Scheller, 1999; Haunerland, 1996). There is no doubt thatthe uptake process occurs by receptor-mediated endocytosis.One goal of the present study was to show that protein–protein interactions between hexamerins and the hexamerinreceptor can be demonstrated in an artificial yeast system.We performed two two-hybrid-screening experiments usingthe full-length hexamerin receptor precursor (ABP130) andthe functional receptor (ABP96) as bait proteins. Theexperiments resulted in 32 plasmids, which were analysed.Seventeen contained hexamerin cDNA fragments; 14 fromarylphorin and three from larval serum protein 2 (LSP-2).LSP-2 is a minor hexamerin molecule, different from aryl-phorin, and typical for cyclorraphan

Diptera

(Burmester

et al

., 1998). The size of the selected arylphorin fragmentsis depicted in Fig. 1B. The shortest fragment screenedincludes amino acids 536–759 of arylphorin. An arylphorinmonomer has three domains (Markl

et al

., 1992) and thereceptor-binding epitope was located in the C-terminaldomain 3. Both bait proteins (ABP130-bait and ABP96-bait) proved to be capable of binding arylphorin and LSP-2.Thus, the yeast-two-hybrid system is suitable to study theinteraction between hexamerin receptor and hexamerins.

Mapping of the receptor-binding domain of

Calliphora

arylphorin

We constructed different shortened arylphorin-prey plas-mids. These plasmids were cotransformed with ABP96-baitand tested for reporter gene activity (Fig. 1C). By combin-ing this data with the data from the screening experiment,the receptor-binding epitope of

Calliphora

arylphorin waslocated within the C-terminal domain 3 of the arylphorinmonomer, at amino acids 536–600. In our homologymodel, constructed from the structural data of

Panulirusinterruptus

haemocyanin (Fig. 2A–C), this region com-prises the beta sheets 3.B

3.E and the connecting loops inbetween. An arylphorin hexamer is built of two trimers(Fig. 2D), that are arranged as an antiprism (Markl

et al

.,1992). In our model this region is freely accessible to pos-sible interacting proteins. Interestingly, the strength of theprotein–protein interaction, measured via reporter geneactivity, reached its highest levels when the completedomain 3 was used as a prey protein. We speculate that thestable beta-barrel conformation of a complete domain 3promotes the ability of the fusion protein to interact with thehexamerin receptor bait. It was found that the amino acidsequence of the receptor-binding domain contains tworegions that are highly conserved between the hexamerinsof various insect species (Fig. 3).

Mapping of the ligand-binding domain of the

Calliphora

arylphorin receptor

We constructed seven bait plasmids encoding differentfragments of the hexamerin receptor. The bait plasmids

Figure 1. Schematic diagram showing the size of the arylphorin fragments screened with hexamerin receptor bait proteins and the regions of arylphorin required for interaction with the hexamerin receptor. (A) The full length arylphorin cDNA encodes a protein of 759 amino acids, which has three domains (pictured with different shades of grey). (B) Length of the arylphorin fragments coded by the library plasmids isolated in the two-hybrid screening experiments. The clones labelled ‘a’ were identified in a screen with ABP96-bait and the clones labelled ‘b’ were screened with ABP130-bait. (C) Analysis of the interaction between ABP96-bait and different arylphorin derivates. The β-galactosidase activity and the competency of the different strains to grow on leucine-deficient media is specified with plus and minus, while the activation of the EGFP-reporter is indicated in relative fluorescence units (RFU).

Hexamerin/hexamerin receptor interactions

429

© 2003 The Royal Entomological Society,

Insect Molecular Biology

,

12

, 427–432

ABP130-bait, ABP96-bait and ABP64-bait represent thenaturally occurring hexamerin receptor fragments whereasthe other three are shorter N-terminal fragments. Thesebait plasmids were cotransformed in the yeast strainEGY48 with a prey plasmid that bears the completedomain 3 of

Calliphora

arylphorin, and tested for reporter

gene activity (Fig. 4). It was found that the ligand-bindingepitope of the hexamerin receptor is located at the very N-terminus of these proteins. We cannot exclude that thereceptor contains more than one arylphorin-bindingdomain but all the arylphorin-binding activity is restricted tothe N-terminal ABP64 peptide.

Figure 2. Structural models from (A) haemocyanin from the lobster Panulirus interruptus, (B) the domain 3 of arylphorin from the blowfly Calliphora vicina and (C) the receptor binding domain of arylphorin, all modelled through the webpage http://www.expasy.ch/spdbv/. The figures were generated by the software SWISSPDB-VIEWER. (D) Schematic model of the molecular interactions between hexamerin monomers in a hexamerin trimer, the interacting domains labelled 1 and 2 and circled.

Figure 3. (A) Amino acid alignment of the receptor-binding domain of arylphorin of Calliphora vicina with hexamerins of various insect species. Identical amino acids are shown by white letters within black or grey boxes. (B) Table of the protein sequences used for the alignment.

430

I. A. Hansen

et al.

© 2003 The Royal Entomological Society,

Insect Molecular Biology

,

12

, 427–432

‘Non hexamerin’ interactors of the hexamerin receptor

We found 15 plasmids containing cDNAs encoding proteinsor protein fragments that are not hexamerins. Seven plas-mids contained open reading frames that exhibit no homol-ogy to any described proteins in our G

EN

B

ANK

search.Three of these plasmids contained the cDNA for glutath-ione transferase zeta1 (all three screened with ABP130-bait; G

EN

B

ANK

accession number AY102625). Three furtherplasmids contained cDNAs encoding the anterior fat bodyprotein (AFP) of

Calliphora

(screened with ABP130-baitonce and with ABP96-bait twice; G

EN

B

ANK

accessionnumber AY028616) (Hansen

et al

., 2002). One plasmidcontained the cDNA for an AP-3 delta-adaptin subunit(screened with ABP96-bait; G

EN

B

ANK

accession numberAF329283). It is worth mentioning that only one obviouslyfalse-positive clone was isolated in our experiments; thisclone, screened with ABP130-bait, contained the cDNA forthe translation-elongation factor 2.

Protein–protein interactions of the hexamerin receptor

Our knowledge concerning the protein interactions of thehexamerin receptor has broadened during recent years.Originally the hexamerin receptor was identified as anarylphorin-binding protein in

Sarcophaga peregrina

and

Calliphora vicina

(Burmester & Scheller, 1995; Chung

et al

., 1995

)

. Furthermore, it could be demonstrated that thesame receptor is responsible even for the uptake of LSP-2,the other hexamerin storage protein of

Calliphora

and

Drosophila

(Burmester

et al

., 1998; Burmester & Scheller,1997a). The occurrence of the receptor cleavage productsP30 and P45 demonstrate that the receptor is cleaved atleast twice by prohormonconvertase-like enzymes (Burm-ester & Scheller, 1997b). Our yeast-two-hybrid analysis ofthe protein interactions of the hexamerin receptor demon-strated some new interactors. We were able to show thatAFP specifically binds to the P30 peptide of the hexamerinreceptor (Hansen

et al

., 2002). Another newly identifiedinteractor is a delta-adaptin subunit, which could be part ofan AP-3 adaptor complex. Adaptins are adaptor moleculesthat connect membrane-spanning receptors with clathrin orother coat proteins, which arbitrate receptor-mediatedendocytosis processes (Kirchhausen, 2002). We could alsoshow that delta-adaptin binds specifically the C-terminalpart of ABP64 and neither P45 nor P30 (unpublishedresults). This is a strong hint that the hexamerin receptor isa true receptor with an N-terminal ligand binding domain, atransmembrane domain and a C-terminal cytoplasmic tail.

Experimental procedures

The two-hybrid strategy

We chose the LexA-based yeast-two-hybrid-system (Origene,Rockville, MD, USA) because of the reduced occurrence of falsepositives in this system compared with the Gal4-based one (Fash-ena, Serebriiskii & Golemis, 2000). All yeast experiments wereperformed following standard protocols (Ausubel

et al

., 2001). Theyeast strain EGY48 was transformed by the lithium acetate method(Gietz

et al

., 1995), using different combinations of plasmids.pEG202 expresses a LexA-fused bait and pJG4-5 expresses aB42 transactivation domain fused to interacting partner proteins.Blue colouration (LacZ-reporter plasmid pSH18-34), fluorescenceactivity (EGFP-reporter plasmid p8op-EGFP, see below) and theability of colonies to grow on leucin deficient medium reflects theoccurrence of interaction between the hybrid proteins.

Construction of the hexamerin receptor bait plasmids

The vector pEG202 was used for the construction of the bait plas-mids. All baits were obtained by PCR amplification using PfuTurboDNA Polymerase (Stratagene, Heidelberg, Germany) and clonedinto pEG202. Six different hexamerin receptor bait plasmids wereconstructed (Fig. 4), using a pBluescript SK+ vector bearing thecomplete hexamerin receptor cDNA sequence (G

EN

B

ANK

acces-sion number X79100) as a template. The primers used aredepicted in Table 1. The primer combinations were [R1/R2], creatinga full length hexamerin receptor precursor fusion protein desig-nated ABP130-bait; [R1/R3], generating a fusion protein coveringthe natural occurring receptor cleavage product ABP96 (ABP96-bait); [R1/R4], yielding a fusion protein covering the shortestnatural occurring receptor cleavage product ABP64 (ABP64 bait);[R8/R2], creating a fusion protein covering the C-terminal P30 andP45 subunits of the hexamerin receptor precursor (P30/45 bait);[R1/R5], bearing a fusion protein covering the first 380 amino acidsof the N-terminus of the hexamerin receptor (ABP64-

178 bait);[R1/R6], generating a fusion protein covering the first 200 aminoacids of the N-terminus of the hexamerin receptor (ABP64-

358

Figure 4. Regions of the hexamerin receptor required for interaction with the domain 3 of arylphorin. (A) The full-length hexamerin receptor comprises a signal peptide of 17 amino acids, a 558 amino acid arylphorin-binding peptide of 64 kDaA (ABP64) and the naturally occurring cleavage fragments P30 and P45. (B) Analysis of the interaction between D3 prey (domain 3 of arylphorin) and different hexamerin receptor derivates. The β-galactosidase activity and the competency of the different strains to grow on leucine-deficient media is specified with plus and minus; the activation of the EGFP-reporter is indicated in relative fluorescence units (RFU).

Hexamerin/hexamerin receptor interactions

431

© 2003 The Royal Entomological Society,

Insect Molecular Biology

,

12

, 427–432

bait); and [R1/R7], producing a fusion protein covering the first24 amino acids of the N-terminus of the hexamerin receptor(ABP64-

534 bait). The N-terminal hydrophobic signal peptide ofthe hexamerin receptor was omitted in all these constructs. Thecorrect insertion of the cDNAs was verified by sequencing the baitplasmids with the sequencing primer S1 (Table 1).

Construction of the prey library

The construction of the two-hybrid library is described elsewhere(Hansen

et al

., 2002).

Construction of arylphorin prey plasmids

The vector pJG4-5 (Origene, Rockville, MD, USA) was used for the con-struction of prey plasmids. We modified the multiple cloning site of thisvector by introducing two different

Sfi

I restriction sites, which allowsfor directed cloning when using this enzyme and the insertion of

Sfi

I-digested cDNA for library construction. Therefore we developed twoprimers that are in part complementary (MCS+, MCS

; Table 1). Fivehundred picomoles of each primer was used in a 100

µ

l reactionand heated to 97

°

C. A 5 min incubation at 97

°

C was followed by suc-cessive cooling (

1

°

C/min) until 4

°

C was reached. The library vectorpJG4-5 was digested with

Eco

RI and

Xho

I and afterwards dephos-phorylated. The double-stranded DNA linker was ligated in thelibrary vector. We named the modified prey vector pB42AD(SfiIA/B).

Various prey vectors were constructed by PCR amplification ofarylphorin cDNA fragments, introducing restriction sites that

allowed cloning into pB42AD(SfiIA/B) and adding stop codons atthe 3

-ends. A selection of constructed prey plasmids is depictedin Fig. 1B. The following combinations of primers (Table 1) wereused for PCR amplification of cDNA fragments that were cloned inthe library plasmid: [A1/A2]

A1 prey; [A3/A4]

A5 prey; [A5/A2]

A6 prey; and [A6/A7]

A7 prey. Three prey plasmids wereobtained by the amplification of arylphorin fragments plus vectorfollowed by circularization: [A8/A9]

D1 prey; [A10/A11]

D2/3 prey; and [A12/A13]

D3 prey. The plasmids A2 prey, A3 preyand A4 prey contain

Eco

RI restriction fragments of the arylphorincDNA. The correct insertion of the cDNAs was checked bysequencing with primers S2 and S3 (Table 1).

Construction and implementation of a two-hybrid EGFP reporter plasmid

A two-hybrid reporter plasmid was constructed by removing theLacZ-cDNA and introducing EGFP-cDNA into the reporter plasmidpSH18-34. Therefore the LacZ-insertion site was sequenced usingthe primers that lie within the LacZ-cDNA-sequence (S4 and S5).Two primers were developed to amplify the reporter plasmid back-bone spacing out the LacZ-cDNA (R1 and R2). The EGFP-cDNAwas amplified from a common EGFP-vector (primer: EGFP5

,EGFP3

; template plasmid: pEGFP-N1, BD Clontech, Heidelberg,Germany) and introduced in the pSH18-34 backbone via a blunt-end ligation. The insertion site was sequenced (primer S6) and thefunction of the new vector was tested by transformation in theyeast strain EGY48 that contained the strong activating bait vector

Primer Sequence

S1 → CGTCAGCAGAGCTTCACCATTGS2 → CCAGCCTCTTGCTGAGTGGAGATGS3 ←←←← GACAAGCCGACAACCTTGATTGGAGS4 → TGGAGCCCGTCAGTATCGGCGGS5 ←←←← TGCAAGGCGATTAAGTTGGGTAACS6 → CCAGTGGTTATATGTACAGTACTG

R1 → CTCGAGGGTGTTATAATGGATCGAGGTGGACGAGTR2 ←←←← CTCGAGATTCAATTATTTAGTACAAATGGCTAAGAGGCATTTR3 ←←←← GCCCTCGAGTTAAGGCAACAACAGACGATGAGGCAACTTATTR4 ←←←← CTCGAGTTAACCAGAGATCTCATCATTATCATTGTAATTR5 ←←←← CTCGAGTTATCTGCCACCCAAAATATTGCCTR6 ←←←← CTCGAGTTATCCAGTGTCCATATGAGCAATCTCATCAATR7 ←←←← CTCGAGTTACTGCATTGTCTGCACACCCAAACCAATATTAGCR8 → CTCGAGAAAGAAGGCAAGGATAATAAGGAAGGACGTCAATGGAATAAA1 → CTCGAGCCGATTCTCAATAGTTTAACACCATTTTTAATGA2 ←←←← CTCGAGTTAATGATAGTAGCTGTAGTCGAAGTGGCCA3 → GAATTCTTCAACAAGGAATCTGTCTTGTCTTA4 ←←←← CTAAACATTAACGTCCTTTATGGTAACACCAGGGAAA5 → GGCCATTATGGCCGGAAATCTGGTGTCAATGAGTTCAAACGCA6 → GGCCATTATGGCCTACACTCACGAAGAATTGTTGTTCCCTGA7 ←←←← GGCCAGGCGGCCTTAGGTGTAGATGAAGCTGTCAATTTCCAA8 ←←←← P-AATATCACGAGTGTAGTCAACAGGCATGAA9 → P-TAACTCGAGAAGCTTTGGACTTCTTCGCA10 ←←←← P-GAATTCGGGAGAGGCATAATCTGGCA11 → P-GAATTCTTCAACAAGGAATCTGTCTTGTCTTA12 ←←←← P-CTATTCACCGTAACCGTGAGTCAAACGTTCCA13 → P-TACACTCACGAAGAATTGTTGTTCCCTGR1 ←←←← GGATCCGGGCTTGGCCAAGCTTR2 → TAATAATAACCGGGCAGGCCATGTCTGEGFP5′ → CGCCACCATGGTGAGCAAGGGCEGFP3′ ←←←← TTACTTGTACAGCTCGTCCATGCC

MCS+ → P-AATTCGGCCATTATGGCCATCGATCCCGGGGGCCGCCTCGGCCCMCS- ←←←← P-TCGAGGGCCGAGGCGGCCCCCGGGATCGATGGCCATAATGGCCG

Table 1. Primers used in this study. Arrows indicate the primer orientation, restriction sites are underlined, and Start and Stop codons are in bold

432 I. A. Hansen et al.

© 2003 The Royal Entomological Society, Insect Molecular Biology, 12, 427–432

pSH17-34. The yeast strain shows a strong green fluorescenceunder UV-light. We named the new reporter plasmid p8op-EGFP.For the determination of fluorescence intensities in different yeaststrains, the strains were grown in liquid culture to an OD600 of 1. Thefluorescence intensity was measured with a Fluoroscan-Ascentphotometer (Labsystems, Farnborough, UK) using an excitationfilter of 485 nm (± 10) and an emission filter of 520 nm (± 20).

Identification of hexamerin receptor interacting proteins (HRIPs)

We performed two interaction-screening experiments using a full-length hexamerin receptor precursor bait (ABP130-bait) andABP96-bait, which represents the functional hexamerin receptor.The bait plasmids were transformed in the yeast strain EGY48.Library plasmid DNA was introduced into the yeast strains with thelithium acetate-based transformation protocol (Gietz et al., 1995).Per experiment, approximately 500 000 colonies were screened tofind clones that would interact with the hexamerin receptor. In eachexperiment 50 primary transformants, which showed growth onleucine-deficient medium and a β-galactosidase-positive pheno-type, were selected and the library plasmids isolated. After aretransformation in yeast strains holding the particular bait plasmidwe chose 17 yeast clones from ABP-130 bait strains and 15 yeastclones of the ABP96-bait strains, and analysed the cDNA insertsby sequencing with primers S2 and S3 (Table 1).

Two-hybrid analysis of the receptor–ligand interaction

Various bait and prey combinations were tested for interaction bytransforming the plasmids into the yeast strain EGY48 and meas-uring the reporter gene activity in these strains (Figs 1 and 4).

Sequence similarities and multiple alignment

Sequence database analysis was performed with the BLAST program(National Centre for Biotechnology Information, National Institutesof Health, Bethesda, MD). Sequence alignment was conductedwith the CLUSTALW algorithm (http://www.clustalw.genome.ad.jp/).

Comparative modelling of Calliphora arylphorin

The structure of Calliphora arylphorin was modelled using theautomated comparative protein modelling server SWISS-MODEL(Guex & Peitsch, 1997; Peitsch, 1996). The model is based uponthe X-ray crystal structure coordinates of lobster haemocyaninfrom Panulirus interruptus (GENBANK accession number 494086)(Volbeda & Hol, 1989).

Acknowledgements

We thank Claudia Koelling and Aneliese Striewe-Conz forcompetent technical assistance and Geoffrey Attardo forlanguage-editing the manuscript. This work was supportedby the Deutsche Forschungsgemeinschaft (Sche 195/13).

References

Ausubel, F., Brent, R., Kingston, R., Moore, D., Seidman, J., Smith, J.and Struhl, K. (2001) Current Protocols in Molecular Biology.John Wiley & Sons, Inc. Hoboken, NJ.

Burmester, T. (2002) Origin and evolution of arthropod hemocy-anins and related proteins. J Comp Physiol 172: 95–107.

Burmester, T., Antoniewski, C. and Lepesant, J. (1999) Ecdysone-regulation of synthesis and processing of fat body protein 1, thelarval serum protein receptor of Drosophila melanogaster. EurJ Biochem 262: 49–55.

Burmester, T., Kolling, C., Schroer, B. and Scheller, K. (1998)Complete sequence, expression, and evolution of the hexam-erin LSP-2 of Calliphora vicina. Insect Biochem Mol Biol 28:11–22.

Burmester, T. and Scheller, K. (1995) Complete cDNA-sequenceof the receptor responsible for arylphorin uptake by the fat bodyof the blowfly, Calliphora vicina. Insect Biochem Mol Biol 25:981–989.

Burmester, T. and Scheller, K. (1996) Common origin of arthropodtyrosinase, arthropod hemocyanin, insect hexamerin, and dip-teran arylphorin receptor. J Mol Evol 42: 713–728.

Burmester, T. and Scheller, K. (1997a) Conservation of hexamerinendocytosis in Diptera. Eur J Biochem 244: 713–720.

Burmester, T. and Scheller, K. (1997b) Developmentally controlledcleavage of the Calliphora arylphorin receptor and posttrans-lational action of the steroid hormone 20-hydroxyecdysone.Eur J Biochem 247: 695–702.

Burmester, T. and Scheller, K. (1999) Ligands and receptors: com-mon theme in insect storage protein transport. Naturwissen-schaften 86: 468–474.

Chung, S., Kubo, T. and Natori, S. (1995) Molecular cloning andsequencing of arylphorin-binding protein in protein granules ofthe Sarcophaga fat body. Implications of a post-translationalprocessing mechanism. J Biol Chem 270: 4624–4631.

Fashena, S.J., Serebriiskii, I.G. and Golemis, E.A. (2000) LexA-based two-hybrid systems. Methods Enzymol 328: 14–26.

Gietz, R.D., Schiestl, R.H., Willems, A.R. and Woods, R.A. (1995)Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11: 355–360.

Guex, N. and Peitsch, M.C. (1997) SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling.Electrophoresis 18: 2714–2723.

Hansen, I.A., Meyer, S.R., Schafer, I. and Scheller, K. (2002)Interaction of the anterior fat body protein with the hexamerinreceptor in the blowfly Calliphora vicina. Eur J Biochem 269:954–960.

Haunerland, N. (1996) Insect storage proteins: gene families andreceptors. Insect Biochem Mol Biol 26: 755–765.

Kirchhausen, T. (2002) Clathrin adaptors really adapt. Cell 109:413–416.

Markl, J., Burmester, T., Decker, H., Savel-Niemann, A., Harris, J.,Suling, M., Naumann, U. and Scheller, K. (1992) Quaternaryand subunit structure of Calliphora arylphorin as deduced fromelectron microscopy, electrophoresis, and sequence similari-ties with arthropod hemocyanin. J Comp Physiol 162: 665–680.

Peitsch, M.C. (1996) ProMod and Swiss-Model: Internet-basedtools for automated comparative protein modelling. BiochemSoc Trans 24: 274–279.

Schneider, W.J. (1996) Vitellogenin receptors: oocyte-specificmembers of the low-density lipoprotein receptor supergenefamily. Int Rev Cytol 166: 103–137.

Volbeda, A. and Hol, W.G. (1989) Crystal structure of hexamerichaemocyanin from Panulirus interruptus refined at 3.2 A reso-lution. J Mol Biol 209: 249–279.