Inhae Ji

Nearly 2000 G protein-coupled receptors (GPCRs)1 have been reported since bovine opsin was cloned in 1983 (1) and the β-adrenergic receptor in 1986 (2). They are classified into over 100 subfamilies according to the sequence homology, ligand structure, and receptor function. A substantial degree of amino acid homology is found among members of a particular subfamily, but comparisons between subfamilies show significantly less or no similarity.

Environmentally friendly toxins of Bacillus thuringiensis are effective in controlling agriculturally and biomedically harmful insects. However, little is known about the insect receptor molecules that bind these toxins and the mechanism of insecticidal activity. We report here for the first time the cloning and expression of a cDNA that encodes a receptor (BT-R1) of the tobacco hornworm Manduca sexta for an insecticidal toxin of B. thuringiensis. The receptor is a 210-kDa membrane glycoprotein that specifically binds the cryIA(b) toxin of B. thuringiensis subsp. berliner and leads to death of the hornworm. BT-R1 shares sequence similarity with the cadherin superfamily of proteins. Environmentally friendly toxins of Bacillus thuringiensis are effective in controlling agriculturally and biomedically harmful insects. However, little is known about the insect receptor molecules that bind these toxins and the mechanism of insecticidal activity. We report here for the first time the cloning and expression of a cDNA that encodes a receptor (BT-R1) of the tobacco hornworm Manduca sexta for an insecticidal toxin of B. thuringiensis. The receptor is a 210-kDa membrane glycoprotein that specifically binds the cryIA(b) toxin of B. thuringiensis subsp. berliner and leads to death of the hornworm. BT-R1 shares sequence similarity with the cadherin superfamily of proteins. INTRODUCTIONBiopesticides based on the bacterium Bacillus thuringiensis currently are being used as safe alternatives to chemical insecticides. B. thuringiensis toxins are environmentally friendly because they kill only those insects susceptible to the toxins, whereas current synthetic chemical pesticides indiscriminately kill pest and beneficial insects alike and are considered to be major toxic pollutants of the environment. Insecticidal properties of B. thuringiensis are manifested in crystalline glycoprotein toxins (cry gene products) (1Höfte H. Whitely H.R. Microbiol. Rev. 1989; 53: 242-255Crossref PubMed Google Scholar) that are produced during the sporulation cycle of this bacterium. The insects affected by B. thuringiensis include many agriculturally and biomedically detrimental pests in the orders Lepidoptera, Coleoptera, and Diptera. The primary action of B. thuringiensis toxins occurs in the brush border of insect midgut epithelial cells(2Gill S.S. Cowles E.A. Pietrantonio P.V. Annu. Rev. Entomol. 1992; 37: 615-636Crossref PubMed Google Scholar). Specific binding of these toxins to midgut brush-border membrane vesicles has been reported(3Hofmann H. Vanderbruggen H. Höfte H. Van Rie J. Jansens S. Van Mellaert H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7844-7848Crossref PubMed Scopus (342) Google Scholar, 4Van Rie J. Jansens S. Höfte H. Degheele D. Van Mellaert H. Eur. J. Biochem. 1989; 186: 239-247Crossref PubMed Scopus (280) Google Scholar, 5Van Rie J. Jansens S. Höfte H. Degheele D. Van Mellaert H. Appl. Environ. Microbiol. 1990; 56: 1378-1385Crossref PubMed Google Scholar). A number of putative receptors have also been identified(6Vadlamudi R.K. Ji T.H. Bulla Jr., L.A. J. Biol. Chem. 1993; 268: 12334-12340Abstract Full Text PDF PubMed Google Scholar, 7Oddou P. Hartmann H. Geiser M. Eur. J. Biochem. 1991; 202: 673-680Crossref PubMed Scopus (44) Google Scholar, 8Sangadala S. Walters F.S. English L.H. Adang M.J. J. Biol. Chem. 1994; 269: 10088-10092Abstract Full Text PDF PubMed Google Scholar, 9Knight P.J.K. Crickmore N. Ellar D.J. Mol. Microbiol. 1994; 11: 429-436Crossref PubMed Scopus (357) Google Scholar).However, little is known about the molecular nature of the insect receptors that bind these toxins and the mechanism of insecticidal activity. Here, we report the cloning and expression of a cDNA that encodes a novel cadherin-like glycoprotein receptor present in the midgut of the tobacco hornworm Manduca sexta. The receptor binds the cryIA(b) toxin of B. thuringiensis subsp. berliner, leading to death of this particular lepidopteran insect. We have named this receptor molecule BT-R1.EXPERIMENTAL PROCEDURESBT-R1 Purification and SequencingNatural BT-R1 of M. sexta was purified, as previously reported(6Vadlamudi R.K. Ji T.H. Bulla Jr., L.A. J. Biol. Chem. 1993; 268: 12334-12340Abstract Full Text PDF PubMed Google Scholar), by immunoprecipitating toxinbinding protein complexes with toxin-specific antisera and separating the complexes by SDS-polyacrylamide gel electrophoresis, followed by electroelution. Purified BT-R1 was subjected to cyanogen bromide digestion. The cyanogen bromide fragments were separated on a 17% high resolution Tricine 1The abbreviation used is: TricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. /SDS-polyacrylamide gel (10Schagger H. Jagow G.W. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10442) Google Scholar) and transferred to Problott membranes (Applied Biosystems Inc.). Five distinct bands (see Fig. 1) were extracted, and peptide sequences were determined by Edman degradation (Applied Biosystems Inc.). The amino acid sequences obtained from microsequencing were (M)LDYEVPEFQSITIRVVATDNNDTRHVGVA, (M)XETYELIIHPFNYYA, (M)XXXHQLPLAQDIKNH, (M)F/PN/IVR/YVDI/G, and (M)NFF/HSVNR/DE.cDNA Cloning and SequencingAn M. sexta cDNA library was constructed in γgt10 using the Superscript Choice System according to the manufacturer's instructions (Life Technologies, Inc.). Synthetic oligonucleotides corresponding to peptides 1-3 were labeled with γ-32P using polynucleotide kinase and were utilized to screen the cDNA library(11Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Approximately 4 × 105 recombinants were screened. A clone hybridizing to all three probes was plaque-purified and subcloned into pBluescript (Stratagene). Double-stranded cDNA in pBluescript was sequenced in both directions by the dideoxy chain termination method with Sequenase (U. S. Biochemical Corp.) according to the manufacturer's instructions.Northern Blot AnalysisTotal RNA from M. sexta midgut (10 μg) was separated on a 0.8% formaldehyde-agarose gel and blotted onto a nylon membrane (Amersham Corp.). The analysis was carried out according to Maniatis et al.(11Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The filter was hybridized with 32P-labeled, random-primed BT-R1 cDNA (SacI fragment, 4.8 kilobases). Filter hybridization was carried out at 42°C in 50% formamide, 5 × Denhardt's reagent, 5 × phosphate-buffered (25 mM KPO4) SSC, and 50 μg/ml salmon sperm DNA. The filter was washed two times with 1 × SSC plus 0.1% SDS and two times with O.25 × SSC plus 0.1% SDS at 42°C. Each wash was for 20 min, and the filter was exposed to x-ray film for 24 h.Expression of BT-R1 cDNA in COS-7 and 293 CellsThe 4810-base pair cDNA that encodes the open reading frame of BT-R1 was subcloned into the pcDNA3 vector (Invitrogen). COS-7 cells and human embryonic kidney 293 cells were transfected with the construct using a modified calcium phosphate method(11Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). For transient expression assays, cells (COS-7 and 293) were harvested 60 h after transfection and washed with phosphate-buffered saline(12Ji I. Ji T.H. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 7167-7170Crossref PubMed Scopus (39) Google Scholar). Cell membranes were prepared by differential centrifugation(13Ji I. Zeng H. Ji T.H. J. Biol. Chem. 1993; 268: 22971-22974Abstract Full Text PDF PubMed Google Scholar). Control cells were transfected with the pcDNA3 vector without insert. For stable expression, 293 cells were subcultured 1 day after transfection into fresh medium containing G418 (400 μg/ml). Surviving cells were assayed for their ability to bind the toxin, and those cells expressing the highest level of receptors were selected.Blotting and Binding StudiesCell membranes (10 μg) were separated on a 7.5% SDS-polyacrylamide gel, blotted onto a nylon membrane, and blocked with Tris-buffered saline containing 5% nonfat dry milk powder, 5% glycerol, and 0.1% Tween 20. The nylon membrane then was incubated with 125I-cryIA(b) toxin (2 × 105 cpm/ml) for 2 h. The nylon membrane was washed four times (20 min each) with blocking buffer, dried, and exposed to x-ray film at −70°C. For competition binding assays, 293 cells transiently expressing the BT-R1 cDNA clone were incubated with 125I-cryIA(b) toxin (0.32 nM) in the presence of increasing concentrations (0-10−6M) of unlabeled cryIA(b) toxin. Nonspecific binding was measured as bound radioactivity in the presence of 1 μM unlabeled toxin. The toxin was radioiodinated as described previously(14Elshourbagy N.A. Korman D.R. Wu H. Sylvester D.R. Lee J.A. Nagulaganti P. Bergsma D.J. Kumar C.S. Nambi P. J. Biol. Chem. 1993; 268: 3873-3879Abstract Full Text PDF PubMed Google Scholar).In Vitro TranslationpBluescript plasmid containing BT-R1 cDNA was linearized and transcribed with T3 polymerase (Pharmacia Biotech Inc.). In vitro translation was carried out according to the manufacturer's instructions with nuclease-treated rabbit reticulolysate (Life Technologies, Inc.). After 1 h of incubation at 30°C, the reaction mixture was either combined with an equal volume of SDS buffer or lysed with 50 mM Tris buffer containing 1% Nonidet P-40 and 250 mM NaCl (pH 8.0) for immunoprecipitation. Water was substituted for mRNA in the control reaction (see Fig. 6, lane1). Immunoprecipitation was carried out with either preimmune serum or anti-BT-R1 serum. Translation and immunoprecipitation products were electrophoresed on a 7.5% SDS-polyacrylamide gel, fixed, treated with ENHANCE (DuPont NEN), dried, and exposed to x-ray film for 12 h.Figure 6:In vitro translation and N-glyconase digestion of BT-R1. A, in vitro translation of the BT-R1 cDNA clone. mRNA produced in vitro was translated in a rabbit reticulolysate system. 35S-Labeled proteins were separated on a 7.5% SDS-polyacrylamide gel and visualized by autoradiography. Lane1, translation products generated in the absence of mRNA; lane2, translation products generated with BT-R1 mRNA; lane3, translation products immunoprecipitated with normal serum; lane4, translation products immunoprecipitated with anti-BT-R1 serum. B, N-glycanase F treatment of BT-R1. Purified BT-R1 protein was digested with N-glycanase F (lane6) and without enzyme (lane5) as described under “Experimental Procedures,” and the digestion products were separated on a 7.5% SDS-polyacrylamide gel and stained with Coomassie Brilliant Blue. Positions of molecular mass markers are indicated in kilodaltons.View Large Image Figure ViewerDownload Hi-res image Download (PPT)N-Glycanase F Treatment of BT-R1Purified BT-R1 protein was denatured by boiling in 1% SDS for 5 min, followed by the addition of Nonidet P-40 to a final concentration of 1% in the presence of 0.1% SDS. The preparation was incubated in sodium phosphate buffer (pH 8.5) at 37°C with N-glycanase F (10 units/ml) and without enzyme for 10 h. Digestion products were separated on a 7.5% SDS-polyacrylamide gel and stained with Coomassie Brilliant Blue.RESULTS AND DISCUSSIONPreviously, we described the identification, purification, and characterization of natural BT-R1, which specifically recognizes the cryIA(b) toxin of B. thuringiensis subsp. berliner(6Vadlamudi R.K. Ji T.H. Bulla Jr., L.A. J. Biol. Chem. 1993; 268: 12334-12340Abstract Full Text PDF PubMed Google Scholar). The binding of the toxin to natural BT-R1 present in brush-border membrane vesicles of M. sexta was specific and high, with a Kd value of 0.71 nM. Immunoprecipitation and 125I-cryIA(b) toxin binding in a ligand blot revealed specific binding of the toxin to a 210-kDa protein (Fig. 1A, arrowheads in lanes2 and 3, respectively). Purified natural BT-R1 was digested with cyanogen bromide (Fig. 1B), and five major peptides were sequenced. Degenerate oligonucleotides were synthesized, based on these peptide sequences (15Zhang S. Zubay G. Goldman E. Gene (Amst.). 1991; 105: 61-72Crossref PubMed Scopus (146) Google Scholar), and were used to screen an M. sexta midgut cDNA library. A single clone hybridized to three of the oligonucleotide probes and contained an insert of 5571 bases. It had an open reading frame of 4584 bases and 1528 amino acids. Amino acid sequences of the cyanogen bromide fragments of natural BT-R1 matched perfectly within the deduced sequence of BT-R1 (Fig. 2A). The deduced polypeptide is 172 kDa and has a pI of 4.5. The translation start site is flanked by the consensus translation initiation sequence (GAGATGG) of eukaryotic mRNAs(16Kozak M. Nucleic Acids Res. 1987; 15: 8125-8132Crossref PubMed Scopus (4151) Google Scholar). A polyadenylation signal(17Manley J.L. Yu H. Ryner L. Mol. Cell. Biol. 1985; 5: 373-379Crossref PubMed Scopus (42) Google Scholar), AATAAA, was observed at position 5561.Figure 2:Deduced amino acid sequence of BT-R1 and alignment of BT-R1 repeats with published cadherin repeats. A, the putative signal sequence and the transmembrane domain are underlined with single and doubleboldfacesolidlines, respectively. Asterisks denote putative N-glycosylation sites. Cysteines are indicated in boldface. Amino acids determined by sequencing of cyanogen bromide fragments of BT-R1 are underlined with thinsolidlines(19von Heijne G.V. J. Mol. Biol. 1985; 184: 99-105Crossref PubMed Scopus (1520) Google Scholar). Arrows delineate boundaries between the repeats. The arrowhead designates the C terminus of repeat 11. B, extracellular repeats of BT-R1 (BT-R1EC 1-11) are aligned with representative extracellular repeats of mouse P-cadherin (mPEC1), Drosophilafat extracellular repeat 18 (fatEC18), protocadherin (PC42EC2), and human intestinal peptide transporter extracellular repeat 1 (HPT-1 EC1). Conserved residues are in boldface.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Total RNA was prepared from midguts of M. sexta and was hybridized by Northern blotting with the antisense 4.8-kilobase SacI fragment of the BT-R1 cDNA clone. The probe hybridized to a single 5.6-kilobase band that corresponds to the 5571-base-long BT-R1 cDNA clone (Fig. 3). This result indicates that the BT-R1 cDNA clone represents the full-length coding sequence of the BT-R1 gene. To demonstrate that the protein encoded by the BT-R1 cDNA is a membrane protein and is capable of binding cryIA(b) toxin, the BT-R1 cDNA was subcloned into the mammalian expression vector pcDNA3 (Invitrogen), and the construct was transfected into COS-7 cells. Membranes from the COS-7 were and with 125I-cryIA(b) toxin. 125I-cryIA(b) toxin bound to a protein of kDa (Fig. It was labeled only in membranes prepared from M. sexta (Fig. and from COS-7 cells transfected with the BT-R1 cDNA construct band was observed in membranes from COS-7 cells embryonic 293 cells were transfected with BT-R1 cDNA and for stable blotting of the transfected expression of the 210-kDa which recognizes the cryIA(b) toxin binding of toxin to control 293 was observed BT-R1 was on the of transfected human embryonic 293 cells and high 1 nM) for the toxin (Fig. as natural Rie J. Jansens S. Höfte H. Degheele D. Van Mellaert H. Eur. J. Biochem. 1989; 186: 239-247Crossref PubMed Scopus (280) Google blot of RNA from M. sexta RNA was on an gel, blotted onto a nylon membrane, and hybridized with random-primed cDNA SacI as described under “Experimental and 18 Large Image Figure ViewerDownload Hi-res image Download blot analysis of BT-R1 A, ligand blot analysis of membranes from M. sexta and COS-7 Membranes (10 μg) were on a 7.5% SDS-polyacrylamide gel, transferred to a nylon membrane, and labeled with 125I-cryIA(b) toxin. Lane1, M. sexta brush-border lane2, lane3, with the BT-R1 cDNA clone. B, ligand blot analysis of 293 cells expressing BT-R1 from 293 cells were with SDS buffer, separated by SDS-polyacrylamide gel electrophoresis, and labeled with 125I-cryIA(b) toxin. protein from control 293 protein from 293 cells expressing BT-R1 Large Image Figure ViewerDownload Hi-res image Download of 125I-cryIA(b) toxin to transfected human embryonic 293 cells expressing BT-R1. The cells were transfected with the BT-R1 cDNA in pcDNA3 and incubated with 125I-cryIA(b) toxin (0.32 nM) in the presence of increasing concentrations (0-10−6M) of unlabeled cryIA(b) toxin. Nonspecific binding was determined as bound radioactivity in the presence of 1 μM unlabeled toxin. The Kd value nM) was determined by Large Image Figure ViewerDownload Hi-res image Download of the 210-kDa protein is 172 the molecular mass of the protein (Fig. To the was to of the the BT-R1 clone was translated in a rabbit reticulolysate that The translated products were immunoprecipitated with natural BT-R1. In vitro translation of the BT-R1 cDNA clone generated two protein bands of and kDa as determined by SDS-polyacrylamide gel (Fig. 6, The two bands were immunoprecipitated specifically by BT-R1 by preimmune serum The presence of the translation was to the initiation of translation from an M. Mol. Cell. Biol. 1989; PubMed Scopus Google Scholar) at amino acid The presence of a band and that this clone represents BT-R1, and the in is to F treatment the molecular mass of BT-R1 from to kDa and N-glycosylation at of the consensus N-glycosylation in the protein (Fig. 2A). Treatment of BT-R1 with and the of the natural protein that the protein from the BT-R1 cDNA clone is the as the natural protein in the midgut of M. sexta because they both have amino acid and molecular and toxin binding and as as pI R.K. Ji T.H. Bulla Jr., L.A. J. Biol. Chem. 1993; 268: 12334-12340Abstract Full Text PDF PubMed Google Scholar). The amino acid sequence (Fig. a putative signal peptide Heijne G.V. J. Mol. Biol. 1985; 184: 99-105Crossref PubMed Scopus (1520) Google Scholar) of 20 amino acids and a transmembrane domain of amino acids J. J. Mol. Biol. PubMed Scopus Google Scholar) at position A C terminus the transmembrane the toxin binds to a extracellular fragment of R.K. Ji T.H. Bulla Jr., L.A. J. Biol. Chem. 1993; 268: 12334-12340Abstract Full Text PDF PubMed Google Scholar), the is to in the This is with the that is only consensus N-glycosylation site in the domain with N-glycosylation in the extracellular similarity and to of the cadherin superfamily of proteins (Fig. D.J. J. Mol. Biol. 1990; PubMed Scopus Google Scholar). are membrane and are to and M. 1991; PubMed Scopus Google Scholar). cadherin-like molecules as Drosophilafat human intestinal peptide and have been D.J. B. J. Cell Biol. 1992; PubMed Scopus Google Scholar, U. P. H. C.S. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar, J. S. D. Jr., 1994; PubMed Scopus Google Scholar, H. S. M. T. S. S. J. 1993; PubMed Scopus Google Scholar). the extracellular domain of BT-R1 is and repeats (Fig. 2A). The of the BT-R1 repeats is to that of amino for repeats 6, and which are amino acids cadherin repeats. The in the repeats of BT-R1 (Fig. include and and 1 and 2 residues (Fig. and are the consensus sequences for calcium M. D. H. J. J. S. J. 1987; PubMed Scopus Google Scholar). The putative domain of amino acids is cadherin amino and to proteins in the sequences the cadherin little with those of all the described This particular with the of a of to a and for BT-R1. the of BT-R1 is is that BT-R1 be in membrane P. B. P. Scopus Google Scholar, B. M. M. P. Biochem. C Biochem. 1993; Scholar). is to that of the cadherin-like human intestinal peptide which peptide epithelial cells that the J. S. D. Jr., 1994; PubMed Scopus Google thuringiensis toxins are to at epithelial cells in the midgut of S.S. Cowles E.A. Pietrantonio P.V. Annu. Rev. Entomol. 1992; 37: 615-636Crossref PubMed Google Scholar). It has been that the toxins bind to a specific membrane receptor and then are into the membrane to a that membrane The is of the epithelial cells and death of the Ellar D.J. 1987; Scopus Google Scholar). competition binding Van Rie et Rie J. Jansens S. Höfte H. Degheele D. Van Mellaert H. Eur. J. Biochem. 1989; 186: 239-247Crossref PubMed Scopus (280) Google Scholar) that M. sexta brush-border membranes have two by all three toxins and and that recognizes only In ligand of M. sexta brush-border membrane the 210-kDa receptor is capable of specifically all three R.K. Ji T.H. Bulla Jr., L.A. J. Biol. Chem. 1993; 268: 12334-12340Abstract Full Text PDF PubMed Google Scholar, S. B. M. Biochem. Res. 1994; PubMed Scopus Google Scholar). This indicates that the BT-R1 a binding site for all toxins by Van Rie et Rie J. Jansens S. Höfte H. Degheele D. Van Mellaert H. Eur. J. Biochem. 1989; 186: 239-247Crossref PubMed Scopus (280) Google this report is the first to the cloning and expression of an insect receptor for a B. thuringiensis toxin. characterization of BT-R1 to a of the molecular of action of B. thuringiensis toxins and to an of the mechanism of insect to B. thuringiensis In the of environmentally friendly for current insect pests as as for insects to B. thuringiensis INTRODUCTIONBiopesticides based on the bacterium Bacillus thuringiensis currently are being used as safe alternatives to chemical insecticides. B. thuringiensis toxins are environmentally friendly because they kill only those insects susceptible to the toxins, whereas current synthetic chemical pesticides indiscriminately kill pest and beneficial insects alike and are considered to be major toxic pollutants of the environment. Insecticidal properties of B. thuringiensis are manifested in crystalline glycoprotein toxins (cry gene products) (1Höfte H. Whitely H.R. Microbiol. Rev. 1989; 53: 242-255Crossref PubMed Google Scholar) that are produced during the sporulation cycle of this bacterium. The insects affected by B. thuringiensis include many agriculturally and biomedically detrimental pests in the orders Lepidoptera, Coleoptera, and Diptera. The primary action of B. thuringiensis toxins occurs in the brush border of insect midgut epithelial cells(2Gill S.S. Cowles E.A. Pietrantonio P.V. Annu. Rev. Entomol. 1992; 37: 615-636Crossref PubMed Google Scholar). Specific binding of these toxins to midgut brush-border membrane vesicles has been reported(3Hofmann H. Vanderbruggen H. Höfte H. Van Rie J. Jansens S. Van Mellaert H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7844-7848Crossref PubMed Scopus (342) Google Scholar, 4Van Rie J. Jansens S. Höfte H. Degheele D. Van Mellaert H. Eur. J. Biochem. 1989; 186: 239-247Crossref PubMed Scopus (280) Google Scholar, 5Van Rie J. Jansens S. Höfte H. Degheele D. Van Mellaert H. Appl. Environ. Microbiol. 1990; 56: 1378-1385Crossref PubMed Google Scholar). A number of putative receptors have also been identified(6Vadlamudi R.K. Ji T.H. Bulla Jr., L.A. J. Biol. Chem. 1993; 268: 12334-12340Abstract Full Text PDF PubMed Google Scholar, 7Oddou P. Hartmann H. Geiser M. Eur. J. Biochem. 1991; 202: 673-680Crossref PubMed Scopus (44) Google Scholar, 8Sangadala S. Walters F.S. English L.H. Adang M.J. J. Biol. Chem. 1994; 269: 10088-10092Abstract Full Text PDF PubMed Google Scholar, 9Knight P.J.K. Crickmore N. Ellar D.J. Mol. Microbiol. 1994; 11: 429-436Crossref PubMed Scopus (357) Google Scholar).However, little is known about the molecular nature of the insect receptors that bind these toxins and the mechanism of insecticidal activity. Here, we report the cloning and expression of a cDNA that encodes a novel cadherin-like glycoprotein receptor present in the midgut of the tobacco hornworm Manduca sexta. The receptor binds the cryIA(b) toxin of B. thuringiensis subsp. berliner, leading to death of this particular lepidopteran insect. We have named this receptor molecule BT-R1.

G protein-coupled receptors (GPCRs) are ubiquitous mediators of signaling of hormones, neurotransmitters, and sensing. The old dogma is that a one ligand/one receptor complex constitutes the functional unit of GPCR signaling. However, there is mounting evidence that some GPCRs form dimers or oligomers during their biosynthesis, activation, inactivation, and/or internalization. This evidence has been obtained exclusively from cell culture experiments, and proof for the physiological significance of GPCR di/oligomerization in vivo is still missing. Using the mouse luteinizing hormone receptor (LHR) as a model GPCR, we demonstrate that transgenic mice coexpressing binding-deficient and signaling-deficient forms of LHR can reestablish normal LH actions through intermolecular functional complementation of the mutant receptors in the absence of functional wild-type receptors. These results provide compelling in vivo evidence for the physiological relevance of intermolecular cooperation in GPCR signaling.

The genomic structure of the LH receptor is important to our understanding of its expression mechanisms, functional domains, relationships with other hormone receptors, and evolution. We have isolated four overlapping cosmid clones and six subgenomic clones of the rat LH receptor gene. They span a total of 95.6 kilobases (kb) and extend from 23 kb upstream of the translation start site to 13 kb down-stream of the stop codon. In addition, part of the human LH receptor gene has been isolated. The coding region of the rat hormone receptor gene spans over 60 kb and consists of 11 exons and 10 introns. Southern blots hybridized with exon 1 and exon 11 probes as well as gene dose analyses demonstrate that a single copy gene encodes the rat LH receptor. Sequence comparison suggests that the porcine and human LH receptor genes have similar, if not identical, exon-intron structures. There is no consensus cAMP-responsive element within 600 basepairs up-stream of the translation start site in spite of the cAMP responsiveness of the LH receptor gene. There are, however, unconventional cAMP-responsive elements in the region: one which is identical, several which are homologous to the activating protein-2-binding elements, CCCCAGGC, and several sequences which are similar to the G-rich cAMP-responsive element found in P450c21, a steroid 21-hydroxylase. The first 10 exons encode the N-terminal half of the molecule, while exon 11 encodes the C-terminal half of the molecule. This last exon is the same in the rat and human genes. The DNA and amino acid sequences of the first 10 exons show significant similarities and reveal repetitive sequence motifs. They have similar sizes which occur in the range of 69 and 183 bases; 8 of them are from 69-81 bases. Despite these remarkable similarities, structural predictions of exons 1-10 show a diversity of structures. The N-terminal half of the LH receptor appears to have a folded structure, with frequent turns and an extensive surface area. Part of the surface is predicted to be covered by amphiphilic helices and beta structures, types of secondary structure frequently found at the interfaces between subunits or between 2 interacting molecules. The introns dividing these exons also share many similarities.(ABSTRACT TRUNCATED AT 400 WORDS)

We have reported that the rat LH receptor is encoded by 11 exons of a single copy gene. Exons 1-10 encode the N-terminal half and exon 11 the C-terminal half. Since exon splice sites often mark structural transitions of multiexon molecules, we have attempted to define the function of the exons by generating mutant receptors with missing exons. As a first step, we have constructed two LH mutant receptors, one containing exons 1-10 (LH receptor (exon)1-10) and the other containing exon 1 and exon 11 (LH receptor(exon)1&11). These mutant receptors were functionally expressed in Cos 7A cells. The LH mutant receptor(exon)1-10, which lacks the membrane associated C-terminal half of the receptor, showed a high affinity for hCG. Surprisingly, the LH mutant receptor(exon)1&11 recognized hCG with a low affinity and stimulated G-proteins and cAMP production. The results demonstrate that exons 1-10 encode a high affinity hCG binding site and proves an important hypothesis that exon 11 encodes the site for receptor-modulation to activate G-proteins. Furthermore, the results raises an intriguing possibility of a second hormone binding site in the C-terminal half and multistep hormone binding.