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Latrophilin-like receptor from the parasitic nematode Haemonchus contortus as target for the anthelmintic depsipeptide PF1022A¹

BEATE SAEGER, HANS-PETER SCHMITT-WREDE, MARKUS DEHNHARDT^*, W. PETER M. BENTEN, JÜRGEN KRÜCKEN, ACHIM HARDER, GEORG VON SAMSON-HIMMELSTJERNA, HERBERT WIEGAND^* and FRANK WUNDERLICH²

Division of Molecular Parasitology and Center for Biological and Medical Research, Heinrich-Heine-University, 40225 Düsseldorf, Germany;
^* Medical Institute of Environmental Hygiene at the Heinrich-Heine University, 40225 Düsseldorf, Germany; and
Business Group Animal Health, Research and Development, Biological Technology Center, Bayer AG, 51368 Leverkusen, Germany

²Correspondence: Division of Molecular Parasitology and Center for Biological and Medical Research, Heinrich-Heine-University, Universitätsstr. 1, 40225 Düsseldorf, Germany; E-mail: frank.wunderlich{at}uni-duesseldorf.de

SPECIFIC AIMS

In this study, we have tried to identify a possible protein target for PF1022A belonging to a new class of cyclodepsipeptides with broad anthelmintic activity in the parasitic nematode Haemonchus contortus.

PRINCIPAL FINDINGS

1. The orphan receptor HC110-R
Using PF1022A ligand immunoscreening of a cDNA library constructed from the parasitic nematode H. contortus, we identified a 3539 bp cDNA encoding a novel orphan heptahelical transmembrane 110 kDa receptor, termed HC110-R (Fig. 1A ). Database and phylogenetic analysis reveal a heptahelical transmembrane protein of 1014 amino acids (B0457.1, protein id: CAA9109.1) of the nematode Caenorhabditis elegans as to be the homolog receptor to HC110-R.

View larger version (67K): [in this window] [in a new window]   Figure 1. The HC110-R protein. A) Alignment of amino acid sequences of H. contortus HC110-R (accession no.: AJ272270) and C. elegans protein B0457.1 (protein id: CAA91091.1). Identical amino acids are marked with asterisks, highly related amino acids with a colon, related amino acids with a single dot. Signal peptide (SP, residues 1–21, boldface); lectin-like sequence (lectin, residues 22–125, dotted); Thr-stretch (T-rich, residues 128–147, grey shaded); Cys-rich signature (C signature, residues 166–221, underlined; Cys and Trp residues additional boldface); 4-Cys motif (4 C region, residues 478–524, underlined; Cys and Trp residues additional boldface); the 7-transmembrane domains (between residues 536–772, boldface and underlined); Pro-rich stretch (P-rich, residues 845–861, grey-shaded); the PEST region (PEST, residues 915–933, grey shaded); N-glycosylation sites (N, residues 26, 499, and 862, boldface); highly conserved putative disulfide binding Cys-Cys-pair among secretin GPCRs (position 595, and 666, boldface and double underlined). B) Structure of HC110-R and rat latrophilin-1. Signal peptide (SP), lectin domain (lectin), Thr-stretch (T-stretch), Cys motif (signature), 4-Cys region (4 C region), 7-transmembrane domains (1–7, black), Pro-rich motif (P-rich), PEST region (PEST). Putative glycosylation sites (N), Cys residues of the 4 C region, Cys residues for a putative disulfide binding (small line without capital letters), olfactomedin binding motif (olfactomedin), Pro-Thr region (P-T region), linker domain (linker), and long region containing several repeats (long). C) Phylogenetic tree. The dendrogram contains bootstrap values only above 90%. The scale bar indicates a phylogenetic distance of 0.2 substitutions per site. HC HC110-R (protein id: CAC01096) H. contortus latrophilin-like receptor HC110-R; CE B0457.1 (protein id: CAA91091.1) C. elegans hypothetical protein B0457.1; RN Lat-1 (accession no.: U72487) Rattus norvegicus latrophilin-1; BT Lat-1 (AF111098) Bos taurus latrophilin-1; RN Lat-2 (AF063102) R. norvegicus latrophilin-2; BT Lat-2 (AF111078) B. taurus latrophilin-2; BT Lat-3 (AF111085) B. taurus latrophilin-3; RN Lat-3 (U72487) R. norvegicus latrophilin-3; HS CD97 (X84700) Homo sapiens CD97; MM EMR1-R (X93328) Mus musculus EMR1 hormone receptor; CE B0286.2 (AAA80689.2) C. elegans hypothetical protein B0286.2; HS CTR (L00587) H. sapiens calcitonin receptor; RN CTR (X70658) R. norvegicus calcitonin-like receptor; SS CTR (M74420) Sus scrofa calcitonin receptor; HS GHRHR (L01406) H. sapiens growth hormone-releasing hormone receptor; RN VIPR2 (Z25885) R. norvegicus vasoactive intestinal polypeptide receptor 2; HS CRFR (L23333) H. sapiens corticotropin releasing factor receptor; RN CRFR (L25438) R. norvegicus corticotropin releasing factor receptor; MM CRFR (X72305) M. musculus corticotropin releasing factor receptor; MM Celsr1 (AF031572) M. musculus neural-specific 7- transmembrane receptor.

2. Similarity of HC110-R with latrophilin
The G-protein-coupled receptor (GPCR) latrophilin-1 of 210 kDa was originally isolated from mammalian brain, and two close homologs latrophilin-2 and latrophilin-3 were recently identified. HC110-R has 31% identity with latrophilin from human, bovine, and rat. Figure 1B depicts the structural domains of HC110-R and the rat 1466 amino acid latrophilin-1. The mammalian latrophilins, HC110-R and CE-B0457.1 form a highly significant monophyletic subfamily within the secretin receptor family (Fig. 1C ).

3. -Latrotoxin (LTX) signaling through HC110-R
LTX is the major vertebrate-specific neurotoxic protein of the black widow spider venom and acts as an ligand to the mammalian orphan latrophilin receptor. LTX also binds to HC110-R. Indeed, when HEK-293 cells transfected with a carboxyl-terminally myc/his-tagged HC110-R were subjected to Western blotting, LTX binding to immobilized HC110-R protein could be detected by an anti-LTX antibody. In particular, LTX binding was confined to the 54 kDa amino-terminal fragment of HC110-R expressed in Escherichia coli, whereas the 21 kDa carboxyl-terminal polypeptide of HC110-R did not bind any LTX.

Binding of LTX to HC110-R also evoked functional responses of transfected HEK-293 cells. A video imaging system at the single cell level was used to analyze the effect of 75 nM LTX on the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) of Fura-2-loaded cells transfected with HC110-R-GFP. LTX caused two subsequent rises in [Ca²⁺]_i: an early, very small elevation by 5 ± 0.2 nM Ca²⁺ beginning after about 2 min; and a delayed, major Ca²⁺ elevation by 220 ± 14.9 nM peaking after about 22 min. HEK-293 cells not transfected with HC110-R did not exhibit any response to LTX, just as cells transiently transfected only with GFP or with two other GPCRs such as the mouse Lß adrenergic receptor and the human M1 muscarinic acetylcholine receptor. LTX-induced rise in [Ca²⁺]_i was predominantly due to an influx of external Ca²⁺. This proceeded through Ca²⁺ channels in the plasma membrane that were blockable by Cd²⁺ and nifedipine.

4. Targeting of HC110-R by BAY44–4400
BAY44–4400 is a more soluble derivative of PF1022A containing two morpholine residues covalently linked to the phenyl rings of the two D-phenyllactyl residues of PF1022A, and therefore was used in the following studies. Binding of BAY44–4400 to HC110-R, particularly to the amino terminus, was shown using an overlay analysis and a pull-down assay (Fig. 2A , B ). Binding of BAY44–4400 to HC110-R also affected LTX signaling through HC110-R. At concentrations up to 400 ng/ml, the very effective nematicide BAY44–4400 and its optical antipode PF1022–001, which exhibits anti-nematodal activity only at 100-fold higher concentrations than BAY44–4400, did not induce any change in [Ca²⁺]_i of HC110-R-GFP-transfected HEK-293 cells (Fig. 2Ca ). However, both substances affected LTX signaling, though to a different extent (Fig. 2Cb , d ). Whereas the almost ineffective PF1022–001 always caused about the same effect on LTX-induced change in [Ca²⁺]_i under different experimental conditions, BAY44–4400 impaired the LTX-induced Ca²⁺ response to varying extents. Specificity of these findings is further substantiated by the fact that 400 ng/ml BAY44–4400 did not affect the Ca²⁺ response of nontransfected HEK-293 cells to 1 mM carbachol, 1 mM isoproterenol, or 1 mM arecoline mediated through the endogenous M1 muscarinic receptors, ß₂-adrenergic receptors or nicotinic receptors in HEK-293 cells, respectively (Fig. 2Ce , f ).

View larger version (48K): [in this window] [in a new window]   Figure 2. Interactions of PF1022A with HC110-R. A) HEK-293 cells nontransfected (1) and transfected with carboxyl-terminally myc/his-tagged HC110-R (2) were subjected to SDS-PAGE. Proteins were then partly renatured before blotting and incubating with PF1022A. Immunodetection of bound PF1022A was done using the anti-PF1022A-KLH antiserum. B) The 54 kDa amino-terminal and 21 kDa carboxyl-terminal regions of HC110-R expressed in E. coli were precipitated with streptavidin-dynabeads coated with biotinylated PF1022A and subjected to Western blotting. 1) 2 mg dynabeads; 2) 4 mg dynabeads; 3) mixture of amino- and carboxyl-terminal regions; 4) control without biotinylated PF1022A. C) HEK-293 cells transfected with GFP-tagged HC110-R were stimulated as indicated by arrows. n = number of cells. a) Addition of 400 ng/ml BAY44–4400 (black line) or optical antipode PF1022–001 (grey line). For control, cells were treated with HEPES-buffer (HBS), the solvent of LTX, after addition of 400 ng/ml BAY44–4400 or PF1022–001. b) LTX signaling in the presence of 4 ng/ml BAY44–4400 and of PF1022–001. c) LTX signaling of cells preincubated for 90 min with 4 ng/ml BAY44–4400 or PF1022–001. Substances were removed 6 min before adding LTX. d) LTX signaling of HC110-R-GFP-transfected cells preincubated with 400 ng/ml BAY44–4400 or PF1022–001 for 90 min; substances were removed before adding LTX. e) Nontransfected HEK-293 cells were stimulated with 1 mM carbachol for 100 s in the presence or absence of BAY44–4400 (400 ng/ml). f) Nontransfected HEK-293 cells were stimulated with 1 mM isoproterenol and with 1 mM arecoline for 100 s in the presence or absence of BAY44–4400 (400 ng/ml).

CONCLUSIONS AND SIGNIFICANCE

PF1022A belongs to a novel class of cyclodepsipeptides with broad anthelmintic activity. In this study, we have isolated HC110-R, a novel heptahelical transmembrane receptor from the parasitic nematode H. contortus, with similarity to the mammalian G-protein coupled receptor latrophilin (Fig. 3) . LTX, the ligand of latrophilin, binds to the extracellular amino-terminal region of HC110-R and induces influx of external Ca²⁺ through Cd²⁺- and nifedipine-blockable Ca²⁺ channels in HC110-R-transfected HEK-293 cells. PF1022A also binds to the amino terminus of HC110-R and acts as an antagonist to LTX signaling in HC110-R-transfected HEK-293 cells. Identification of the natural ligand to HC110-R will reveal which physiological signaling pathways are impaired in H. contortus and other parasitic nematodes by PF1022A.

View larger version (52K): [in this window] [in a new window]   Figure 3. Schematic view of HC110-R disposition in plasma membrane (cf. also Fig. 1A , B ).

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0664fje ; to cite this article, use FASEB J. (March 27, 2001) 10.1096/fj.00-0664fje

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