First published online December 3, 2004
Journal of Experimental Biology 207, 4573-4586 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01317
Functional characterisation of the Anopheles leucokinins and their cognate G-protein coupled receptor
Jonathan C. Radford,
Selim Terhzaz,
Pablo Cabrero,
Shireen-A. Davies and
Julian A. T. Dow*
Institute of Biomedical and Life Sciences, Division of Molecular
Genetics, University of Glasgow, Glasgow G11 6NU, UK
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Fig. 1. The cDNA and protein sequences of the putative Anopheles gambiae
preproleucokinin gene. Nucleotide and amino acid residue numbers are indicated
at the end of each line, amino acids are centred on their codons. The start
codon, following stop codon and putative polyadenylation signal are indicated
in bold type. Upstream in-frame stop codons are double underlined. A putative
TATA-box sequence is underlined, the initiation of transcription consensus
sequence is indicated by a shaded box, and the position of the single intron
is indicated by ><. Within the putative preproleucokinin the location of
the three A. gambiae leucokinin peptides are indicated by shaded
boxes and a possible signal peptide is underlined. Proteolytic processing
sites thought to be used are underlined. The Gly (G) residues that are
presumed to be processed to C-terminal amides in the mature A.
gambiae leucokinins are indicated in bold and double underlined. Four Cys
(C) residues within the preproleucokinin are also indicated by shaded
boxes.
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Fig. 2. Comparison of the fruit fly and mosquito leucokinin peptides. The sequences
of the Drosophila melanogaster (Drosophila leucokinin),
Aedes aegypti (Aedes leucokinin), Culex salinarius
(Culex leucokinin) and Anopheles gambiae (Anopheles
leucokinin) leucokinin peptides are compared. Residues conserved with
Drosophila leucokinin are indicated in bold. Analysis of the D.
pseudoobscura genome sequence suggests that a single leucokinin identical
to that of D. melanogaster is encoded.
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Fig. 4. Alignment of the known leucokinin receptor protein sequences. Amino acid
residue numbers are indicated at the end of each line. The predicted TM
domains are underlined. TM domains were predicted using the TMHMM 2.0
prediction program. Identical residues with a threshold limit of 75% are
indicated by a shaded box. Sequence alignments were performed using CLUSTAL X,
and annotated using BioEdit. Key conserved residues, such as the
Asp-Arg-Tyr/His (DRY/H) triplet motif after TM III, Cys
(C) residues in the second and third extracellular loops and potential
N-glycosylation sites are in bold type and underlined.
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Fig. 5. Dendrogram of the known leucokinin receptors. A CLUSTAL X protein alignment
was performed using the putative TM spanning regions of the known leucokinin
receptors. From this a phylogram was produced using the TREEVIEW program. TM
domains were predicted using the TMHMM 2.0 program. The scale bar gives an
approximation of the number of substitutions per site. The Lymnaea
stagnalis (pond snail) receptor is used as an outgroup.
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Fig. 6. Real-time measurement of the [Ca2+]i response by
A. stephensi leucokinin receptor. S2 cells were co-transfected with
the A. stephensi leucokinin receptor ORF and apoaequorin ORF
constructs, and expression induced. Peptide was injected at 15 s. Samples were
stimulated with either Anopheles leucokinin I (A), II (B) or III (C)
peptide at a concentration of 10–7 mol l–1.
(D) Comparison of the responses to the three Anopheles leucokinin
peptides applied at a concentration of 10–7 mol
l–1. Data are expressed as [Ca2+]i
(nmol l–1) against time (s); measurements were taken at 0.1 s
intervals. The traces shown are average responses ± S.E.M.
(N=8). Error bars are negligible for all panels.
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Fig. 7. Dose–response curves for the action of the Anopheles
leucokinins on the A. stephensi leucokinin receptor. S2 cells were
co-transfected with the A. stephensi leucokinin receptor ORF and
apoaequorin ORF constructs, and expression induced.
Peptide-stimulated [Ca2+]i increases were measured in S2
cell aequorin-based assays, at different concentrations of peptide as
indicated. Values were expressed as maximal [Ca2+]–background
[Ca2+] (nmol l–1; mean ± S.E.M.,
N=5–8). Where error bars are not visible they are too small to
reproduce.
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Fig. 8. Real-time measurement of the [Ca2+]i response in S2
cells expressing D. melanogaster LKR, CG10626. S2 cells were
co-transfected with the D. melanogaster LKR, CG10626 ORF
(Radford et al., 2002 ) and
apoaequorin ORF constructs, and expression induced. Data are
expressed as [Ca2+]i (nmol l–1) against
time (s); measurements were taken at 0.1 s intervals. The traces shown are
average responses (N=5). Peptide was injected at 15 s. (A–C)
Samples were stimulated with either Anopheles leucokinin I (A), II
(B) or III (C) peptide at a concentration of 10–6 mol
l–1 (blue) or 10–7 mol l–1
(red). (D) Comparison of the responses to the three Anopheles
leucokinin peptides applied at a concentration of 10–6 mol
l–1. Error bars are negligible for all panels.
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Fig. 9. Cross-specific activation of the A. stephensi leucokinin receptor
with Drosophila leucokinin (drosokinin). (A) Real-time measurements
of [Ca2+]i in S2 cells expressing the A.
stephensi leucokinin receptor and apoaequorin in response to
Drosophila leucokinin. Data are expressed as
[Ca2+]i (nmol l–1) against time (s);
measurements were taken at 0.1 s intervals. The trace shown is an average
response (N=5). Peptide was injected at 15 s. (B) Dose–response
curve. Values were expressed as maximal [Ca2+]–background
[Ca2+] (nmol l–1; mean ± S.E.M.,
N=5). Where error bars are not visible they are too small to
reproduce.
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Fig. 10. Western blot analysis of the Anopheles leucokinin receptor.
Western blot of adult Malpighian tubule and head proteins using
Anopheles leucokinin receptor purified IgG. The antibody recognises
both a protein of the expected size (65 kDa) and a heavier band, of
approximately 72 kDa. The control lane is blotted with pre-immune serum.
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Fig. 11. Anopheles leucokinin receptor is expressed in stellate cells of
the Malpighian tubule. Tubules were stained with anti-Anopheles
leucokinin peptide purified IgG, raised as described in the text. Texas Red
secondary antibodies were used to visualize the primary antiserum and DAPI was
used to stain nuclei (blue). (A,B), Fluorescence microscopy of immunostained
whole-mount tubules revealed staining in the secondary stellate cell type
(arrows), concentrated in the basolateral membrane. (C) Tubules were processed
as for A and B, but with pre-immune serum, confirming the specificity of the
antibody. Only low-level non-specific staining of apical microvilli was
observed. All images were captured on a Zeiss 510 Meta confocal microscope
using a 63 x objective and the approximate scale can be determined from
the tubule diameter, which can be taken to be 35 µm.
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© The Company of Biologists Ltd 2004
Potential N-glycosylation sites in the
protein sequence. The conserved GPCR Asp-Arg-Tyr/His
(DRY/H) motif just after TM III and conserved Cys (C)
residues in extracellular loops 1 and 2 are also marked in bold type. The
position of binding sites for primers used in the analysis and construction of
the cDNA sequence are indicated. Nucleotides that are different between the
cDNA and primer sequences are indicated in red type.