Molecular cloning of a functional allatostatin gut/brain receptor and an allatostatin preprohormone from the silkworm Bombyx mori.
The cockroach-type or A-type allatostatins are inhibitory insect neuropeptides with the C-terminal sequence Tyr/Phe-X-Phe-Gly-Leu-NH(2). Here, we have cloned an A-type allatostatin receptor from the silkworm Bombyx mori (BAR). BAR is 361 amino acid residues long, has seven transmembrane domains, shows 60% amino acid residue identity with the first Drosophila allatostatin receptor (DAR-1), and 48% identity with the second Drosophila allatostatin receptor (DAR-2). The BAR gene has two introns and three exons. These two introns coincide with and have the same intron phasing as two introns in the DAR-1 and DAR-2 genes, showing that the three receptors are not only structurally but also evolutionarily related. Furthermore, we have cloned a Bombyx allatostatin preprohormone that contains eight different A-type allatostatins. Chinese hamster ovary cells permanently transfected with BAR DNA react on the addition of 4 x 10(-9)M Bombyx A-type allatostatins with a second messenger cascade (measured as bioluminescence), showing that BAR is a functional A-type allatostatin receptor. Southern blots suggest that Bombyx has at least one other BAR-related gene in addition to the BAR gene described in this paper. Northern blots and quantitative reverse transcriptase-polymerase chain reaction of different larval tissues show that BAR mRNA is mainly expressed in the gut and to a much lesser extent in the brain. To our knowledge, this is the first report on the molecular cloning and functional expression of an insect gut/brain peptide hormone receptor.
Secher
T
T
Department of Cell Biology, Zoological Institute, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen, Denmark.
Lenz
C
C
Cazzamali
G
G
Sørensen
G
G
Williamson
M
M
Hansen
G N
GN
Svane
P
P
Grimmelikhuijzen
C J
CJ
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D012831:Bombyx; D001921:Brain / Q000378:metabolism*; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004064:Digestive System / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004330:Drosophila; D029721:Drosophila Proteins; D019143:Evolution, Molecular; D005091:Exons; D006728:Hormones / Q000235:genetics*; D019476:Insect Proteins; D007438:Introns; D007700:Kinetics; D008969:Molecular Sequence Data; D009479:Neuropeptides; D010802:Phylogeny; D011498:Protein Precursors / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014018:Tissue Distribution; D014162:Transfection
C096426:AstA-R1 protein, Drosophila; C543272:AstA-R2 protein, Drosophila; D018076:DNA, Complementary; D029721:Drosophila Proteins; D006728:Hormones; D019476:Insect Proteins; D009479:Neuropeptides; D011498:Protein Precursors; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide