Molecular Cloning of a Functional Allatostatin Gut/Brain Receptor and an Allatostatin Preprohormone from the Silkworm Bombyx mori

Abstract

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.