Insulin-mediated secretion of ecdysteroids from mosquito ovaries and molecular cloning of the insulin receptor homologue from ovaries of bloodfed Aedes aegypti.
The reproductive cycle of female mosquitoes is activated by ingestion of blood from vertebrate hosts. Shortly after feeding, neurohormones are released from the brain neurosecretory system and stimulate the ovaries to secrete ecdysteroids, which are necessary for vitellogenesis by the fat body. Because bombyxins, which are insulin-like peptides, stimulate ecdysteriodgenesis in silkworm larvae, we tested porcine insulin and found that it activates ecdysteroidogenesis and protein synthesis in ovaries isolated from unfed mosquitoes. To further characterize the regulation of ecdysteroidogenesis in female mosquitoes, we cloned the mosquito insulin receptor (MIR) homologue from ovarian mRNA. The sequence of the extracellular domain shows moderate homologies with vertebrate and Drosophila insulin receptor homologues, as well as with the insulin receptor-related receptor, the latter being an "orphan" receptor with an unknown function. In the intracellular domain, high homologies are observed, particularly in those subdomains that are responsible for ATP binding and kinase activity. Northern blot analysis of MIR demonstrated a highly specific expression in ovaries, and cloning experiments indicated its presence in the brain. Recombinant MIR extracts from a baculovirus expression system contained high constitutive kinase activity in the presence of manganese or magnesium. Activation was independent of a ligand. SDS-gel analysis suggested that the recombinant receptor was not post-translationally processed into an alpha- and beta-subunit as was expected from a putative cleavage signal. Enzymatic properties of the proreceptor are presented: the Km for ATP was between 15 and 50 microM in the presence of a synthetic substrate: maximal kinase activity to 100-fold over basic activity was reached in the presence of 1 mM manganese. Stimulation of key oogenic processes by porcine insulin and identification of a MIR indicate that insulin-like neurohormones may have an important regulatory role in mosquito oogenesis.
Graf
R
R
Zoologisches Insitut, Universität Zürich, Switzerland.
Neuenschwander
S
S
Brown
M R
MR
Ackermann
U
U
D000330:Aedes / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004247:DNA; D026461:Ecdysteroids; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors; D019476:Insect Proteins / Q000235:genetics*; D007328:Insulin / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010053:Ovary / Q000378:metabolism; D011972:Receptor, Insulin / Q000235:genetics*; D011995:Recombination, Genetic; D017386:Sequence Homology, Amino Acid; D013256:Steroids / Q000096:biosynthesis*
D026461:Ecdysteroids; D019476:Insect Proteins; D007328:Insulin; D013256:Steroids; D004247:DNA; D011972:Receptor, Insulin