INSULIN-MEDIATED SECRETION OF ECDYSTEROIDS FROM MOSQUITO OVARIES AND MOLECULAR-CLONING OF THE INSULIN-RECEPTOR HOMOLOG 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 a re released from the brain neurosecretory system and stimulate the ova ries to secrete ecdysteroids, which are necessary for vitellogenesis b y the fat body. Because bombyxins, which are insulin-like peptides, st imulate ecdysteroidogenesis in silkworm larvae, we tested porcine insu lin and found that it activates ecdysteroidogenesis and protein synthe sis 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 ve rtebrate and Drosophila insulin receptor homologues, as well as with t he insulin receptor-related receptor, the latter being an 'orphan' rec eptor with an unknown function. In the intracellular domain, high homo logies are observed, particularly in those subdomains that are respons ible for ATP binding and kinase activity. Northern blot analysis of MI R demonstrated a highly specific expression in ovaries, and cloning ex periments indicated its presence in the brain. Recombinant MIR extract s from a baculovirus expression system contained high constitutive kin ase activity in the presence of manganese or magnesium. Activation was independent of a ligand. SDS-gel analysis suggested that the recombin ant receptor was not post-translationally processed into an alpha- and beta-subunit as was expected from a putative cleavage signal. Enzymat ic properties of the proreceptor are presented: the k(m) for ATP was b etween 15 and 50 mu M in the presence of a synthetic substrate: maxima l kinase activity to 100-fold over basic activity was reached in the p resence of 1 mM manganese. Stimulation of key oogenic processes by por cine insulin and identification of a MIR indicate that insulin-like ne urohormones may have an important regulatory role in mosquito oogenesi s.