MOSQUITO VITELLOGENIN RECEPTOR - PURIFICATION, DEVELOPMENTAL AND BIOCHEMICAL-CHARACTERIZATION

Vitellogenin receptors (VgRs) play a critical role in egg development of oviparous animals by mediating endocytosis of the major yolk protei n precursor, vitellogenin, A modification of the method for extracting the mosquito (Aedes aegypti) VgR from ovary membranes resulted in an 11-fold higher yield and 56-fold increase in relative purity of the Vg R, in turn permitting purification, antibody production, and microsequ encing. A K-d of 15 nM was estimated from binding assays for the enric hed VgR, indicating a very high affinity for its ligand. Immunoprecipi tation of [C-14]VgR using anti-VgR polyclonal antibodies followed by S DS-PAGE under reducing conditions and fluorography demonstrated that t he 205 kDa VgR does not consist of subunits held together with disulfi de bonds. However, an immunoblot of the native VgR suggests that it ex ists as a similar to 390 kDa noncovalent homodimer in its native state , Immunoblot assays confirmed that the VgR is present only in ovarian tissue. A quantitative immunoassay of VgR extracts showed that VgR was present in previtellogenic ovaries on the day of emergence, increasin g from 2 ng to more than 10 ng per ovary by day 5, After initiation of vitellogenesis and onset of Vg uptake, VgR quantity increased rapidly between 8 and 24 h after a blood meal, then began to decline between 24 and 36 h. Immunocytochemistry confirmed the presence of substantial amounts of the VgR in 4-day-old previtellogenic oocytes, In both prev itellogenic and vitellogenic ovaries, the VgR was present only in the oocyte, primarily in the cortex.