Drosophila INAD, which contains five tandem protein interaction PDZ domains, plays an important role in the G protein-coupled visual signal transduction. Mutations in InaD alleles display mislocalization of signaling molecules of phototransduction which include the essential effector, phospholipase C-β (PLC-β), which is also known as NORPA. The molecular and biochemical details of this functional link are unknown. We report that INAD directly binds to NORPA via two terminally positioned PDZ1 and PDZ5 domains. PDZ1 binds to the C-terminus of NORPA, while PDZ5 binds to an internal region overlapping with the G box-homology region (a putative G protein-interacting site). The NORPA proteins lacking binding sites, which display normal basal PLC activity, can no longer associate with INAD in vivo. These truncations cause significant reduction of NORPA protein expression in rhabdomeres and severe defects in phototransduction. Thus, the two terminal PDZ domains of INAD, through intermolecular and/or intramolecular interactions, are brought into proximity in vivo. Such domain organization allows for the multivalent INAD-NORPA interactions which are essential for G protein-coupled phototransduction.
Interphotoreceptor retinoid binding protein (IRBP) is a retinoid and fatty acid binding glycoprotein secreted by rod and cone photoreceptors in all vertebrates. IRBP is believed to serve as a carrier for retinoids in the bleaching and regeneration cycle of rhodopsin. IRBP protein has been found to be decreased in vitamin A-deprived rats; it is rapidly recovered after retinol repletion. To understand the mechanism for this recovery, we determined whether vitamin A affects transcription and translation of the IRBP gene. Wild-type and transgenic mice harboring the IRBP promoter-CAT reporter fusion gene were maintained on a retinol-deficient diet supplemented with retinoic acid (-A) or on a control diet (+A) for up to 60 wk postweaning. Some of the -A mice were given retinol repletion for 7 days (-A+A). Electroretinography analysis revealed alterations in waveform and a 2 log unit decrease in b-wave sensitivity in the -A mice over a broad range of stimulus wavelengths. Retinol repletion effected a full recovery. Immunochemistry showed a significant decrease in the immunogold-labeled IRBP between the retinal pigment epithelium and the outer segments of the -A mice compared with +A and -A+A mice. Northern blots showed no differences in the amounts of IRBP or CAT mRNA between these three treatment groups. These results suggest that the regulation of IRBP by retinol is not transcriptional.
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