The Role of the Scaffolding Protein INAD in Localization of Signaling Complexes to the Rhabdomeres of Drosophil Photoreceptors
Hahn, Adrienne R.
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Organization of proteins into macromolecular complexes is one way cells maximize the speed, specificity, and efficiency of signal transduction. In fruit fly photoreceptors InaD, a scaffolding protein containing 5 PDZ domains, organizes proteins involved in the visual signaling pathway into complexes within a microvillar stack of membranes known as the rhabdomere. Light activation of rhodopsin activates a signaling cascade via a Gq-coupled reaction that quickly opens Ca++-selective TRP channels. Subsequent Ca++ influx activates eye protein kinase C (ePKC), and calmodulin, which in turn modulate the activity of other visual proteins. Mutants in which the InaD / TRP association has been disrupted (inaD215) phenocopy the delayed inactivation of mutants lacking ePKC, suggesting that one of the functions of InaD includes localizing ePKC to its downstream targets such as the TRP channel. There are currently two different models for scaffolding proteins: the "beads on a string" or "tethering" model where the order of the binding domains and their respective binding partners is unimportant, and the "specific quaternary structure" model where the specific stereochemical orientation of the domains is vital for proper signaling. The latter model also allows for allosteric regulation of binding. We assess the "beads on a string" vs. the "specific quaternary structure" model for InaD, a scaffolding protein found in the photoreceptor cells of fruit flies, by analyzing the characteristics of the light response in flies expressing two InaD constructs where the order of the PDZ domains has been shuffled. Based on biochemical and electrophysiological data on these mutants, we conclude that the "specific quaternary structure" model applies best to InaD. In addition, we investigate whether the inaD215 phenotype is due to displacement of ePKC from microdomains of calcium initiated by TRP channels, by calcium imaging of photoreceptors expressing visual proteins tagged with CaMgaroo, a Ca++-sensitive derivative of yellow fluorescent protein. After conducting these experiments, we conclude that this is not the case. Rather, the inaD215 phenotype is most likely due to the inability of ePKC to phosphorylate TRP and and attenuate its activity. These results also support the "specific quaternary structure" model for InaD.