A newly independent applicant plans to continue work on mechanisms underlying the defects in rhodopsin mutations. Earlier work by the applicant established two different types of defects in rhodopsin mutants implicated in ADRP. One class is defective in protein exit from the ER/Golgi complex; the second class is incapable of polarized sorting and fails to targeting the rhodopsin to the rod outer segment. In this proposal, the applicant will follow up the observations on this second class of mutants in hopes of deciphering the mechanisms involved in rhodopsin localization. In the first specific aim, three complementary systems will be used to identify the signals responsible for the targeting of rhodopsin to the appropriate membrane. A polarized epithelial cell from dog kidney, the MDCK cell, will be used to analyze large numbers of rhodopsin mutants. To determine the suitability of this system for reflecting photoreceptor behavior, a salamander primary retinal culture will be examined for suitability as an experimental system. Finally, transgenic mice will be used to determine the reliability of both experimental systems in reflecting the in vivo situation. The work will establish the requirement and the sufficiency of the C-terminus domain as well as search for other important sequences in the sorting process. In the second specific aim, other molecular components of the sorting process will be sought by identifying proteins that bind to rhodopsin sorting sequences. Both the yeast two hybrid system and a direct protein/protein filter binding assays will be used. Identified genes will be sequenced to determine the nature of the encoded gene and their subcellular location will be determined.
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