Intracellular trafficking of melanosomes in the retinal pigmented epithelium: mapping of interactions between Rab27a, Myrip and MyoVIIa

Abstract

Abstract: : Purpose:Usher syndrome type 1B (USH1B) is an autosomal recessive disorder characterised by progressive retinitis pigmentosa leading to blindness, profound congenital deafness and constant vestibular dysfunction. The genetic product of this locus has been implicated in the trafficking of rhodopsin to rod outer segments and in phagocytosis of rod outer segments by the RPE. Recently, Rab27a, Myrip and MyoVIIa were shown to form a complex involved in intracelullar movement of melanosomes within the RPE. Methods: We have characterised the Rab27a/Myrip/MyoVIIa complex binding–determinants by yeast two–hybrid and immunoprecipitation techniques. Results: The two MyoVII isoforms, MyoVIIa and MyoVIIb interact with Myrip in a yeast two–hybrid system and this direct interaction was confirmed by biochemical immunoprecipitation techniques and by co–immunoprecipitation following over expression in COS–7 cells. Myrip also interacted with the closely–related motor protein MyoVa. On the other hand, MyoVIIa/b did not interact with other members of the Myrip family or members of the synaptotagmin–like family, further supporting the idea of specificity of MyoVIIa/Myrip interaction. Our data indicate that the C–terminal tail of MyoVIIa interacts with two domains of Myrip, corresponding to the medial and C–terminal regions. MyoVIIb interacts preferentially with the second domain corresponding to C–terminus of Myrip. Conclusions: Our studies suggest MyoVII interact with two distinct regions of Myrip and that MyoVII isoforms bind differentially to these two regions. Characterization of the binding determinants for formation of the Rab27a/Myrip/MyoVIIa complex may reveal clues to the pathogenesis of Usher syndrome and related congenital retinal degenerations.

Keywords: protein structure/function • melanocytes • retinal pigment epithelium