Ocular albinism type 1 (OA1) is an X-Iinked disorder characterized by severe reduction of visual acuity, retinal hypopigmentation, foveal hypoplasia, and the presence of giant melanosomes (macromelanosomes) in skin melanocytes and retinal pigment epithelium. The protein product of the OA1 gene is a pigment cell specific membrane glycoprotein, displaying structural and functional features of G protein-coupled receptors (GPCRs). However, in contrast to all other previously characterized GPCRs, OA1 is not localized to the plasma membrane, but is targeted to intracellular organelles, namely the melanosomes in pigment cells and the Iysosomes in transfected non-melanocytic cells. These unique characteristics suggest that OA1 represents the first example described so far of an exclusively intracellular GPCR, supporting previous hypotheses that GPCR-mediated signal transduction systems might also operate at the internal membranes in mammalian cells. In particular, OA1 could function as a """"""""sensor"""""""" of a yet unidentified intra-melanosomal ligand, regulating melanosome maturation and/or movements through activation of heterotrimeric G proteins on the cytoplasmic side of the melanosomal membrane. To test these hypotheses, the present project focuses on the understanding of the molecular function of OA1 and on the study of its upstream and downstream pathways.
The first aim of the proposal concerns the formal demonstration that OA1 functions as a GPCR by using different approaches: definition of the membrane topology of OA1; identification of the OA1 ligand; generation of constitutively active OA1 mutants; characterization of the sorting determinants of OA1, which could facilitate ligand identification.
The second aim of the proposal focuses on the dissection of the OA1 downstream pathway, including the testing of the microtubule cytoskeleton and of other melanosomal proteins as candidate effectors, and the search for unsuspected interactors by the yeast two-hybrid system and mass spectrometry-based approaches. Given the roles of intracellular heterotrimeric G proteins in membrane traffic, these studies will hopefully shed light not only on the pathogenesis of ocular albinism, but also on more general mechanisms of intracellular signal transduction possibly mediated by GPCR-G protein systems, providing important clues for understanding fundamental processes both in cell biology and molecular medicine.
