Subcellular mislocalization and loss of function of mutant Gpnmb proteins

Abstract

Purpose: Pigmentary glaucoma involves optic nerve degeneration after accumulation of debris originating from pigmented cells within the eye, a frequent endpoint for Pigment Dispersion Syndrome (PDS) patients . The molecular events within pigmented ocular cells that precipitate this initial pigment dispersion remain uncharacterized. The DBA/2J mouse is an established model for human PDS and harbors a truncated mutant Gpnmb^R150X allele which has been proposed to be a functional null. To date the impact of C-terminal truncation on the intracellular sorting and biology of Gpnmb has yet to be determined.

Methods: We have characterized the intracellular transport pathway followed by wild-type Gpnmb and compared this to both Gpnmb^R150X and cytoplasmic domain mutants, including C-terminal truncation mutants that lack candidate sorting signals within the cytoplasmic domain of this integral membrane protein. We have assessed the intracellular transport of these mutant proteins by ectopic expression in transfected tissue culture cells followed by immunofluorescence microscopy. Early secretory pathway localization of protein at steady-state has been determined by endoglycosidase-H(endoH)-sensitivity in Western blotting experiments. Defects in endocytic sorting have been further analyzed by antibody uptake experiments.

Results: The severe truncation mutant Gpnmb^R150X is unable to leave the endoplasmic reticulum after synthesis as shown by intracellular steady-state localization to calnexin-positive membranes and complete endoH sensitivity. Cytoplasmic truncation mutants that retain the single transmembrane domain are able to progress through the secretory pathway but fail to be internalized upon delivery to the cell surface. Efficient endocytosis is dependent upon a functional cytoplasmic dileucine sorting signal, as shown by steady-state indirect immunofluorescence microscopy as well as antibody uptake endocytosis assays.

Conclusions: The wild-type function of Gpnmb in healthy pigmented cells within the eye requires the efficient intracellular targeting of Gpnmb to endosome-derived organelles. Loss of critical sorting signals, including a conserved cytoplasmic dileucine, results in missorting and so loss of function leading to aberrant melanosome morpohology in DBA/2J animals. Our data support a model for a protective function of Gpnmb within the internal environment of the melanosome.

Keywords: 448 cell membrane/membrane specializations • 599 microscopy: light/fluorescence/immunohistochemistry • 588 melanocytes