Dysbindin promotes the post-endocytic sorting of G protein-coupled receptors to lysosomes.
BACKGROUND
Dysbindin, a cytoplasmic protein long known to function in the biogenesis of specialized lysosome-related organelles (LROs), has been reported to reduce surface expression of D2 dopamine receptors in neurons. Dysbindin is broadly expressed, and dopamine receptors are members of the large family of G protein-coupled receptors (GPCRs) that function in diverse cell types. Thus we asked if dysbindin regulates receptor number in non-neural cells, and further investigated the cellular basis of this regulation.


RESULTS
We used RNA interference to deplete endogenous dysbindin in HEK293 and HeLa cells, then used immunochemical and biochemical methods to assess expression and endocytic trafficking of epitope-tagged GPCRs. Dysbindin knockdown up-regulated surface expression of D2 receptors compared to D1 receptors, as reported previously in neurons. This regulation was not mediated by a change in D2 receptor endocytosis. Instead, dysbindin knockdown specifically reduced the subsequent trafficking of internalized D2 receptors to lysosomes. This distinct post-endocytic sorting function explained the minimal effect of dysbindin depletion on D1 receptors, which recycle efficiently and traverse the lysosomal pathway to only a small degree. Moreover, dysbindin regulated the delta opioid receptor, a more distantly related GPCR that is also sorted to lysosomes after endocytosis. Dysbindin was not required for lysosomal trafficking of all signaling receptors, however, as its depletion did not detectably affect down-regulation of the EGF receptor tyrosine kinase. Dysbindin co-immunoprecipitated with GASP-1 (or GPRASP-1), a cytoplasmic protein shown previously to modulate lysosomal trafficking of D2 dopamine and delta opioid receptors by direct interaction, and with HRS that is a core component of the conserved ESCRT machinery mediating lysosome biogenesis and sorting.


CONCLUSIONS
These results identify a distinct, and potentially widespread function of dysbindin in promoting the sorting of specific GPCRs to lysosomes after endocytosis.
Marley
Aaron
A
Department of Psychiatry, University of California San Francisco, San Francisco, California, United States of America.
von Zastrow
Mark
M
D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D000074765:Dysbindin; D049029:Dystrophin-Associated Proteins; D004705:Endocytosis / Q000502:physiology*; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D036341:Intercellular Signaling Peptides and Proteins; D008247:Lysosomes / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D021381:Protein Transport / Q000502:physiology; D011506:Proteins / Q000378:metabolism; D034622:RNA Interference; D017448:Receptors, Dopamine D2 / Q000235:genetics / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors
D002352:Carrier Proteins; C432416:DTNBP1 protein, human; D000074765:Dysbindin; D049029:Dystrophin-Associated Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D036341:Intercellular Signaling Peptides and Proteins; D011506:Proteins; D017448:Receptors, Dopamine D2; D043562:Receptors, G-Protein-Coupled; C424444:WFIKKN2 protein, human; D049452:Green Fluorescent Proteins; D066246:ErbB Receptors