The regulatory subunit of PDE6 interacts with PACSIN in photoreceptors.
OBJECTIVE
In photoreceptors, phosphodiesterase 6 (PDE6) is regulated in response to light, due to the shuttling of a regulatory subunit (PDE6gamma) between the catalytic subunits of PDE6 and the activated form of transducin. We showed previously that PDE6gamma is able to interact with the Src-homology type 3 (SH3) domain of formin-binding protein 17 (FBP17), a protein involved in membrane receptor endocytosis. FBP17 was not detected in rat retina. Therefore, we looked for other SH3 domain-containing proteins that might interact with PDE6gamma in rat photoreceptors.


METHODS
Several SH3 domains highly homologous to this domain of FBP17 were found by structural alignment. Yeast two-hybrid system and GST pull-downs were used to test interaction of PDE6gamma with these putative partners. Expression patterns in rat retina of the SH3 containing candidates were also determined by immunohistochemistry and western blotting. GST pull-downs and co-immunopreciptations were then used to test in vivo interaction with PDE6gamma in rat retina extracts. Colocalization and light translocation of PDE6gamma and one of its partner were studied by confocal microscopy.


RESULTS
PDE6gamma interacts in vitro with a number of SH3 domains. These interactions involve a polyprolin motif located between amino acids 20 and 28 of PDE6gamma. Several of the putative partners of PDE6gamma are expressed in photoreceptor cells and might therefore interact in vivo with PDE6gamma. Our results show that only PACSIN, a protein implicated in endocytosis, was found to interact with PDE6gamma in rat retina extracts. The colocalization of the two proteins occurs in photoreceptor inner segments and synapses and is greatly enhanced upon illumination of the retina.


CONCLUSIONS
PDE6gamma function is mostly documented in the regulation of phototransduction. Our results provide evidence that in vitro PDE6gamma has a broad pattern of SH3 containing partners expressed in photoreceptors. PDE6gamma interaction with PACSIN points to a possible role of PDE6gamma in endocytosis. Further studies will be needed to understand the exact role of PDE6gamma-PACSIN interactions in photoreceptors. The description of this new function of PDE6gamma might help to understand the molecular mechanism of the severe retinal degeneration observed in PDE6gamma knock-out mice.
Houdart
Florent
F
Laboratoire de Neurobiologie Cellulaire de l'UMR CNRS 6187, Faculte des Sciences, Universite de Poitiers, Poitiers, France.
Girard-Nau
Nathalie
N
Morin
Fabrice
F
Voisin
Pierre
P
Vannier
Brigitte
B
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002531:Cerebellum / Q000378:metabolism; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D003598:Cytoskeletal Proteins; D003623:Dark Adaptation; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism*; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism* / Q000528:radiation effects; D016133:Polymerase Chain Reaction; D017729:Presynaptic Terminals / Q000378:metabolism; D011485:Protein Binding; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D020798:Two-Hybrid System Techniques; D018909:src Homology Domains / Q000235:genetics
D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; C117738:Pacsin1 protein, rat; D012333:RNA, Messenger; D012313:RNA; D010727:Phosphoric Diester Hydrolases; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6