A proline-rich domain in the gamma subunit of phosphodiesterase 6 mediates interaction with SH3-containing proteins.
OBJECTIVE
Phosphodiesterase 6 (PDE6) is the primary effector of phototransduction in vertebrate photoreceptors. Previous studies described the expression of the regulatory subunit of rod PDE6 (Pgamma-rod) in non-photosensitive tissues of the adult rat and the effects of this protein on MAP kinase pathways. Upon examination of the Pgamma-rod sequence, we detected a proline-rich domain that might reveal its ability to interact with SH3-containing proteins. Therefore, the present study was initiated to identify new protein partners of Pgamma-rod.


METHODS
A yeast two-hybrid screen of a rat brain cDNA library was performed using Pgamma-rod as a bait. Pgamma-rod-SH3 interaction was confirmed by GST pull-down of in vitro-translated proteins. The aminoacids involved in the interaction were mapped by site-directed mutagenesis. Rnase protection assay, RT-PCR and western blot analysis were used to detect Pgamma-rod expression in various rat tissues.


RESULTS
A clone was isolated twice, that consisted essentially of the SH3 domain of the formin-binding protein 17 (FBP17). This interaction was confirmed by GST pull-down. Mutational analysis of the Pgamma-rod-FBP17 interaction confirmed it involved the proline-rich domain of Pgamma-rod and the SH3 domain of FBP17. This proline-rich domain also allowed Pgamma-rod to interact with Cdc42-interacting protein 4 (CIP4), another SH3-containing protein. RT-PCR and Rnase protection assay detected different amounts of Pgamma-rod mRNA in adult and embryonic rat tissues. Western blots confirmed the presence of low levels of Pgamma-rod protein only in embryonic tissues.


CONCLUSIONS
Our data suggest that Pgamma-rod participates in SH3-mediated cellular pathways and may therefore play a wider role than previously appreciated. One possibility is that FBP17 interaction with sorting nexin 2 might connect Pgamma-rod to receptor tyrosine kinase recycling. However, further studies are still required to identify the diversity of SH3-containing proteins that interact with Pgamma-rod. This effort should provide a rationale to understand how Pgamma-rod can affect receptor internalization-dependent MAP kinase activity.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D004252:DNA Mutational Analysis; D050556:Fatty Acid-Binding Proteins; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011392:Proline / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D017948:Retinal Rod Photoreceptor Cells / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020798:Two-Hybrid System Techniques
D002352:Carrier Proteins; C433770:FNBP1 protein, human; D050556:Fatty Acid-Binding Proteins; D008869:Microtubule-Associated Proteins; D021122:Protein Subunits; D012333:RNA, Messenger; D012313:RNA; D011392:Proline; C490205:Sprk protein, rat; D017346:Protein Serine-Threonine Kinases; D010727:Phosphoric Diester Hydrolases; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; C517431:PDE6B protein, human