A proline-rich domain in the gamma subunit of phosphodiesterase 6 mediates interaction with SH3-containing proteins.
PURPOSE: 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.
Mesh Terms:
Amino Acid Sequence, Animals, Base Sequence, Blotting, Western, Carrier Proteins, Cyclic Nucleotide Phosphodiesterases, Type 6, DNA Mutational Analysis, Microtubule-Associated Proteins, Molecular Sequence Data, Mutagenesis, Site-Directed, Phosphoric Diester Hydrolases, Proline, Protein Binding, Protein Subunits, Protein-Serine-Threonine Kinases, RNA, RNA, Messenger, Rats, Rats, Wistar, Retinal Rod Photoreceptor Cells, Reverse Transcriptase Polymerase Chain Reaction, Two-Hybrid System Techniques
Amino Acid Sequence, Animals, Base Sequence, Blotting, Western, Carrier Proteins, Cyclic Nucleotide Phosphodiesterases, Type 6, DNA Mutational Analysis, Microtubule-Associated Proteins, Molecular Sequence Data, Mutagenesis, Site-Directed, Phosphoric Diester Hydrolases, Proline, Protein Binding, Protein Subunits, Protein-Serine-Threonine Kinases, RNA, RNA, Messenger, Rats, Rats, Wistar, Retinal Rod Photoreceptor Cells, Reverse Transcriptase Polymerase Chain Reaction, Two-Hybrid System Techniques
Mol. Vis.
Date: Sep. 18, 2003
PubMed ID: 14502124
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