Molecular Vision 2005; 11:1061-70 <http://www.molvis.org/molvis/v11/a125/> Received 18 May 2005 | Accepted 21 November 2005 | Published 9 December 2005 Phopshodiesterase 6 is the primary effector of phototransduction in vertebrate photoreceptors [1,2]. This enzyme is composed of two catalytic subunits, PDE6α (88 kDa) and PDE6β (84 kDa) and two inhibitory subunits, PDE6γ (11 kDa). In photoreceptor outer segments, photolyzed rhodop- sin activates the GTP-binding protein transducin, which in turn activates PDE6 by chelating its PDE6γ subunits [1]. The PDE6γ domains that mediate its interactions with the cata- lytic subunits of PDE6 and with the GTP-binding subunit of transducin have been mapped. They are comprised of an in- ternal basic region (amino acids 24-45), a stretch of 10-20 aa upstream of position 76 and the C-terminal end of the protein (aa 77-87) [3-9]. In a recent study [10], we observed that PDE6γ also contains a functional polyproline motif (aa 20-28) that mediated its interaction with the Src homology type 3 (SH3) domain of formin-binding protein 17 (FBP17), a protein that is involved in the functional network linking endocytosis, cy- toskeleton dynamics and MAP kinase signaling [11], through its interactions with sorting nexin 2 and with dynamin [12,13]. Our results fell in good agreement with other studies indicat- ing that, when overexpressed in HEK293 cells, PDE6γ inter- fered with MAP kinase signalling and co-precipitated with the endocytosis-related protein, dynamin [14]. These obser- vations called for further studies aimed at identifying the SH3- containing partners of endogenously-expressed PDE6γ. Al- though several studies have reported the presence of PDE6γ protein and/or mRNA in non-photosensitive tissues or cell lines [10,14], expression levels appeared to be quite low and ill- adapted for the identification of PDE6γ-associating proteins. In contrast, PDE6γ is naturally expressed at high levels in photoreceptors, which makes them an obvious site in which to examine the in vivo interactions of PDE6γ with SH3-con- taining proteins. In the present work, we investigate the abil- ity of PDE6γ to associate with a variety of SH3-containing proteins, functionally related to endocytosis and to receptor tyrosine kinase signalling. As PACSIN was found to interact with PDE6γ in vivo and to colocalize with it in photorecep- tors inner segments and synaptic pedicles, our interest pro- gressively focused on this protein of the endocytosis machin- ery. METHODS Antibodies: The antibodies used in this study were, a mouse monoclonal anti-PACSIN (anti-syndapin I; Transduction Labo- ratories, Lexington, KY), a mouse monoclonal anti- AMPHIPHYSIN (Bramp2; Transduction Laboratories), a ©2005 Molecular Vision The regulatory subunit of PDE6 interacts with PACSIN in photoreceptors Florent Houdart, Nathalie Girard-Nau, Fabrice Morin, Pierre Voisin, Brigitte Vannier Laboratoire de Neurobiologie Cellulaire de l’UMR CNRS 6187, Faculte des Sciences, Universite de Poitiers, Poitiers, France Purpose: In photoreceptors, phosphodiesterase 6 (PDE6) is regulated in response to light, due to the shuttling of a regu- latory subunit (PDE6γ) between the catalytic subunits of PDE6 and the activated form of transducin. We showed previ- ously that PDE6γ 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 PDE6γ 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 PDE6γ 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 PDE6γ in rat retina extracts. Colocalization and light translocation of PDE6γ and one of its partner were studied by confocal microscopy. Results: PDE6γ interacts in vitro with a number of SH3 domains. These interactions involve a polyprolin motif located between amino acids 20 and 28 of PDE6γ. Several of the putative partners of PDE6γ are expressed in photoreceptor cells and might therefore interact in vivo with PDE6γ. Our results show that only PACSIN, a protein implicated in endocytosis, was found to interact with PDE6γ 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: PDE6γ function is mostly documented in the regulation of phototransduction. Our results provide evidence that in vitro PDE6γ has a broad pattern of SH3 containing partners expressed in photoreceptors. PDE6γ interaction with PACSIN points to a possible role of PDE6γ in endocytosis. Further studies will be needed to understand the exact role of PDE6γ-PACSIN interactions in photoreceptors. The description of this new function of PDE6γ might help to understand the molecular mechanism of the severe retinal degeneration observed in PDE6γ knock-out mice. Correspondence to: Brigitte Vannier, Laboratoire de Neurobiologie Cellulaire, UMR CNRS 6187, UFR Sciences, 40 Avenue du Recteur Pineau, 86022 Poitiers cedex, France; Phone: (33) 549 45 39 77; FAX: (33) 549 45 40 51; email: brigitte.vannier@univ-poitiers.fr 1061