Modulation of CRX transactivation activity by phosducin isoforms

Phosducin (Phd) and Phd-like proteins (PhLPs) selectively bind guanine nucl eotide protein (G protein) beta gamma subunits (G beta gamma), while Phd-li ke orphan proteins (PhLOPs) lack the major functional domain for the bindin g of G beta gamma. A retina- and pineal gland-specific transcription factor , cone-rod homeobox (CRX), was identified by a yeast two-hybrid screen usin g PhLOP1 as the bait. Direct protein-protein interactions between Phd or Ph LOP1 and CRX were demonstrated using a beta-galactosidase quantitative assa y in the yeast two-hybrid system and were confirmed by an in vitro binding assay and a glutathione S-transferase (GST) pull-down assay. To determine i f the interaction with Phd or PhLOP1 affected CRX transactivation, a 120-bp interphotoreceptor retinoid binding protein (IRBP) promoter-luciferase rep orter construct containing a CRX consensus element (GATTAA) was cotransfect ed into either COS-7 or retinoblastoma Weri-Rb-1 cells with expression cons tructs for CRX and either Phd or PhLOP1, Phd and PhLOP1 inhibited the trans criptional activation activity of CRX by 50% during transient cotransfectio n in COS-7 cells and by 70% in Weri-Rb-1 cells and COS-7 cells stably trans fected with CRX. Phd inhibited CRX transactivation in a dose-dependent mann er. Whereas Phd is a cytoplasmic phosphoprotein, coexpression of Phd with C RX results in Phd being localized both in the cytoplasm and nucleus. By con trast, PhLOP1 is found in the nucleus even without CRX coexpression. To add ress the physiological relevance of these potential protein interacting par tners, we identified immunoreactive proteins for Phd and CRX in retinal cyt osolic and nuclear fractions. Immunohistochemical analysis of bovine retina s reveals colocalization of Phd isoforms with CRX predominantly in the inne r segment of cone cells, with additional costaining in the outer nuclear la yer and the synaptic region. Our findings demonstrate that both Phd and PhL OP1 interact directly with CRX and that each diminishes the transactivation activity of CRX on the IRBP promoter. A domain that interacts with CRX is found in the carboxyl terminus of the Phd isoforms. Phd antibody-immunoreac tive peptides are seen in light adapted mouse retinal cytosolic and nuclear extracts. Neither Phd nor PhLOP1 affected CRX binding to its consensus DNA element in electrophoretic mobility shift assays. A model that illustrates separate functional roles for interactions between Phd and either SUG1 or CRX is proposed. The model suggests further a mechanism by which Phd isofor ms could inhibit CRX transcriptional activation.