Phosducin (Phd) and Phd-like
proteins (PhLPs) selectively bind
guanine nucleotide protein (
G protein) betagamma subunits (Gbetagamma), while Phd-like orphan
proteins (PhLOPs) lack the major functional domain for the binding of Gbetagamma. A retina- and pineal gland-specific
transcription factor, cone-rod homeobox (CRX), was identified by a yeast two-hybrid screen using
PhLOP1 as the bait. Direct
protein-
protein interactions between Phd or
PhLOP1 and CRX were demonstrated using a
beta-galactosidase quantitative assay 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 if the interaction with Phd or
PhLOP1 affected CRX transactivation, a 120-bp
interphotoreceptor retinoid binding protein (IRBP) promoter-
luciferase reporter construct containing a CRX consensus
element (GATTAA) was cotransfected into either COS-7 or
retinoblastoma Weri-Rb-1 cells with expression constructs for CRX and either Phd or
PhLOP1. Phd and
PhLOP1 inhibited the transcriptional activation activity of CRX by 50% during transient cotransfection in COS-7 cells and by 70% in Weri-Rb-1 cells and COS-7 cells stably transfected with CRX. Phd inhibited CRX transactivation in a dose-dependent manner. Whereas Phd is a cytoplasmic
phosphoprotein, coexpression of Phd with CRX results in Phd being localized both in the cytoplasm and nucleus. By contrast,
PhLOP1 is found in the nucleus even without CRX coexpression. To address the physiological relevance of these potential
protein interacting partners, we identified immunoreactive
proteins for Phd and CRX in
retinal cytosolic and nuclear fractions. Immunohistochemical analysis of bovine retinas reveals colocalization of Phd
isoforms with CRX predominantly in the inner segment of cone cells, with additional costaining in the outer nuclear layer and the synaptic region. Our findings demonstrate that both Phd and
PhLOP1 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-immunoreactive
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
isoforms could inhibit CRX transcriptional activation.