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Molecular and Cellular Biology, July 2000, p. 5216-5226, Vol. 20, No. 14
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Modulation of CRX Transactivation Activity by Phosducin Isoforms†

Xuemei Zhu and Cheryl M. Craft*

The Mary D. Allen Laboratory for Vision Research, Doheny Eye Institute, and Department of Cell & Neurobiology, the Keck School of Medicine of the University of Southern California, Los Angeles, California 90089-9112

Received 15 November 1999/Returned for modification 23 December 1999/Accepted 9 March 2000

Phosducin (Phd) and Phd-like proteins (PhLPs) selectively bind guanine nucleotide protein (G protein) beta gamma subunits (Gbeta gamma ), while Phd-like orphan proteins (PhLOPs) lack the major functional domain for the binding of Gbeta gamma . 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.

* Corresponding author. Mailing address: Doheny Eye Institute, Department of Cell & Neurobiology, The Keck School of Medicine of the University of Southern California, BMT 401, 1333 San Pablo St., Los Angeles, CA 90089-9112. Phone: (323) 442-6692. Fax: (323) 442-2709. E-mail: ccraft{at}hsc.usc.edu.

dagger Dedicated to Mary D. Allen for her generous support of vision research and to the memory of Richard N. Lolley, who died on 3 April 2000.

Molecular and Cellular Biology, July 2000, p. 5216-5226, Vol. 20, No. 14
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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