The retinitis pigmentosa GTPase regulator, RPGR, interacts with the delta subunit of rod cyclic GMP phosphodiesterase

Abstract

Recently, the retinitis pigmentosa 3 (RP3) gene has been cloned and named retinitis pigmentosa GTPase regulator (RPGR). The amino-terminal half of RPGR is homologous to regulator of chromosome condensation (RCC1), the nucleotide exchange factor for the small GTPbinding protein Ran. In a yeast two-hybrid screen we identified the delta subunit of rod cyclic GMP phosphodiesterase (PDE␦) as interacting with the RCC1-like domain (RLD) of RPGR (RPGR 392 ). The interaction of RPGR with PDE␦ was confirmed by pull-down assays and plasmon surface resonance. The binding affinity was determined to be 90 nM. Six missense mutations at evolutionary conserved residues within the RLD, which were found in RP3 patients, were analyzed by using the two-hybrid system. All missense mutations showed reduced interaction with PDE␦. A non-RP3-associated missense substitution outside the RLD, V36F, did not abolish the interaction with PDE␦. PDE␦ is widely expressed and highly conserved across evolution and is proposed to regulate the membrane insertion or solubilization of prenylated proteins, including the catalytic subunits of the PDE holoenzyme involved in phototransduction and small GTP-binding proteins of the Rab family. These results suggest that RPGR mutations give rise to retinal degeneration by dysregulation of intracellular processes that determine protein localization and protein transport.Retinitis pigmentosa (RP) designates a heterogeneous group of hereditary retinal dystrophies characterized by impaired dark adaptation, progressive visual field defects, and severe reduction in visual acuity. X-linked RP (xlRP) is the most severe form of RP, which affects about 1 in 25,000 and leads to blindness by the third or fourth decade (1). Clinical surveys indicate that at least 16-33% of all RP patients show X-linked inheritance (2). The most common form of xlRP is RP type 3 (RP3), which affects about 70% of xlRP patients and is caused by mutations in a gene named RPGR.The RP GTPase regulator (RPGR) gene codes for a 90-kDa protein that contains a weakly conserved nucleotide binding motif at the N terminus and a potential isoprenylation site at the carboxyl terminus (3). Most significantly, the N-terminal half of RPGR is homologous to the regulator of chromosome condensation (RCC1) (3, 4). RCC1 is a chromatin binding protein that is involved in cell cycle regulation. Biochemically, RCC1 is the guanine nucleotide exchange factor (GEF) for Ran (5, 6), a small GTP-binding protein that is essential for nucleo-cytoplasmic transport (7,8). Recently, the threedimensional structure of RCC1 has been determined (9). It consists of a seven-bladed propeller formed by internal repeats. This structural motif also is found in p532, a GEF for Rab and Arf proteins (10), indicating that RCC1-like proteins might be general guanine nucleotide exchange proteins. To investigate the role of the RCC1-like domain (RLD) of RPGR in RP3, we applied the yeast two-hybrid system to identify proteins interacting with the RLD of RPGR. We were able to identify t...

Mutations ofRPGR in X-linked retinitis pigmentosa (RP3)

“…Since RCC1 domains may have guanine nucleotide exchange factor activity for small GTPases such as Ran [Bischoff and Ponstingl, 1991] or ARF1 and Rab proteins [Rosa et al, 1996], it was postulated that RPGR may also be an exchange factor for an unknown GTPase. Yeast two hybrid studies identified two proteins interact- ing with the RCC1 domain of RPGR: the phosphodiesterase delta subunit (PDE δ) [Linari et al, 1999a;Hong et al, 2001] and a novel protein called RPGRIP1 [Boylan and Wright, 2000;Roepman et al, 2000;Hong et al, 2001]. Interestingly, PDEδ not only interacts with retinal phosphodiesterase subunits [Florio et al, 1996], but also with the small GTPases Rab13 [Marzesco et al, 1998] and Arl3 [Linari et al, 1999b].…”

“…The effect on these interactions of a growing number of missense mutations in the RCC1 domain, many of which affect conserved residues, should be tested [Linari et al, 1999a;Boylan and Wright, 2000;Roepman et al, 2000].…”

Deletion of PrBP/δ impedes transport of GRK1 and PDE6 catalytic subunits to photoreceptor outer segments

“…Interactions with prenylated small GTPases of the Ras superfamily (12,(14)(15)(16), with a prenylated prostacyclin receptor, a G proteincoupled receptor located on blood vessels (17), as well as with nonprenylated small GTPases (Arl2 and Arl3) (18) are well established, but the physiological significance of these interactions is largely undefined. In the retina, PrBP/␦ was shown to interact in vitro with the RCC1-like domain of the retinitis pigmentosa G protein regulator (RPGR) (19,20), the prenyl chains of rhodopsin kinase (GRK1) (10), and PDE6␣ and PDE6␤ subunits (10,13).…”

PDE 6D binds to the C‐terminus of RPGR in a prenylation‐dependent manner

“…PDE6D (also known as PDEd and PrBP) is a prenylbinding protein involved in ciliary targeting of prenylated proteins [1][2][3][4]. Previous studies uncovered that PDE6D interacts with RPGR [5,6], mutations of which cause retinitis pigmentosa [7][8][9][10]. More recently, the structure of the PDE6D and RPGR (the N-terminal half) complex was determined and RPGR was proposed as a scaffold protein that recruits cargo-loaded PDE6D to the ciliary base [6].…”