In vitro isoprenylation and membrane association of mouse rod photoreceptor cGMP phosphodiesterase alpha and beta subunits expressed in bacteria.

Qin, Ning; Pittler, Steven J; Baehr, Wolfgang

doi:10.1016/s0021-9258(18)42466-0

Cited by 72 publications

(7 citation statements)

References 46 publications

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“…The catalytic subunits of the PDE, R and β, undergo posttranslational C-terminal prenylation. The R subunit has been shown to be farnesylated on the cysteine of the C-terminal motif CCVQ, whereas the β subunit has been shown to be geranylgeranylated on the cysteine of CRIL (3,4), consistent with the specificity rules for protein prenyltransferases (5,6). Some evidence suggests that the C-termini of the PDE catalytic subunits are required in order for the PDE to remain membrane-bound.…”

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confidence: 78%

Binding of the Delta Subunit to Rod Phosphodiesterase Catalytic Subunits Requires Methylated, Prenylated C-Termini of the Catalytic Subunits

et al. 2000

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PDE6 (type 6 phosphodiesterase) from rod outer segments consists of two types of catalytic subunits, alpha and beta; two inhibitory gamma subunits; and one or more delta subunits found only on the soluble form of the enzyme. About 70% of the phosphodiesterase activity found in rod outer segments is membrane-bound, and is thought to be anchored to the membrane through C-terminal prenyl groups. The recombinant delta subunit has been shown to solubilize the membrane-bound form of the enzyme. This paper describes the site and mechanism of this interaction in more detail. In isolated rod outer segments, the delta subunit was found exclusively in the soluble fraction, and about 30% of it did not coimmunoprecipitate with the catalytic subunits. The delta subunit that was bound to the catalytic subunits dissociated slowly, with a half-life of about 3.5 h. To determine whether the site of this strong binding was the C-termini of the phosphodiesterase catalytic subunits, peptides corresponding to the C-terminal ends of the alpha and beta subunits were synthesized. Micromolar concentrations of these peptides blocked the phosphodiesterase/delta subunit interaction. Interestingly, this blockade only occurred if the peptides were both prenylated and methylated. These results suggested that a major site of interaction of the delta subunit is the methylated, prenylated C-terminus of the phosphodiesterase catalytic subunits. To determine whether the catalytic subunits of the full-length enzyme are methylated in situ when bound to the delta subunit, we labeled rod outer segments with a tritiated methyl donor. Soluble phosphodiesterase from these rod outer segments was more highly methylated (4.5 +/- 0.3-fold) than the membrane-bound phosphodiesterase, suggesting that the delta subunit bound preferentially to the methylated enzyme in the outer segment. Together these results suggest that the delta subunit/phosphodiesterase catalytic subunit interaction may be regulated by the C-terminal methylation of the catalytic subunits.

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confidence: 78%

Binding of the Delta Subunit to Rod Phosphodiesterase Catalytic Subunits Requires Methylated, Prenylated C-Termini of the Catalytic Subunits

et al. 2000

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“…Specific receptors may be involved in localizing some peripheral membrane proteins, such as p60src (Resh, 1989;Resh & Ling, 1990), to specific membranes and may recognize lipids covalently attached to these proteins. However, if the isoprenyl groups on PDEa|s (Anant et al, 1992) or the N-linked myristic acid that has recently been found on Ta (Wensel & Yang, 1992) is important for these proteins to bind membranes, as has been suggested for PDE (Ong et al, 1989;Catty & Deterre, 1991;Qin et al, 1992), their contributions to membrane binding clearly do not require specific receptor proteins. Nor do membranes enhance activation by serving as protein concentrators.…”

Section: P-i-t-mmentioning

confidence: 97%

Membrane stimulation of cGMP phosphodiesterase activation by transducin: comparison of phospholipid bilayers to rod outer segment membranes

Malinski¹,

Wensel²

1992

Biochemistry

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To clarify the role of phospholipids in G protein-effector interactions of vertebrate phototransduction, transducin activation of cGMP phosphodiesterase (PDE) has been reconstituted on the surface of well-defined phosphatidylcholine (PC) vesicles, using purified proteins from bovine rod outer segments (ROS). PC vesicles enhanced PDE stimulation by the GTP-gamma S-bound transducin alpha subunit (T alpha-GTP gamma S) as much as 17-fold over activation in the absence of membranes. In the presence of 3.5 microM accessible PC in the form of large (100 nm) unilamellar vesicles, 500 nM T alpha-GTP gamma S stimulated PDE activity to more than 70% of the maximum activity induced by trypsin. Activation required PC, PDE, and T alpha-GTP gamma S, but did not require prior incubation of any of the components, and occurred within 4 s of mixing. The PC vesicles were somewhat more efficient than urea-washed ROS membranes in enhancing PDE activation. Half-maximal activation occurred at accessible phospholipid concentrations of 3.8 microM for PC vesicles, and 13 microM for ROS membranes. Titrations of PDE with T alpha-GTP gamma S in the presence of membranes indicated a high-affinity (Kact less than 250 pM) activation of PDE by a small fraction (0.5-5%) of active T alpha-GTP gamma S, as did titrations of ROS with GTP gamma S. When activation by PC vesicles was compared to PDE binding to membranes, the results were consistent with activation enhancement resulting from formation of a T alpha-GTP gamma S-dependent PDE-membrane complex with half-maximal binding at phospholipid concentrations in the micromolar range. The value of the apparent dissociation constant, KPL, associated with the activation enhancement was estimated to be in the range of 2.5 nM (assuming an upper limit value of 1600 phospholipids/site) to 80 nM (for a lower limit value of 50 phospholipids/site). Another component of membrane binding was more than 100-fold weaker and was not correlated with activation by T alpha-GTP gamma S. Low ionic strength disrupted the ability of ROS membranes, but not PC vesicles, to bind and activate PDE. Removal of PDE's membrane-binding domain by limited trypsin digestion eliminated both the binding of PDE to vesicles and the ability of PDE to be activated by T alpha-GTP gamma S and membranes. These results suggest that ROS membrane stimulation of PDE activation by T alpha-GTP gamma S is due almost exclusively to the phospholipids in the disk membrane.

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“…PDE is bound through the isoprenylated carboxy termini of the catalytic a and p subunits to rod disc membranes (Qin, Pittler, and Baehr, 1992;Anant et al, 1992). The isoprenylated membrane-bound PDE is recognised and solubilised by PDE5 under physiological conditions .…”

Section: 61viii -Cone Pigmentsmentioning

confidence: 99%

Identification of a novel protein interacting with RPGR

Boylan

2000

Human Molecular Genetics

113

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A novel protein, called RPGRIP, has been identified as interacting with the RPGR protein, which is mutated in a severe form of human retinal degeneration, X-linked retinitis pigmentosa (RP3 type). The bovine RPGRIP was identified initially by screening for RPGR-interacting proteins with a bovine retina cDNA library using the yeast two-hybrid system. The specificity of the interaction was confirmed by co-immunoprecipitation of in vitro translated protein and using RPGR mutants. The human RPGRIP gene was isolated and shown to be expressed in retina and testis. Human RPGRIP spans a genomic interval of 34 kb, and consists of 15 exons, some of which are alternatively spliced. It was mapped using monochromosomal and radiation hybrid cell lines to chromosomal region 14q11. The function of RPGRIP is unknown; it shows no homology to proteins of known function, although it is predicted to form two coiled-coil domains at the N-terminus. RPGRIP is a strong candidate gene for causing human retinal degeneration.

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In vitro isoprenylation and membrane association of mouse rod photoreceptor cGMP phosphodiesterase alpha and beta subunits expressed in bacteria.

Cited by 72 publications

References 46 publications

Binding of the Delta Subunit to Rod Phosphodiesterase Catalytic Subunits Requires Methylated, Prenylated C-Termini of the Catalytic Subunits

Binding of the Delta Subunit to Rod Phosphodiesterase Catalytic Subunits Requires Methylated, Prenylated C-Termini of the Catalytic Subunits

Membrane stimulation of cGMP phosphodiesterase activation by transducin: comparison of phospholipid bilayers to rod outer segment membranes

Identification of a novel protein interacting with RPGR

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