[63] Purification of rod outer segment GTP-Binding protein subunits and cgmp phosphodiesterase by single-step column chromatography

Yamazaki, Akio; Tatsumi, Masahiro; Bitensky, Mark W.

doi:10.1016/0076-6879(88)59065-1

Cited by 43 publications

(28 citation statements)

References 5 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The proteins were concentrated, and glutathione was removed by buffer exchange with PBS using centrifugal concentrators (Millipore). G␤ 1 ␥ 1 was purified from bovine rod outer segments as described (21).…”

Section: Methodsmentioning

confidence: 99%

Gβγ Binds Histone Deacetylase 5 (HDAC5) and Inhibits Its Transcriptional Co-repression Activity

Spiegelberg

Hamm

2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

In a yeast two-hybrid screen designed to identify novel effectors of the G␤␥ subunit of heterotrimeric G proteins, we found that G␤␥ binds to histone deacetylase 5 (HDAC5), an enzyme involved in a pathway not previously recognized to be directly impacted by G proteins. Formation of the G␤ 1 ␥ 2 -HDAC5 complex in mammalian cells can be blocked by overexpression of G␣ o , and this inhibition is relieved by activation of ␣ 2A -adrenergic receptor, suggesting that the interaction occurs in a signal-dependent manner. The C-terminal domain of HDAC5 binds directly to G␤␥ through multiple motifs, and overexpression of this domain mimics the C terminus of G protein-coupled receptor kinase 2, a known G␤␥ scavenger, in its ability to inhibit the G␤␥/HDAC5 interaction. The C terminus of HDAC4 shares significant similarity with that of HDAC5, and accordingly, HDAC4 is also able to form complexes with G␤ 1 ␥ 2 in cultured cells, suggesting that the C-terminal domain of class II HDACs is a general G␤␥ binding motif. Activation of a G i/o -coupled receptor results in a time-dependent activation of MEF2C, an HDAC5-regulated transcription factor, whereas inhibition of the interaction with a G␤␥ scavenger inhibits MEF2C activity, suggesting a reduced potency of HDAC5-mediated inhibition. Taken together, these data imply that HDAC5 and possibly other class II HDACs can be added to the growing list of G␤␥ effectors.

show abstract

Section: Methodsmentioning

confidence: 99%

Gβγ Binds Histone Deacetylase 5 (HDAC5) and Inhibits Its Transcriptional Co-repression Activity

Spiegelberg

Hamm

2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Preparation of retGC and GCAPs-Bovine ROS were prepared from dark-adapted frozen retinas as described (49). Bleached ROS membranes from 20 retinas were suspended in 3 ml of Buffer A (10 mM HEPES (pH 7.5), 5 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 5 M leupeptin, 5 M pepstatin A, and 100 M CaCl 2 ), homogenized by passing through a No.…”

Section: Methodsmentioning

confidence: 99%

Activation of Retinal Guanylyl Cyclase-1 by Ca2+-binding Proteins Involves Its Dimerization

Yu¹,

Olshevskaya²,

Duda³

et al. 1999

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Retinal guanylyl cyclase-1 (retGC-1), a key enzyme in phototransduction, is activated by guanylyl cyclase-activating proteins (GCAPs) if [Ca 2؉ ] is less than 300 nM. The activation is believed to be essential for the recovery of photoreceptors to the dark state; however, the molecular mechanism of the activation is unknown. Here, we report that dimerization of retGC-1 is involved in its activation by GCAPs. The GC activity and the formation of a 210-kDa cross-linked product of retGC-1 were monitored in bovine rod outer segment homogenates, GCAPs-free bovine rod outer segment membranes and recombinant bovine retGC-1 expressed in COS-7 cells. In addition to recombinant bovine GCAPs, constitutively active mutants of GCAPs that activate retGC-1 in a [Ca 2؉ ]-independent manner and bovine brain S100b that activates retGC-1 in the presence of ϳ10 M [Ca 2؉ ] were used to investigate whether these activations take place through a similar mechanism, and whether [Ca 2؉ ] is directly involved in the dimerization. We found that a monomeric form of retGC-1 (ϳ110 kDa) was mainly observed whenever GC activity was at basal or low levels. However, the 210-kDa product was increased whenever the GC activity was stimulated by any Ca 2؉ -binding proteins used. We also found that [Ca 2؉ ] did not directly regulate the formation of the 210-kDa product. The 210-kDa product was detected in a purified GC preparation and did not contain GCAPs even when the formation of the 210-kDa product was stimulated by GCAPs. These data strongly suggest that the 210-kDa cross-linked product is a homodimer of retGC-1. We conclude that inactive retGC-1 is predominantly a monomeric form, and that dimerization of retGC-1 may be an essential step for its activation by active forms of GCAPs.

show abstract

“…After preparation of ROS from frozen retinas and extensive washing of ROS membranes, crude PDE preparations were extracted by further washing ROS membranes as described (17). The crude PDE preparation was applied to a TSK DEAE-5PW column that had been equilibrated with buffer A (10 mM Tris HC1, pH 7.5/1 mM dithiothreitol/5 mM MgSO4).…”

Section: Methodsmentioning

confidence: 99%

Phosphatidylinositol-stimulated phosphorylation of an inhibitory subunit of cGMP phosphodiesterase in vertebrate rod photoreceptors.

Hayashi

Lin

Matsumoto

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

An inhibitory subunit (Py) of cGMP phosphodiesterase from vertebrate rod photoreceptors (frog, toad, and bovine) was phosphorylated by cytosolic protein kinase(s) derived from intact frog rod outer segments. The phosphorylation offrog Py was stimulated by phosphatidylinositol but not by cAMP or cGMP. One-and two-dimensional gel electrophoresis revealed that 70-80% of Py was phosphorylated with 1 mol of phosphate per frog Py under optimal conditions. A peptide that derived from an active domain of bovine Py was also phosphorylated. Phosphorylation of frog Py was inhibited by addition of the peptide to the reaction mixture. Phosphorylation of frog Py was also inhibited by addition of transducin subunits or active (Py-less) cGMP phosphodiesterase. Okadaic acid, on the other hand, enhanced Py phosphorylation, suggesting the presence of protein phosphatase(s) in the cytosolic fraction. These data suggest another mechanism for the regulation of cGMP phosphodiesterase in vertebrate rod photoreceptors.

show abstract

[63] Purification of rod outer segment GTP-Binding protein subunits and cgmp phosphodiesterase by single-step column chromatography

Cited by 43 publications

References 5 publications

Gβγ Binds Histone Deacetylase 5 (HDAC5) and Inhibits Its Transcriptional Co-repression Activity

Gβγ Binds Histone Deacetylase 5 (HDAC5) and Inhibits Its Transcriptional Co-repression Activity

Activation of Retinal Guanylyl Cyclase-1 by Ca2+-binding Proteins Involves Its Dimerization

Phosphatidylinositol-stimulated phosphorylation of an inhibitory subunit of cGMP phosphodiesterase in vertebrate rod photoreceptors.

Contact Info

Product

Resources

About