A Molecular Mechanism for the Phosphorylation-Dependent Regulation of Heterotrimeric G Proteins by Phosducin

Gaudet, Rachelle; Savage, Justin R.; McLaughlin, Joseph N.; Willardson, Barry M.; Sigler, Paul B.

doi:10.1016/s1097-2765(00)80358-5

Cited by 83 publications

(78 citation statements)

References 31 publications

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“…These observations were consistent with the finding that Pdc formed a soluble complex with G t βγ by blocking its binding to rod disc membranes [14]. Third, phosphorylation of Pdc at the major PKA site (serine 73 (S73) in most mammals, serine 71 in the mouse) resulted in destabilization of helix 2 of Pdc, which uncovered residues of G t βγ involved in the G t α interaction that might allow G t α to displace Pdc from G t βγ [40]. This later finding provided a possible mechanism by which Pdc phosphorylation could regulate the interaction between G t βγ and G t α.…”

Section: Early Observations -The Gβγ Sequestration Hypothesissupporting

confidence: 84%

“…However, careful measurements of Pdc binding to G t βγ showed that PKA phosphorylation only decreased the binding by 3-fold [47,48]. In fact, the complex between PKA phosphorylated Pdc and G t βγ was crystallized and its structure determined as mentioned above [40]. These findings raised questions about whether Pdc and G t βγ actually dissociated upon PKA phosphorylation in the dark.…”

Section: Modifying the Model -New Clues About Pdc Functionmentioning

confidence: 99%

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Function of phosducin-like proteins in G protein signaling and chaperone-assisted protein folding

Willardson

Howlett

2007

Cellular Signalling

100

101

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Members of the phosducin gene family were initially proposed to act as down-regulators of G protein signaling by binding G protein βγ dimers (Gβγ) and inhibiting their ability to interact with G protein α subunits (Gα) and effectors. However, recent findings have overturned this hypothesis by showing that most members of the phosducin family act as cochaperones with the cytosolic chaperonin complex (CCT) to assist in the folding of a variety of proteins from their nascent polypeptides. In fact rather than inhibiting G protein pathways, phosducin-like protein 1 (PhLP1) has been shown to be essential for G protein signaling by catalyzing the folding and assembly of the Gβγ dimer. PhLP2 and PhLP3 have no role in G protein signaling, but they appear to assist in the folding of proteins essential in regulating cell cycle progression as well as actin and tubulin. Phosducin itself is the only family member that does not participate with CCT in protein folding, but it is believed to have a specific role in visual signal transduction to chaperone Gβγ subunits as they translocate to and from the outer and inner segments of photoreceptor cells during light-adaptation.

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Section: Early Observations -The Gβγ Sequestration Hypothesissupporting

confidence: 84%

Section: Modifying the Model -New Clues About Pdc Functionmentioning

confidence: 99%

Function of phosducin-like proteins in G protein signaling and chaperone-assisted protein folding

Willardson

Howlett

2007

Cellular Signalling

100

101

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“…Upon exposure to light, phosducin is dephosphorylated by PP2A (19, 21). The dephosphorylated species of phosducin, no longer binding 14-3-3 protein, is more efficient in sequestering the γ subunits of transducin (65). Therefore, dephosphorylation of phosducin by PP2A establishes the conditions for phosducin to modulate the amplification of cGMP hydrolysis by preventing the recycling of transducin with the activated rhodopsin.…”

Section: Discussionmentioning

confidence: 99%

Light-Driven Translocation of the Protein Phosphatase 2A Complex Regulates Light/Dark Dephosphorylation of Phosducin and Rhodopsin

et al. 2002

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In steps of protein purification of bovine retinal protein phosphatase 2A (PP2A), phosducin dephosphorylation activity peaks coelute with a PP2A enzyme complex, shown by peptide sequence analysis to contain a B′ subunit, B56epsilon. Other PP2A complexes with a slightly larger (56.5 kDa) B′ subunit (sequenced to be B56alpha) or with the BR regulatory subunit have no phosducin dephosphorylation activity. Upon exposure to light, a significant increase in the immunoreactive protein level of the A, C, and B56epsilon PP2A subunits is observed in the cytosolic fraction of mouse retina, the phosducin dephosphorylation of which occurs rapidly. During dark exposure, these subunits translocate to the membrane fraction where rhodopsin is slowly dephosphorylated. This PP2A redistribution occurs in less than 1.5 min and is dependent upon light and not upon an intrinsic circadian rhythm. Forty times more of the A subunit (∼20 ng/mouse retina) and 9 times more of the C subunit (∼4 ng/mouse retina) than of the B56epsilon subunit (∼0.45 ng/mouse retina) redistribute, which suggests that the predominant form of the PP2A enzyme complex on the membrane in the dark is a dimer, consisting of only A and C subunits. We observe that the dimer favors phosphorylated opsin as a substrate, while the trimer, particularly the enzyme complex with the B56epsilon subunit, greatly prefers phosphorylated phosducin, with an activity several hundred times those of other substrates that were tested. This light-driven PP2A translocation provides a potential mechanism for efficient dephosphorylation of two critical photoreceptor transduction proteins, cytosolic phosducin and membrane-bound rhodopsin, by the same enzyme.

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“…These effects have been studied extensively in vitro (16,18,20,21,34), but have not been examined in vivo. To address this issue, the binding of Pdc to G t ␤␥ was measured in light-or dark-adapted retina and was compared with the phosphorylation of Ser-54 and Ser-73.…”

Section: Stoichiometry Of Ser-54 and Ser-73 Phosphorylation-ifmentioning

confidence: 99%

Site-specific Phosphorylation of Phosducin in Intact Retina

Lee

Thulin

Willardson

2004

Journal of Biological Chemistry

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Phosducin (Pdc) is a G protein ␤␥ dimer (G␤␥) binding protein, highly expressed in retinal photoreceptor and pineal cells, yet whose physiological role remains elusive. Light controls the phosphorylation of Pdc in a cAMP and Ca 2؉ -dependent manner, and phosphorylation in turn regulates the binding of Pdc to G t ␤␥ or 14-3-3 proteins in vitro. To directly examine the phosphorylation of Pdc in intact retina, we prepared antibodies specific to the three principal phosphorylation sites (Ser-54, Ser-73, and Ser-106) and measured the kinetics of phosphorylation/dephosphorylation during light/dark adaptation and the subsequent effects on G t ␤␥ binding. Ser-54 phosphorylation increased slowly (t1 ⁄2 ϳ 90 min) during dark adaptation to ϳ70% phosphorylated and decreased rapidly (t1 ⁄2 ϳ 2 min) during light adaptation to less than 20% phosphorylated. Ser-73 phosphorylation increased much faster during dark adaptation (t1 ⁄2 ϳ 3 min) to ϳ50% phosphorylated and decreased more slowly during light adaptation (t1 ⁄2 ϳ 9 min) to less than 20% phosphorylated. The Ca 2؉ chelator BAPTA-AM blocked Ser-54 phosphorylation during dark adaptation but had no effect on Ser-73 phosphorylation. In contrast, Ser-106 was not phosphorylated in either the light or dark. Importantly, G␤␥ binding to Pdc was enhanced by Ca 2؉ chelation and the binding kinetics closely paralleled those of Ser-54 dephosphorylation, indicating that Ser-54 phosphorylation controls G t ␤␥ binding in vivo. These results suggest a pivotal role of Ser-54 and Ser-73 phosphorylation in determining the interactions of Pdc with its binding partners, G t ␤␥ and 14-3-3 protein, which may regulate the light-dependent translocation of the photoreceptor G protein.G protein-coupled receptors mediate cellular responses to a wide variety of signals including hormones, neurotransmitters, odorants, chemoattractants, and photons of light. The conversion of light into a neural response by the photoreceptor cells of the vertebrate retina is initiated by a canonical G protein signaling pathway involving the receptor rhodopsin, the photoreceptor G protein, transducin (G t ), 1 cGMP phosphodiesterase, and cGMP-gated cation channels (1). Closure of the channels in response to light causes a hyperpolarization of the photoreceptor cell membrane, which triggers the neural response. In addition, channel closure also results in a decrease in Ca 2ϩ in the photoreceptor cell, which orchestrates a number of molecular events that lead to a decrease in sensitivity of the photoreceptor to light, a process termed light adaptation (2).The role of the G t ␣ subunit in activation of the phosphodiesterase has been extensively studied (3-5), as has regulation of its GTPase activity by RGS-9 (6 -8). In contrast, the only known function of the G t ␤␥ subunit complex is to mediate the interaction of G t ␣ with rhodopsin, yet in other G protein signaling systems G␤␥ plays significant roles in downstream effector activation (9). Besides G t ␣, the only other reported binding partner for G t ␤␥ in photoreceptor c...

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A Molecular Mechanism for the Phosphorylation-Dependent Regulation of Heterotrimeric G Proteins by Phosducin

Cited by 83 publications

References 31 publications

Function of phosducin-like proteins in G protein signaling and chaperone-assisted protein folding

Function of phosducin-like proteins in G protein signaling and chaperone-assisted protein folding

Light-Driven Translocation of the Protein Phosphatase 2A Complex Regulates Light/Dark Dephosphorylation of Phosducin and Rhodopsin

Site-specific Phosphorylation of Phosducin in Intact Retina

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