Phosducin expression in NG 108-15 hybrid cells enhances prostaglandin E1 stimulated adenylate cyclase activity

Wehmeyer, Andrea; Schulz, Rüdiger

doi:10.1016/s0024-3205(97)01190-9

Cited by 8 publications

(5 citation statements)

References 18 publications

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“…Since Phd is predominantly expressed in the cytoplasm of rod photoreceptors and pinealocytes, we sought to determine the subcellular localization of Phd and PhLOP1 in either control COS-7 cells, immortalized rat neuroretinal cells (R28) or NGF-primed neuroblastoma PC12 cells. EGFP fusion of Phd has been shown to have the normal cytoplasmic localization and function in transiently transfected neuronal NG 108-15 hybrid cells (43,44). Our results confirmed that Phd is localized to the cytoplasm; however, PhLOP1 is FIG.…”

Section: Discussionsupporting

confidence: 78%

The Carboxyl Terminal Domain of Phosducin Functions as a Transcriptional Activator

Zhu

Craft

2000

Biochemical and Biophysical Research Communications

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In previous work, we identified a set of phosducin (Phd) isoforms with unknown function including the phosducin (Phd)-like orphan protein 1 (PhLOP1), an amino terminal truncated isoform of the retinal Phd lacking the Gbetagamma binding domain. To investigate the potential biological function of PhLOP1, PhLOP1 was fused at its amino terminus with the DNA binding domain (BD) of the yeast transcriptional factor, GAL4, and used as bait in a yeast two-hybrid screen. Two potential functional protein partners were identified during the screen: SUG1, a subunit of the 26S proteasome and a putative transcriptional mediator, and CRX, a retina- and pineal-specific transcription factor. Upon localizing the interacting domain of PhLOP1 with one of the new partners, SUG1, we found that a domain of 40 amino acids at the carboxyl terminus of Phd and PhLOP1 had intrinsic transcriptional activation activity in yeast. The transactivation activity was further confirmed in mammalian cells. This region contains an acidic domain that has been shown to be involved in the function of several transcriptional activators. In addition, we showed that Phd is cytoplasmic while PhLOP1 is localized predominantly to the nucleus when fused to an enhanced green fluorescent protein (EGFP) and transiently expressed in transfected cells, suggesting that PhLOP1 may play a distinct functional role in transcriptional regulation independent of the known Phd interaction/regulation of Gbetagamma transduction.

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Section: Discussionsupporting

confidence: 78%

The Carboxyl Terminal Domain of Phosducin Functions as a Transcriptional Activator

Zhu

Craft

2000

Biochemical and Biophysical Research Communications

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“…Searching for factors that modulate R-G coupling is an area of intense current interest, and several proteins that regulate coupling have been identified. These include arrestin [22], phosducin [23], recoverin [24] and regulators of G-protein signalling (' RGSs ') [25]. In the case of the A " AR, a putative ' coupling cofactor ' partially purified from cortical brain membranes ($ 150 kDa) was reported to stabilize A " AR-G-protein complexes and to trap the A " AR in a high-affinity agonistbinding state [26].…”

Section: Discussionmentioning

confidence: 99%

Purification of A1 adenosine receptor‒G-protein complexes: effects of receptor down-regulation and phosphorylation on coupling

Gao

Linden

1999

Biochem. J.

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We examined the effects of exposing A1 adenosine receptors (A1ARs) to an agonist on the stability and phosphorylation state of receptor-guanine nucleotide-binding regulatory protein (R-G-protein) complexes. Non-denatured recombinant human A1ARs extended on the N-terminus with hexahistidine (His6) and the FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) epitope (H/F) were purified to near homogeneity from stably transfected Chinese-hamster ovary (CHO)-K1 cells. Purified receptors have pharmacological properties similar to receptors in membranes. G-proteins were co-purified with 15+/-2% of H/F-A1AR unless receptor-G-protein (R-G) complexes were uncoupled by pre-treating cell membranes with GTP. By silver staining, purified A1AR-G-protein complexes contain receptors, G-protein alpha and beta subunits and an unidentified 97 kDa protein. Pretreating intact cells with N6-cyclopentyladenosine (CPA) for 24 h decreased both the total number of receptors measured in membranes and the number of purified A1ARs by about 50%. In contrast, pretreating cells with CPA decreased the number of R-G complexes measured in membranes (54+/-6%) significantly less than it decreased the number of purified R-G complexes (78+/-3%) as detected by 125I-N6-(4-aminobenzyl)adenosine binding or by Western blotting Gialpha2. The effect of CPA to decrease the fraction of receptors purified as R-G complexes was not associated with any change in low-level A1AR phosphorylation (found on serine), or low-level phosphorylation of G-protein alpha or beta subunits or the 97 kDa protein. These experiments reveal a novel aspect of agonist-induced down-regulation, namely a diminished stability of receptor-G-protein complexes that is manifested as uncoupling during receptor purification.

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“…These include arrestin [22], phosducin [23], recoverin [24] and regulators of G-protein signalling (' RGSs ') [25]. In the case of the A " AR, a putative ' coupling cofactor ' partially purified from cortical brain membranes ($ 150 kDa) was reported to stabilize A " AR-G-protein complexes and to trap the A " AR in a high-affinity agonistbinding state [26].…”

Section: Discussionmentioning

confidence: 99%

Purification of A1 adenosine receptor–G-protein complexes: effects of receptor down-regulation and phosphorylation on coupling

Gao

Robeva

Linden

1999

Biochemical Journal

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We examined the effects of exposing A1 adenosine receptors (A1ARs) to an agonist on the stability and phosphorylation state of receptor–guanine nucleotide-binding regulatory protein (R–G-protein) complexes. Non-denatured recombinant human A1ARs extended on the N-terminus with hexahistidine (His6) and the FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) epitope (H/F) were purified to near homogeneity from stably transfected Chinese-hamster ovary (CHO)-K1 cells. Purified receptors have pharmacological properties similar to receptors in membranes. G-proteins were co-purified with 15±2% of H/F-A1AR unless receptor–G-protein (R–G) complexes were uncoupled by pre-treating cell membranes with GTP. By silver staining, purified A1AR–G-protein complexes contain receptors, G-protein α and β subunits and an unidentified 97 kDa protein. Pretreating intact cells with N6-cyclopentyladenosine (CPA) for 24 h decreased both the total number of receptors measured in membranes and the number of purified A1ARs by about 50%. In contrast, pretreating cells with CPA decreased the number of R–G complexes measured in membranes (54±6%) significantly less than it decreased the number of purified R–G complexes (78±3%) as detected by 125I-N6-(4-aminobenzyl)adenosine binding or by Western blotting Giα2. The effect of CPA to decrease the fraction of receptors purified as R–G complexes was not associated with any change in low-level A1AR phosphorylation (found on serine), or low-level phosphorylation of G-protein α or β subunits or the 97 kDa protein. These experiments reveal a novel aspect of agonist-induced down-regulation, namely a diminished stability of receptor–G-protein complexes that is manifested as uncoupling during receptor purification.

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Phosducin expression in NG 108-15 hybrid cells enhances prostaglandin E1 stimulated adenylate cyclase activity

Cited by 8 publications

References 18 publications

The Carboxyl Terminal Domain of Phosducin Functions as a Transcriptional Activator

The Carboxyl Terminal Domain of Phosducin Functions as a Transcriptional Activator

Purification of A1 adenosine receptor‒G-protein complexes: effects of receptor down-regulation and phosphorylation on coupling

Purification of A1 adenosine receptor–G-protein complexes: effects of receptor down-regulation and phosphorylation on coupling

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