Translocation of phosducin in living neuroblastoma × glioma hybrid cells (NG 108-15) monitored by red-shifted green fluorescent protein

Schulz, Rüdiger; Schulz, Karin; Wehmeyer, Andrea; Murphy, John J.

doi:10.1016/s0006-8993(98)00114-0

Cited by 16 publications

(11 citation statements)

References 42 publications

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“…2 and 3). Schulz et al (1998) describe a mechanism whereby a PDC-EGFP fusion protein concentrates at the plasma membrane within 2 to 3 min of prostaglandin receptor stimulation in NG108 neuroblastoma cells-a result similar to the time course we observed in SCG neurons. The lack of an immediate effect on Ca 2ϩ channel modulation provides evidence that heterologously expressed PDC and PDCL were not competing with G␣-GDP for G␤␥ and thereby uncoupling GPCRs from G-proteins before receptor stimulation.…”

Section: Discussionsupporting

confidence: 78%

Phosducin and Phosducin-like Protein Attenuate G-Protein-Coupled Receptor-Mediated Inhibition of Voltage-Gated Calcium Channels in Rat Sympathetic Neurons

Partridge¹,

Puhl²,

Ikeda³

2006

Mol Pharmacol

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Phosducin (PDC) has been shown in structural and biochemical experiments to bind the G␤␥ subunit of heterotrimeric G-proteins. A proposed function of PDC and phosducin-like protein (PDCL) is the sequestration of "free" G␤␥ from the plasma membrane, thereby terminating signaling by G␤␥. The functional impact of heterologously expressed PDC and PDCL on N-type calcium channel (Ca V 2.2) modulation was examined in sympathetic neurons, isolated from rat superior cervical ganglia, using whole-cell voltage clamp. Expression of PDC and PDCL attenuated voltage-dependent inhibition of N-type calcium channels, a G␤␥-dependent process, in a time-dependent fashion. Calcium current inhibition after short-term exposure to norepinephrine was minimally altered by PDC or PDCL expression. However, in the continued presence of norepinephrine, PDC or PDCL relieved calcium channel inhibition compared with control neurons. We observed similar results after activation of heterologously expressed metabotropic glutamate receptors with 100 M L-glutamate. Neurons expressing PDC or PDCL maintained suppression of inhibition after re-exposure to agonist. Unlike other G␤␥ sequestering proteins that abolish the short-term inhibition of Ca 2ϩ channels, PDC and PDCL require prolonged agonist exposure before effects on modulation are realized.G-protein-coupled receptors (GPCRs) are expressed throughout the nervous system and influence systems important for homeostasis. The canonical G-protein signaling pathways consists of a ligand binding to the receptor, exchange of GDP for GTP on the heterotrimeric G-protein ␣-subunit (G␣), separation of the G␤␥ dimer (G␤␥) from the G␣ subunit, and modulation of divergent downstream effector proteins by G␣-GTP and G␤␥. For example, free G␤␥ can associate with N-type voltage-gated Ca 2ϩ channels to reduce the probability of channel opening upon membrane depolarization. The strength and duration of this interaction will be influenced by competition with proteins possessing a high affinity for G␤␥, including G␣-GDP. Therefore, the duration of G-protein signaling is dependent upon the GTP hydrolysis rate because coalescence of the G␣␤␥ heterotrimer terminates signaling. Other proteins that bind G␤␥ with high affinity, such as the carboxyl terminus of GRK2 (ctGRK2) or phospholipase C-␤2, attenuate G␤␥ signaling (Ford et al., 1998). In this study, we examined how the expression of phosducins, a protein family known to interact with G␤␥, influenced GPCR-mediated Ntype Ca 2ϩ channel modulation in sympathetic neurons. The goals of the study were 1) to characterize the effects of PDC and PDCL expression within the context of protein-based optical sensor development and 2) to provide insight into possible physiological roles for PDC/PDCL modification of GPCR responses in neurons.Phosducin (PDC) is a 28-kDa soluble protein expressed primarily in the retina and pineal gland (Schulz, 2001;Sokolov et al., 2004). Retinal PDC has been hypothesized to adjust the gain of luminosity perception in the vertebrate eye (Thulin...

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Section: Discussionsupporting

confidence: 78%

Phosducin and Phosducin-like Protein Attenuate G-Protein-Coupled Receptor-Mediated Inhibition of Voltage-Gated Calcium Channels in Rat Sympathetic Neurons

Partridge¹,

Puhl²,

Ikeda³

2006

Mol Pharmacol

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“…The construction of the phosducin-EGFP fusion protein has been reported (Schulz et al, 1998b). Each fusion protein (DNA sequence) was sequenced to control in-frame cloning and correctness of inserts.…”

Section: Construction Of Expression Vectorsmentioning

confidence: 99%

Visualizing Preference of G Protein-Coupled Receptor Kinase 3 for the Process of κ-Opioid Receptor Sequestration

Schulz¹,

Wehmeyer²,

Schulz³

2002

Mol Pharmacol

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G protein-coupled receptor kinases (GRKs) phosphorylate opioid receptors, which eventually results in receptor sequestration. With respect to -opioid receptors, it is known that internalization occurs in a species-specific manner. That is, the agonist-occupied human -receptors will sequester whereas murine receptors fail to do so. This investigation concentrates on the internalization of -opioid receptors, employing laser scanning microscopy as a major technique to examine receptor internalization in living cells. For this reason, we fused green fluorescence protein to -receptors, and DsRed-fluorescent protein to GRK2 and GRK3. All fusion proteins retained their biologic activities. Permanent cell lines (HEK 293, were transfected to express either green fluorescent -receptors or to coexpress the tagged receptor and a specific GRKDsRed construct. The localization of fluorescent receptors and GRKs was monitored by confocal microscopy before and after opioid exposure of transfected cells. Activation of the murine -receptors triggers rapid translocation of tagged GRKs toward the cell membrane, but receptor internalization was not observed. The agonist-occupied human -receptor also causes translocation of GRK2-and GRK3-DsRed, which was followed by the formation of vesicles carrying the green fluorescent -receptors. Moreover, the green fluorescent vesicles consistently harbour red fluorescent GRK2 and GRK3, respectively. The phenomenon of -receptor internalization as well as cointernalization of GRKs is blocked by phosducin, indicating a critical role of G protein-␤␥ subunits for -receptor sequestration. Comparing the effect of over-expressed GRK2 and GRK3 on sequestration of -receptors, we conclude that GRK3 more strongly induces -receptor internalization than GRK2.

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“…Indeed, -opioid receptors are controlled by GRK2 (Zhang et al, 1998;Ferguson, 2001), GRK3 (Carman and Benovic, 1998;Hurle, 2001), and even by GRK6 (Hurle, 2001). ␦-Opioid receptors are phosphorylated by GRK2 (Schulz et al, 1998b;Harrison et al, 1998), GRK3 (Carman and Benovic, 1998), and GRK5 (Harrison et al, 1998). Additional information links the function of GRKs to chronic opioid effects, as an increased abundance and activity of distinct GRKs was communicated for the state of opioid dependence and tolerance (Terwillinger et al, 1994;Kovoor et al, 1998;Ozaita et al, 1998;Hurle, 2001).…”

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confidence: 99%

“…The general procedure for the construction of fusion proteins is described in a previous publication (Schulz et al, 1998b), using the CLONTECH vectors pEGFP, encoding the red-shifted variant of wt GFP (enhanced GFP, EGFP), and pDsRed. The cDNAs of individual opioid receptors and of GRKs were amplified by polymerase chain reaction, and after removal of the respective stop codon, they were inserted into the multiple cloning sites of pEGFP and pDsRed, respectively.…”

mentioning

confidence: 99%

“…For excitation, the 488 nm argon ion laser was used, and the emission was collected with a band pass 505-530 filter that allowed simultaneous monitoring of EGFP-tagged receptors as well as DsRed-tagged GRKs (excitation 543 nm, emission long pass filter 560 nm) with no bleed through between channels. The generation of phosducin-EGFP fusion protein has been described (Schulz et al, 1998b). Images from EGFP and DsRed fluorescence patterns were processed as one-color images or merged as two-color overlays.…”

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confidence: 99%

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Opioid Receptor Types Selectively Cointernalize with G Protein-Coupled Receptor Kinases 2 and 3

Schulz¹,

Wehmeyer²,

Schulz³

2002

J Pharmacol Exp Ther

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Activation of G protein-coupled receptors (GPCRs) may bring about their disappearance from the cell membrane, and it is commonly accepted that G protein-coupled receptor kinases (GRKs) play a key function in this mechanism. Opioid receptors belong to the family of GPCRs and are substrates of GRKs. We examined the fate of ␦-and -opioid receptors and GRK2 and 3 in living cells during the process of receptor sequestration, using laser scanning microscopy. For visualization purposes, receptors and kinases were tagged at their respective C terminus with a fluorophore. The opioid receptors were fused to enhanced green fluorescence protein (EGFP), and the GRKs were linked to red fluorescence protein (DsRed). The cDNAs of these constructs served for transfection of human embryonic kidney 293 cells and neuroblastoma-glioma hybrid cells (NG 108-15), respectively. We report that activation of ␦-opioid-EGFP receptors triggers a rapid translocation of cytosolic GRKDsRed toward the cell membrane, which in turn releases vesicles carrying both green fluorescent ␦-receptors and red fluorescent GRKs. Phosducin, a G␤␥ scavenger, completely prevents translocation of GRKs and the formation of vesicles. In analogous experiments with -opioid

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Translocation of phosducin in living neuroblastoma × glioma hybrid cells (NG 108-15) monitored by red-shifted green fluorescent protein

Cited by 16 publications

References 42 publications

Phosducin and Phosducin-like Protein Attenuate G-Protein-Coupled Receptor-Mediated Inhibition of Voltage-Gated Calcium Channels in Rat Sympathetic Neurons

Phosducin and Phosducin-like Protein Attenuate G-Protein-Coupled Receptor-Mediated Inhibition of Voltage-Gated Calcium Channels in Rat Sympathetic Neurons

Visualizing Preference of G Protein-Coupled Receptor Kinase 3 for the Process of κ-Opioid Receptor Sequestration

Opioid Receptor Types Selectively Cointernalize with G Protein-Coupled Receptor Kinases 2 and 3

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