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

Schulz, Rüdiger; Wehmeyer, Andrea; Schulz, Karin

doi:10.1124/jpet.300.2.376

Cited by 41 publications

(25 citation statements)

References 31 publications

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“…All these cells carry -receptors but transient cotransfection with GRK-DsRed is achieved only for an estimated 20 to 30% cells per batch with variable expression levels per cell. We confirmed findings (Li et al, 1999), that the endogenous GRK2 level of HEK cells and even over expressed GRK2 proved poorly effective to internalize -receptors, although internalization of ␦-and -receptors is strong in these cells (Schulz et al, 2002). Elevating the kinase activity by over-expression of GRK2-DsRed enhances -receptor internalization but the effect was of a moderate degree.…”

Section: Discussionsupporting

confidence: 78%

“…There is sufficient reason to assume that GRK3-DsRed accounts for the observed internalization of -opioid receptors. Moreover, DsRed is known to exist both in the cytosol and the nucleus (Schulz et al, 2002), but confocal images of cells expressing GRK3-DsRed revealed the absence of red fluorescence in the nucleus. Thus, the findings reported here strongly support the view that DsRed and DsRed constructs exist as monomers in HEK cells.…”

Section: Discussionmentioning

confidence: 99%

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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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Section: Discussionsupporting

confidence: 78%

Section: Discussionmentioning

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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“…Data shown are representative of three independent experiments. desensitization, and internalization of MORs by full agonists (Zhang et al, 1998;Wang, 2000;Celver et al, 2001Celver et al, , 2004Li and Wang, 2001;Schulz et al, 2002). HEK293 cells also express GRK6 endogenously (Iwata et al, 2005), but a dominant negative mutant of GRK6 was unable to modify DAMGO-induced MOR1 desensitization, indicating the specificity of action of GRK2-K220R.…”

Section: Discussionmentioning

confidence: 98%

Agonist-Selective Mechanisms of μ-Opioid Receptor Desensitization in Human Embryonic Kidney 293 Cells

Johnson

Oldfield²,

Braksator³

et al. 2006

Mol Pharmacol

144

179

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The ability of two opioid agonists, [D-Ala 2 ,N-Me-Phe 4 ,Gly 5 -ol]-enkephalin (DAMGO) and morphine, to induce -opioid receptor (MOR) phosphorylation, desensitization, and internalization was examined in human embryonic kidney (HEK) 293 cells expressing rat MOR1 as well G protein-coupled inwardly rectifying potassium channel (GIRK) channel subunits. Both DAMGO and morphine activated GIRK currents, but the maximum response to DAMGO was greater than that of morphine, indicating that morphine is a partial agonist. The responses to DAMGO and morphine desensitized rapidly in the presence of either drug. Expression of a dominant negative mutant G protein-coupled receptor kinase 2 (GRK2), GRK2-K220R, markedly attenuated the DAMGO-induced desensitization of MOR1, but it had no effect on morphine-induced MOR1 desensitization. In contrast, inhibition of protein kinase C (PKC) either by the PKC inhibitory peptide PKC (19-31) or staurosporine reduced MOR1 desensitization by morphine but not that induced by DAMGO. Morphine and DAMGO enhanced MOR1 phosphorylation over basal. The PKC inhibitor bisindolylmaleimide 1 (GF109203X) inhibited MOR1 phosphorylation under basal conditions and in the presence of morphine, but it did not inhibit DAMGO-induced phosphorylation. DAMGO induced arrestin-2 translocation to the plasma membrane and considerable MOR1 internalization, whereas morphine did not induce arrestin-2 translocation and induced very little MOR1 internalization. Thus, DAMGO and morphine each induce desensitization of MOR1 signaling in HEK293 cells but by different molecular mechanisms; DAMGO-induced desensitization is GRK2-dependent, whereas morphine-induced desensitization is in part PKC-dependent. MORs desensitized by DAMGO activation are then readily internalized by an arrestin-dependent mechanism, whereas those desensitized by morphine are not. These data suggest that opioid agonists induce different conformations of the MOR that are susceptible to different desensitizing and internalization processes.

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“…This agonist-dependent desensitization is referred to as homologous desensitization. It involves a phosphorylation of the receptor through various GPCR kinases (GRK) Schulz et al, 2002), subsequent uncoupling from the G-protein (Whistler and von Zastrow, 1998;Liu et al, 1999), binding to ␤-arrestin 2 Law et al, 2000;McDonald et al, 2000;Pierce and Lefkowitz, 2001;Xiang et al, 2001) and sequestration into clathrin-coated vesicles with help of the GTPase dynamin (Chu et al, 1997;Li et al, 1999). In addition to agonist-specific desensitization, functions of GPCRs are regulated by agonist-independent mechanisms, namely heterologous desensitization.…”

Section: Is Cell Death Initiated By Receptor Desensitizationmentioning

confidence: 99%

Opioids As Modulators of Cell Death and Survival—Unraveling Mechanisms and Revealing New Indications

2004

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

Cited by 41 publications

References 31 publications

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

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

Agonist-Selective Mechanisms of μ-Opioid Receptor Desensitization in Human Embryonic Kidney 293 Cells

Opioids As Modulators of Cell Death and Survival—Unraveling Mechanisms and Revealing New Indications

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