Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Several studies using selective opioid agonists or mice with a deletion of the mu-opioid receptor, have shown that morphine dependence is essentially due to chronic stimulation of mu- but not delta-opioid receptors. Because dependence is assumed to be related to persistent intracellular modifications, we have investigated modifications putatively induced by chronic activation of mu receptors with morphine or selective agonists in vitro in SH-SY5Y cells and in vivo in different strains of mice, including mice lacking the mu-opioid receptor gene. The results show a similar down-regulation and desensitization of mu and delta binding sites, whereas an overexpression of dynamin occurred only with mu agonists, strongly suggesting the relevance of this up-regulation with the opiate dependence. Moreover, translocation of overexpressed dynamin from intracellular pools to plasma membranes was observed in chronic morphine-treated rats. This recruitment could be critically involved in long-lasting changes such as alterations of axonal transport observed in opioid dependence.
Mol Pharmacol 2000 Jul
PMID:Overexpression of dynamin is induced by chronic stimulation of mu- but not delta-opioid receptors: relationships with mu-related morphine dependence. 1086 Sep 38

Previously, we showed that the human kappa-opioid receptor (hkor) stably expressed in Chinese hamster ovary (CHO) cells underwent down-regulation after prolonged U50,488H treatment. In the present study, we determined the mechanisms underlying this process. U50, 488H caused a significant down-regulation of the hkor, although etorphine did not. Neither U50,488H nor etorphine caused down-regulation of the rat kappa-opioid receptor. Thus, similar to internalization, there are agonist and species differences in down-regulation of kappa-opioid receptors. Expression of the dominant negative mutants arrestin-2(319-418) or dynamin I-K44A significantly reduced U50,488H-induced down-regulation of the hkor. Coexpression of GRK2 or GRK2 and arrestin-2 permitted etorphine to induce down-regulation of the hkor, although expression of arrestin-2 or dynamin I alone did not. Expression of the dominant negative mutants rab5A-N133I or rab7-N125I blunted U50,488H-induced down-regulation. Pretreatment with lysosomal enzyme inhibitors [(2S, 3S)trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester or chloroquine] or proteasome inhibitors (proteasome inhibitor I, MG-132, or lactacystin) decreased the extent of U50,488H-induced down-regulation. A combination of chloroquine and proteasome inhibitor I abolished U50,488H-induced down-regulation. These results indicate that U50,488H-induced down-regulation of the hkor involves GRK-, arrestin-2-, dynamin-, rab5-, and rab7-dependent mechanisms and receptors seem to be trafficked to lysosomes and proteasomes for degradation. Thus, U50,488H-induced internalization and down-regulation of the hkor share initial common mechanisms. To the best of our knowledge, these results represent the first report on the involvement of both rab5 and rab7 in agonist-induced down-regulation of a G protein-coupled receptor. In addition, this study is among the first to show the involvement of proteasomes in agonist-induced down-regulation of a G protein-coupled receptor.
Mol Pharmacol 2000 Oct
PMID:Mechanisms of agonist-induced down-regulation of the human kappa-opioid receptor: internalization is required for down-regulation. 1099 50

To elucidate the physiological significance of the translocation of Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II), we investigated substrates of CaM kinase II in the postsynaptic density (PSD). PSD proteins were phosphorylated by CaM kinase II of its PSD complex, and separated by two-dimensional gel electrophoresis. More than 28 proteins were phosphorylated under experimental conditions. Proteins corresponding to CaM kinase II substrates were excised from the gels, eluted electrophoretically, and then sequenced. Several substrates were identified, including PSD95, SAP90, alpha-internexin, neurofilament L chain, cAMP phosphodiesterase, and alpha- and beta-tubulin. Some substrates were also identified by immunoblotting, including N-methyl-D-aspartic acid (NMDA) receptor 2B subunit, 1-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor 1 (GluR1), neurofilament H chain and dynamin. PSD95, SAP90, dynamin, and alpha-internexin were demonstrated for the first time to be substrates of CaM kinase II. NMDA receptor 2B subunit and GluR1 existed as major substrates in the PSD. Moreover, translocation of CaM kinase II was inhibited by phosphorylation of PSD proteins. These results suggest that CaM kinase II plays important roles in the regulation of synaptic functions through phosphorylation of PSD proteins.
Brain Res Mol Brain Res 2000 Sep 30
PMID:Investigation of protein substrates of Ca(2+)/calmodulin-dependent protein kinase II translocated to the postsynaptic density. 1100 Apr 84

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a prominent substrate for activated tyrosine kinase receptors that has been proposed to play a role in endosomal membrane trafficking. The protein contains a FYVE domain, which specifically binds to the lipid phosphatidylinositol (PI) 3-phosphate (PI 3-P). We show that this interaction is required both for correct localization of the protein to endosomes that only partially coincides with early endosomal autoantigen 1 and for efficient tyrosine phosphorylation of the protein in response to epidermal growth factor stimulation. Treatment with wortmannin reveals that Hrs phosphorylation also requires PI 3-kinase activity, which is necessary to generate the PI 3-P required for localization. We have used both hypertonic media and expression of a dominant-negative form of dynamin (K44A) to inhibit endocytosis; under which conditions, receptor stimulation fails to elicit phosphorylation of Hrs. Our results provide a clear example of the coupling of a signal transduction pathway to endocytosis, from which we propose that activated receptor (or associated factor) must be delivered to the appropriate endocytic compartment in order for Hrs phosphorylation to occur.
Mol Cell Biol 2000 Oct
PMID:Endosomal localization and receptor dynamics determine tyrosine phosphorylation of hepatocyte growth factor-regulated tyrosine kinase substrate. 1100 64

Beta-arrestins target G protein-coupled receptors (GPCRs) for endocytosis via clathrin-coated vesicles. Beta-arrestins also become detectable on endocytic vesicles in response to angiotensin II type 1A receptor (AT1AR), but not beta2-adrenergic receptor (beta2AR), activation. The carboxyl-terminal tails of these receptors contribute directly to this phenotype, since a beta2AR bearing the AT1AR tail acquired the capacity to stimulate beta-arrestin redistribution to endosomes, whereas this property was lost for an AT1AR bearing the beta2AR tail. Using beta2AR/AT1AR chimeras, we tested whether the beta2AR and AT1AR carboxyl-terminal tails, in part via their association with beta-arrestins, might regulate differences in the intracellular trafficking and resensitization patterns of these receptors. In the present study, we find that beta-arrestin formed a stable complex with the AT1AR tail in endocytic vesicles and that the internalization of this complex was dynamin dependent. Internalization of the beta2AR chimera bearing the AT1AR tail was observed in the absence of agonist and was inhibited by a dominant-negative beta-arrestin1 mutant. Agonist-independent AT1AR internalization was also observed after beta-arrestin2 overexpression. After internalization, the beta2AR, but not the AT1AR, was dephosphorylated and recycled back to the cell surface. However, the AT1AR tail prevented beta2AR dephosphorylation and recycling. In contrast, although the beta2AR-tail promoted AT1AR recycling, the chimeric receptor remained both phosphorylated and desensitized, suggesting that receptor dephosphorylation is not a property common to all receptors. In summary, we show that the carboxyl-terminal tails of GPCRs not only contribute to regulating the patterns of receptor desensitization, but also modulate receptor intracellular trafficking and resensitization patterns.
Mol Endocrinol 2000 Dec
PMID:Receptor/beta-arrestin complex formation and the differential trafficking and resensitization of beta2-adrenergic and angiotensin II type 1A receptors. 1111 33

The major mechanism of agonist-induced internalization of G protein-coupled receptors (GPCRs) is beta-arrestin- and dynamin-dependent endocytosis via clathrin-coated vesicles. However, recent reports have suggested that some GPCRs, exemplified by the AT1 angiotensin receptor expressed in human embryonic kidney (HEK) 293 cells, are internalized by a beta-arrestin- and dynamin-independent mechanism, and possibly via a clathrin-independent pathway. In this study, agonist-induced endocytosis of the rat AT1A receptor expressed in Chinese hamster ovary (CHO) cells was abolished by clathrin depletion during treatment with hyperosmotic sucrose and was unaffected by inhibition of endocytosis via caveolae with filipin. In addition, internalized fluorescein-conjugated angiotensin II appeared in endosomes, as demonstrated by colocalization with transferrin. Overexpression of beta-arrestin1(V53D) and beta-arrestin1(1-349) exerted dominant negative inhibitory effects on the endocytosis of radioiodinated angiotensin II in CHO cells. GTPase-deficient (K44A) mutant forms of dynamin-1 and dynamin-2, and a pleckstrin homology domain-mutant (K535A) dynamin-2 with impaired phosphoinositide binding, also inhibited the endocytosis of AT(1) receptors in CHO cells. Similar results were obtained in COS-7 and HEK 293 cells. Confocal microscopy using fluorescein-conjugated angiotensin II showed that overexpression of dynamin-1(K44A) and dynamin-2(K44A) isoforms likewise inhibited agonist-induced AT1 receptor endocytosis in CHO cells. Studies on the angiotensin II concentration-dependence of AT1 receptor endocytosis showed that at higher agonist concentrations its rate constant was reduced and the inhibitory effects of dominant negative dynamin constructs were abolished. These data demonstrate the importance of beta-arrestin- and dynamin-dependent endocytosis of the AT1 receptor via clathrin-coated vesicles at physiological angiotensin II concentrations.
Mol Pharmacol 2001 Feb
PMID:Beta-arrestin- and dynamin-dependent endocytosis of the AT1 angiotensin receptor. 1116 Aug 59

Mitochondria are dynamic organelles that undergo frequent division and fusion, but the molecular mechanisms of these two events are not well understood. Dnm1p, a mitochondria-associated, dynamin-related GTPase was previously shown to mediate mitochondrial fission. Recently, a genome-wide yeast two-hybrid screen identified an uncharacterized protein that interacts with Dnm1p. Cells disrupted in this new gene, which we call NET2, contain a single mitochondrion that consists of a network formed by interconnected tubules, similar to the phenotype of dnm1 Delta cells. NET2 encodes a mitochondria-associated protein with a predicted coiled-coil region and six WD-40 repeats. Immunofluorescence microscopy indicates that Net2p is located in distinct, dot-like structures along the mitochondrial surface, many of which colocalize with the Dnm1 protein. Fluorescence and immunoelectron microscopy shows that Dnm1p and Net2p preferentially colocalize at constriction sites along mitochondrial tubules. Our results suggest that Net2p is a new component of the mitochondrial division machinery.
Mol Biol Cell 2001 02
PMID:Division of mitochondria requires a novel DNM1-interacting protein, Net2p. 1117 17

The establishment of cadherin-dependent cell-cell contacts in human epidermal keratinocytes are known to be regulated by the Rac1 small GTP-binding protein, although the mechanisms by which Rac1 participates in the assembly or disruption of cell-cell adhesion are not well understood. In this study we utilized green fluorescent protein (GFP)-tagged Rac1 expression vectors to examine the subcellular distribution of Rac1 and its effects on E-cadherin-mediated cell-cell adhesion. Microinjection of keratinocytes with constitutively active Rac1 resulted in cell spreading and disruption of cell-cell contacts. The ability of Rac1 to disrupt cell-cell adhesion was dependent on colony size, with large established colonies being resistant to the effects of active Rac1. Disruption of cell-cell contacts in small preconfluent colonies was achieved through the selective recruitment of E-cadherin-catenin complexes to the perimeter of multiple large intracellular vesicles, which were bounded by GFP-tagged L61Rac1. Similar vesicles were observed in noninjected keratinocytes when cell-cell adhesion was disrupted by removal of extracellular calcium or with the use of an E-cadherin blocking antibody. Moreover, formation of these structures in noninjected keratinocytes was dependent on endogenous Rac1 activity. Expression of GFP-tagged effector mutants of Rac1 in keratinocytes demonstrated that reorganization of the actin cytoskeleton was important for vesicle formation. Characterization of these Rac1-induced vesicles revealed that they were endosomal in nature and tightly colocalized with the transferrin receptor, a marker for recycling endosomes. Expression of GFP-L61Rac1 inhibited uptake of transferrin-biotin, suggesting that the endocytosis of E-cadherin was a clathrin-independent mechanism. This was supported by the observation that caveolin, but not clathrin, localized around these structures. Furthermore, an inhibitory form of dynamin, known to inhibit internalization of caveolae, inhibited formation of cadherin vesicles. Our data suggest that Rac1 regulates adherens junctions via clathrin independent endocytosis of E-cadherin.
Mol Biol Cell 2001 Apr
PMID:RAC1 regulates adherens junctions through endocytosis of E-cadherin. 1129 91

We have demonstrated that the plasmalemmal vesicles (caveolae) of the continuous microvascular endothelium function as transcytotic vesicular carriers for protein molecules > 20 A and that transcytosis is an N-ethylmaleimide-sensitive factor (NSF)-dependent, N-ethylmaleimide-sensitive process. We have further investigated NSF interactions with endothelial proteins to find out 1) whether a complete set of fusion and targeting proteins is present in the endothelium; 2) whether they are organized in multimolecular complexes as in neurons; and 3) whether the endothelial multimolecular complexes differ from their neuronal counterparts, because of their specialized role in transcytosis. To generate the complexes, we have used myc-NSF, cultured pulmonary endothelial cells, and rat lung cytosol and membrane preparations; to detect them we have applied coimmunoprecipitation with myc antibodies; and to characterize them we have used velocity sedimentation and cross-linking procedures. We have found that both cytosolic and membrane fractions contain complexes that comprise beside soluble NSF attachment proteins and SNAREs (soluble NSF attachment protein receptor), rab 5, dynamin, caveolin, and lipids. By immunogold labeling and negative staining we have detected in these complexes, myc-NSF, syntaxin, dynamin, caveolin, and endogenous NSF. Similar complexes are formed by endogenous NSF. The results indicate that complexes with a distinct protein-lipid composition exist and suggest that they participate in targeting, fusion, and fission of caveolae with the endothelial plasmalemma.
Mol Biol Cell 2001 Apr
PMID:Endothelial transcytotic machinery involves supramolecular protein-lipid complexes. 1129 4

The internalization of the M(2) muscarinic cholinergic receptor (mAChR) proceeds through an atypical pathway that is independent of arrestin and clathrin function and shows a unique sensitivity to dynamin when the receptor is expressed in human embryonic kidney 293 cells. In this report we demonstrate that the internalization of the M(2) mAChR was modulated by activation of heterotrimeric G proteins, because treatment with pertussis toxin, which ADP-ribosylates G proteins of the G(i/o) family, caused a significant delay in the onset of internalization of the M(2) mAChR. The effects of pertussis toxin could not be explained by alteration of the agonist-dependent phosphorylation of the M(2) mAChR. The modulation of internalization by pertussis toxin was revealed to be due to recruitment of intracellular receptors to the cell surface upon agonist treatment. Pretreatment with pertussis toxin also greatly increased both the rate and extent of recovery of M(2) mAChRs to the cell surface after agonist-mediated internalization. These results demonstrate a novel aspect involved in the regulation of GPCRs. As with the tightly controlled internalization of GPCRs, the delivery of GPCRs to the cell surface is also highly regulated.
Mol Pharmacol 2001 May
PMID:Agonist-dependent delivery of M(2) muscarinic acetylcholine receptors to the cell surface after pertussis toxin treatment. 1130 11


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