We examined the agonist-dependent sequestration/internalization of dopamine D2 receptor (the long form D2L and short form D2S), which were transiently expressed in COS-7 and HEK 293 cells with or without G-protein-coupled receptor kinases (GRK2 or GRK5). Sequestration was assessed quantitatively by loss of [ 3 H] sulpiride-binding activity from the cell surface and by transfer of [ 3 H] spiperone-binding activity from the membrane fraction to the light vesicle fraction in sucrose-density gradients. In COS-7 cells expressing D2 receptors alone, virtually no sequestration was observed with or without dopamine (, 4%). When GRK2 was coexpressed, 50% of D2S receptors and 36% of D2L receptors were sequestered by treatment with 10 ±4 m dopamine for 2 h, whereas no sequestration was observed in cells expressing the dominant negative form of GRK2 (DN-GRK2). When GRK5 was coexpressed, 36% of D2S receptors were sequestered following the same treatment. The agonist-dependent and GRK2-dependent sequestration of D2S receptors was reduced markedly in the presence of hypertonic medium containing 0.45 m sucrose, suggesting that the sequestration follows the clathrin pathway. Internalization of D2S receptors was also assessed by immunofluorescence confocal microscopy. Translocation of D2 receptors from the cell membrane to intracellular vesicles was observed following the treatment with dopamine from HEK 293 cells only when GRK2 was coexpressed. D2S receptors expressed in HEK 293 cells were shown to be phosphorylated by GRK2 in an agonist-dependent manner. These results indicate that the sequestration of D2 receptors occurs only through a GRK-mediated pathway.Keywords: desensitization; dopamine D2 receptor; G protein-coupled receptor kinase; internalization; sequestration.Guanine nucleotide-binding regulatory protein (G-protein)-coupled receptors are known to be sequestered or internalized when exposed to agonists, and thereby become inaccessible to extracellular ligand [1±5]. Sequestered receptors are recycled back into the plasma membrane, while a portion of sequestered receptors is thought to be transported to lysosomes and degraded. On the one hand, sequestration may reduce G-protein activation when the number of receptors is a limiting factor [6]. On the other hand, sequestration was suggested to be necessary for resensitization of desensitized receptors in the case of b2 adrenergic receptors (b receptors), where desensitized and resensitized receptors are assumed, although not proven, to correspond to phosphorylated and dephosphorylated receptor states, respectively [7±9].Tsuga et al. [10,11] have shown that sequestration of muscarinic receptor m2, m3, m4 and m5 subtypes (m2±m5 receptors) expressed in COS-7 cells is facilitated by coexpression of G-protein-coupled receptor kinase 2 (GRK2) and that sequestration of m2 and m4, but not m3 and m5, receptors is attenuated by coexpression of a dominant-negative mutant of GRK2 (DN-GRK2

Dopamine D2 receptors (D2Rs; short form, which is one of the alternative splicing variants) expressed in COS-7 cells are internalized in an agonist-dependent manner only when G protein-coupled receptor kinase 2 (GRK2) is coexpressed [Ito, K., Haga, T., Lameh, J. & Sade Âe, W., (1999) Eur. J. Biochem. 260, 112±119]. We have examined the effects of coexpression of dynamin, a small molecular mass GTP-binding protein, rab5A, and their mutants on the internalization of D2Rs in the presence of both dopamine (10 or 100 mm) and GRK2. The rate and extent of D2R internalization was increased or decreased by coexpression of dynamin I or a dominant-negative form of dynamin I (dynamin I K44E), respectively. The effects of coexpressing these two dynamins were more prominent at 10 mm dopamine than at 100 mm. In the presence of 10 mm dopamine, internalization of D2R was completely suppressed when dynamin I K44E was coexpressed, and the half-life (t1 2 ) of D2R internalization decreased relative to cells not expressing dynamin from 82 to 29 min when dynamin I was coexpressed. Internalization of D2Rs was facilitated or suppressed by coexpression of a constitutively active form of rab5A (rab5A Q79L) or a dominant-negative form of rab5A (rab5A S34N), respectively. The t1 2 of D2R internalization at 10 mm dopamine decreased from 82 to 16 min in cells coexpressing rab5A Q79L. The effect of coexpression of rab5A S34N was more apparent at 100 mm dopamine than at 10 mm; the t1 2 of D2R internalization at 100 mm dopamine increased from 20 to 56 min and the proportion of internalized D2Rs after 120 min decreased from 53 to 28%. These results indicate that the internalization of D2Rs is dependent on the action of dynamin as well as GRK2, and is regulated by the action of rab5A.Keywords: G protein-coupled-receptor kinase 2; dopamine D2 receptor; rab5; dynamin; internalization.Many kinds of G protein-coupled receptors (GPCRs) are transferred to intracellular compartments from the plasma membrane within minutes of stimulation by agonists, a process that is termed sequestration, or internalization of receptors. This internalization serves to clear receptors from the cell surface, thereby modulating the responsiveness of the cell to further stimulation and/or resensitizing desensitized receptors [1±3]. The molecular mechanism of receptor internalization has not yet been fully elucidated, but several lines of evidence indicate that G protein-coupled receptor kinases (GRKs), arrestin, clathrin, and dynamin, are involved in the internalization of GPCRs, particularly of b 2 adrenergic and muscarinic acetylcholine receptors. Muscarinic m2 receptors expressed in COS-7 and the chemokine receptor CCR-5 expressed in HEK293 cells have been shown to be phosphorylated in an agonist-induced manner by GRK2, and their consequent internalization is facilitated by coexpression of GRK2 [4,5]. In addition, coexpression of a dominant-negative mutant of GRK2 (DN-GRK2) has been reported to inhibit agonist-dependent internalization and phosphorylation of b 2 adrenerg...

Photooxygenation quantum yields are dependent upon oxygen concentration light intensity and conversion, and these values (1 atm of 02,1°= 7.4 X 10s einstein s~•) are not optimized.

Waveforms of acoustic emission (AE) events come close and sometimes overlap each other when AE activity is very high. Conventional AE measurement systems which handle discrete AE events are not suitable for this situation because miss-detection of AE event occurs frequently. A new AE measurement system named as Continuous Wave Memory (CWM) was developed to solve this problem by recording the AE waveforms continuously to hard disks for several hours throughout the testing time. This new system enabled multiple analysis of one waveform with different filtering parameters. Short time Fourier transform (STFT) gave the time-frequency-magnitude characteristic of continuous AE waveforms and useful information for evaluation of degradation of materials. In this study, the degradation of ceramic fiber mat during cyclic compression test and the effect of binder-addition were evaluated by this new system. STFT results clearly showed the classification of degradation of the mat; breakage of fibers was the main source in the early compression cycles and sporadic friction between fibers became the main source of AE in the later compression cycles. The effect of organic binder to prevent the degradation of the mat was also estimated. It was observed that the friction signal disappeared and the breakage signal weakened in the binder-added specimens.

Impurity effects on the stability of a ferromagnetic metallic state in a bicritical-state manganite, (La0.7Pr0.3)0.65Ca0.35MnO3, on the verge of metal-insulator transition have been investigated by substituting a variety of transition-metal atoms for Mn ones. Among them, Fe doping exhibits the exceptional ability to dramatically decrease the ferromagnetic transition temperature. Systematic studies on the magnetotransport properties and x-ray diffraction for the Fe-doped crystals have revealed that charge-orbital ordering evolves down to low temperatures, which strongly suppresses the ferromagnetic metallic state. The observed glassy magnetic and transport properties as well as diffuse phase transition can be attributed to the phase-separated state where short-range chargeorbital-ordered clusters are embedded in the ferromagnetic metallic matrix. Such a behavior in the Fe-doped manganites form a marked contrast to the Cr-doping effects on charge-orbital-ordered manganites known as impurity-induced collapse of charge-orbital ordering.