Real-Time Optical Monitoring of Ligand-Mediated Internalization of α1b-Adrenoceptor with Green Fluorescent Protein

Awaji, Takeo; Hirasawa, Akira; Kataoka, Masakazu; Shinoura, Hitomi; Nakayama, Yasuhisa; Sugawara, Tatsuo; Izumi, Shun-ichiro; Tsujimoto, Gozoh

doi:10.1210/mend.12.8.0149

Cited by 30 publications

(26 citation statements)

References 34 publications

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“…As a central role for ligand-stimulated receptor internalization appears to be to allow separation of the fate of the receptor between resensitization, often by removal of phosphate groups from serine and threonine residues in its C-terminal tail, and its trafficking towards lysosomes for degradation, these mechanisms have been actively studied for some time [16,[27][28][29]. Construction of green-fluorescent-protein-tagged forms of receptors has recently allowed a novel, non-invasive means of observing aspects of these processes in real time [20,[30][31][32][33]. Furthermore, a series of ELISA-based approaches may be used if an antibody to extracellular elements of a receptor or to an introduced epitope tag is available, and the disappearance and reappearance of receptor at the cell surface is all that is to be monitored [34].…”

Section: Discussionmentioning

confidence: 99%

Kinetic analysis of the internalization and recycling of [3H]TRH and C-terminal truncations of the long isoform of the rat thyrotropin-releasing hormone receptor-1

Drmota

Milligan

2000

Biochemical Journal

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The C-terminal tail of the long splice variant of the rat thyrotropin-releasing hormone (TRH) receptor-1 (TRHR-1L) comprises around 93 amino acids. A series of C-terminal truncations was constructed and expressed transiently in HEK-293 cells. The extent of steady-state internalization of these in response to [$H]TRH was dependent upon the degree of truncation. Little effect was produced by deletion of the C-terminal to 50 amino acids, although there was a substantial decrease in the extent of internalization by deletion to 45-46 amino acids. The rate of internalization of TRHR-1L in response to ligand was substantially decreased by the acid-wash procedures often used in the analysis of cellular distribution of receptors with peptide ligands, and thus an alternative procedure using a Mes-containing buffer was employed in the present study. Apart from a truncation

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Section: Discussionmentioning

confidence: 99%

Kinetic analysis of the internalization and recycling of [3H]TRH and C-terminal truncations of the long isoform of the rat thyrotropin-releasing hormone receptor-1

Drmota

Milligan

2000

Biochemical Journal

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“…We identified medium-to long-chain FFAs as the endogenous ligands of GPR120 using a receptor internalization assay (24). Apparent stimulatory activities were detected for saturated FFAs with a carbon chain length of 14 to 18 and for unsaturated FFAs with a chain length of 16 to 22.…”

Section: Ligandsmentioning

confidence: 99%

New Frontiers in Gut Nutrient Sensor Research: Free Fatty Acid Sensing in the Gastrointestinal Tract

et al. 2010

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Abstract. Utilizing the human genome database, the recently developed G-protein-coupled receptors (GPCRs) deorphanizing strategy successfully identified multiple receptors of free fatty acids (FFAs). FFAs have been demonstrated to act as ligands of several GPCRs (FFAR1, FFAR2, FFAR3, and GPR120). These fatty acid receptors are proposed to play critical roles in various types of physiological homeostases. FFAR1 and GPR120 are activated by medium-and long-chain FFAs. In contrast, FFAR2 and FFAR3 are activated by short-chain FFAs.

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“…GFP fusion in this manner does not perturb normal ligand binding nor the subcellular localization of ␣ 1B -adrenergic receptor (22). Flow cytometry analysis of GFP fluorescence enables us to detect the ␣ 1B -adrenergic receptors.…”

Section: Resultsmentioning

confidence: 97%

Interaction of the α1B-Adrenergic Receptor with gC1q-R, a Multifunctional Protein

Xu¹,

Hirasawa

Shinoura

et al. 1999

Journal of Biological Chemistry

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gC1q-R, a multifunctional protein, was found to bind with the carboxyl-terminal cytoplasmic domain of the ␣ 1B -adrenergic receptor (173 amino acids, amino acids 344 -516) in a yeast two-hybrid screen of a cDNA library prepared from the rat liver. In a series of studies with deletion mutants in this region, the ten arginine-rich amino acids (amino acids 369 -378) were identified as the site of interaction. The interaction was confirmed by specific co-immunoprecipitation of gC1q-R with fulllength ␣ 1B -adrenergic receptors expressed on transfected COS-7 cells, as well as by fluorescence confocal laser scanning microscopy, which showed co-localization of these proteins in intact cells. Interestingly, the ␣ 1B -adrenergic receptors were exclusively localized to the region of the plasma membrane in COS-7 cells that expressed the ␣ 1B -adrenergic receptor alone, whereas gC1q-R was localized in the cytoplasm in COS-7 cells that expressed gC1q-R alone; however, in cells that coexpressed ␣ 1B -adrenergic receptors and gC1q-R, most of the ␣ 1B -adrenergic receptors were co-localized with gC1q-R in the intracellular region, and a remarkable down-regulation of receptor expression was observed. These observations suggest a new role for the previously identified complement regulatory molecule, gC1q-R, in regulating the cellular localization and expression of the ␣ 1B -adrenergic receptors.G protein-coupled receptors interact with several classes of cytoplasmic proteins including heterotrimeric G proteins, kinases, phosphatases, and arrestins, and the binding of cytoplasmic protein with the receptor regulates receptor signaling (1-4). These interactions were first inferred from the functional effects of cytoplasmic proteins on receptor signaling and desensitization and were later confirmed by biochemical observation of the binding of the protein with receptor (5-8). Very recently, however, several unexpected interactions between cytoplasmic proteins and receptors have been observed; for instance, the adrenergic receptor interacts with the ␣-subunit of the eukaryotic initiation factor 2B (9) and with the Na ϩ /H ϩ -exchange regulatory factor (10). These raise the possibility that receptors may interact with other types of cellular proteins that could play unanticipated roles in regulating the function of the receptor.We conducted a search for novel proteins that interact with the ␣ 1B -adrenergic receptor, specifically focusing on the carboxyl-terminal cytoplasmic domain, because mutations within this domain have pleiotropic effects on receptor physiology (11)(12)(13)(14). Using interaction cloning and biochemical techniques, we demonstrate that gC1q-R 1 interacts with ␣ 1B -adrenergic receptors in vitro and in vivo through the specific site and that in cells that co-express ␣ 1B -adrenergic receptors and gC1q-R, the subcellular localization of ␣ 1B -adrenergic receptors is markedly altered and its expression is down-regulated. These results suggest that gC1q-R plays a role in the regulation of the subcellular localization as ...

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Real-Time Optical Monitoring of Ligand-Mediated Internalization of α1b-Adrenoceptor with Green Fluorescent Protein

Cited by 30 publications

References 34 publications

Kinetic analysis of the internalization and recycling of [3H]TRH and C-terminal truncations of the long isoform of the rat thyrotropin-releasing hormone receptor-1

Kinetic analysis of the internalization and recycling of [3H]TRH and C-terminal truncations of the long isoform of the rat thyrotropin-releasing hormone receptor-1

New Frontiers in Gut Nutrient Sensor Research: Free Fatty Acid Sensing in the Gastrointestinal Tract

Interaction of the α1B-Adrenergic Receptor with gC1q-R, a Multifunctional Protein

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