28 results on '"GPCR, G protein–coupled receptor"'
Search Results
2. Role of the fourth intracellular loop of D1-like dopaminergic receptors in conferring subtype-specific signaling properties
- Author
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Tumova, Katerina, Zhang, Dongling, and Tiberi, Mario
- Subjects
DOPAMINE ,NEUROTRANSMITTERS ,G proteins ,DNA polymerases - Abstract
We investigate whether the fourth intracellular loop (IL4) of D1 and D5 dopaminergic receptors (D1R, D5R) confers D1-like subtype-specific signaling properties. Using chimeric receptors (D1R-IL4B and D5R-IL4A), we show that swapping of IL4 leads to a switch in dopamine affinity and constitutive activity of D1R and D5R. Dopamine potency was reduced for both chimeras in comparison with wild-type receptors. Moreover, dopamine-mediated maximal activation was drastically increased in cells expressing D1R-IL4B when compared with those harboring D5R-IL4A or wild-type receptors. In conclusion, IL4 plays a pivotal role in imparting subtype-specific ligand binding and activation properties to highly homologous seven-transmembrane receptors. [Copyright &y& Elsevier]
- Published
- 2004
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3. Hexa-histidin tag position influences disulfide structure but not binding behavior of in vitro folded N-terminal domain of rat corticotropin-releasing factor receptor type 2a.
- Author
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Klose, Jana, Wendt, Norbert, Kubald, Sybille, Krause, Eberhard, Fechner, Klaus, Beyermann, Michael, Bienert, Michael, Rudolph, Rainer, and Rothemund, Sven
- Abstract
The oxidative folding, particularly the arrangement of disulfide bonds of recombinant extracellular N-terminal domains of the corticotropin-releasing factor receptor type 2a bearing five cysteines (C2 to C6), was investigated. Depending on the position of a His-tag, two types of disulfide patterns were found. In the case of an N-terminal His-tag, the disulfide bonds C2-C3 and C4-C6 were found, leaving C5 free, whereas the C-terminal position of the His-tag led to the disulfide pattern C2-C5 and C4-C6, and leaving C3 free. The latter pattern is consistent with the disulfide arrangement of the extracellular N-terminal domain of the corticotropin-releasing factor (CRF) receptor type 1, which has six cysteines (C1 to C6) and in which C1 is paired with C3. However, binding data of the two differently disulfide-bridged domains show no significant differences in binding affinities to selected ligands, indicating the importance of the C-terminal portion of the N-terminal receptor domains, particularly the disulfide bond C4-C6 for ligand binding. [ABSTRACT FROM AUTHOR]
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- 2004
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4. Two threonine residues and two serine residues in the second and third intracellular loops are both involved in histamine H1 receptor downregulation
- Author
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Horio, Shuhei, Kato, Toshiyuki, Ogawa, Maki, Fujimoto, Katsumi, and Fukui, Hiroyuki
- Subjects
INFLAMMATORY mediators ,ANTIHISTAMINES ,MEMBRANE proteins ,AMINO acids - Abstract
Human histamine H
1 receptor (H1 R) contains five possible phosphorylation residues (Thr140 , Thr142 , Ser396 , Ser398 and Thr478 ) and the substitution of all these five residues to alanine completely impairs agonist-induced receptor downregulation. In the present study, to determine which residue(s) are responsible for receptor downregulation, we used mutant H1 Rs in which single or multiple residues were substituted with alanine. The results suggested that two groups, i.e., residues Thr140 and Thr142 , and residues Ser396 and Ser398 , independently contributed to H1 R downregulation. Thr140 and Ser398 mainly contributed to downregulation, and Thr142 or Ser396 had a slight inhibitory effect on Thr140 - or Ser398 -mediated process, respectively. [Copyright &y& Elsevier]- Published
- 2004
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5. Prostatic acid phosphatase degrades lysophosphatidic acid in seminal plasma
- Author
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Tanaka, Masayuki, Kishi, Yasuhiro, Takanezawa, Yasukazu, Kakehi, Yoshiyuki, Aoki, Junken, and Arai, Hiroyuki
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PROSTATE cancer ,MECHANICS (Physics) ,CELL proliferation ,CELL division - Abstract
Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological activities and is detected in various biological fluids, including human seminal plasma. Due to its cell proliferation stimulatory and anti-apoptotic activities, LPA has been implicated in the progression of some cancers such as ovarian cancer and prostate cancer. Here, we show that prostatic acid phosphatase, which is a non-specific phosphatase and which has been implicated in the progression of prostate cancer, inactivates LPA in human seminal plasma. Human seminal plasma contains both an LPA-synthetic enzyme, lysoPLD, which converts lysophospholipids to LPA and is responsible for LPA production in serum, and its major substrate, lysophosphatidylcholine. In serum, LPA accumulated during incubation at 37 °C. However, in seminal plasma, LPA did not accumulate. This discrepancy is explained by the presence of a strong LPA-degrading activity. Incubation of LPA with seminal plasma resulted in the disappearance of LPA and an accompanying accumulation of monoglyceride showing that LPA is degraded by phosphatase activity present in the seminal plasma. When seminal plasma was incubated in the presence of a phosphatase inhibitor, sodium orthovanadate, LPA accumulated, indicating that LPA is produced and degraded in the fluid. Biochemical characterization of the LPA-phosphatase activity identified two phosphatase activities in human seminal plasma. By Western blotting analysis in combination with several column chromatographies, the major activity was revealed to be identical to prostatic acid phosphatase. The present study demonstrates active LPA metabolism in seminal plasma and indicates the possible role of LPA signaling in male sexual organs including prostate cancer. [Copyright &y& Elsevier]
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- 2004
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6. The G protein-coupled receptor rhodopsin in the native membrane
- Author
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Fotiadis, Dimitrios, Liang, Yan, Filipek, Slawomir, Saperstein, David A., Engel, Andreas, and Palczewski, Krzysztof
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G proteins ,BINDING sites ,DIMERS ,RHODOPSIN - Abstract
The higher-order structure of G protein-coupled receptors (GPCRs) in membranes may involve dimerization and formation of even larger oligomeric complexes. Here, we have investigated the organization of the prototypical GPCR rhodopsin in its native membrane by electron and atomic force microscopy (AFM). Disc membranes from mice were isolated and observed by AFM at room temperature. In all experimental conditions, rhodopsin forms structural dimers organized in paracrystalline arrays. A semi-empirical molecular model for the rhodopsin paracrystal is presented validating our previously reported results. Finally, we compare our model with other currently available models describing the supramolecular structure of GPCRs in the membrane. [Copyright &y& Elsevier]
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- 2004
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7. Automated large-scale purification of a G protein-coupled receptor for neurotensin
- Author
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White, Jim F., Trinh, Loc B., Shiloach, Joseph, and Grisshammer, Reinhard
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MEMBRANE proteins ,AFFINITY chromatography ,MOLECULAR structure ,CRYSTALLIZATION - Abstract
Structure determination of integral membrane proteins requires milligram amounts of purified, functional protein on a regular basis. Here, we describe a protocol for the purification of a G protein-coupled neurotensin receptor fusion protein at the 3-mg or 10-mg level using immobilized metal affinity chromatography and a neurotensin column in a fully automated mode. Fermentation at a 200-l scale of Escherichia coli expressing functional receptors provides the material needed to feed into the purification routine. Constructs with tobacco etch virus protease recognition sites at either end of the receptor allow the isolation of neurotensin receptor devoid of its fusion partners. The presented expression and purification procedures are simple and robust, and provide the basis for crystallization experiments of receptors on a routine basis. [Copyright &y& Elsevier]
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- 2004
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8. G protein-activated K+ channels: a reporter for rapid activation of G proteins by lysophosphatidic acid in Xenopus oocytes
- Author
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Itzhaki Van-Ham, Irit, Peleg, Sagit, Dascal, Nathan, Shapira, Hagit, and Oron, Yoram
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MEMBRANE proteins ,G proteins ,THYROTROPIN ,POLYMERASE chain reaction - Abstract
Threshold concentrations of lysophosphatidic acid (LPA) or acetylcholine (ACh) induce pertussis toxin (PTX)-sensitive rapid desensitization of responses to LPA in Xenopus oocytes. To demonstrate that threshold [LPA] rapidly activates Gi/o proteins, we used the G protein-activated K
+ channel (GIRK) as a reporter. Low [LPA] inducedI in <3 s of the agonist addition with little or no activation of chloride current. Depletion of Gαo/Gαo1 each decreased the LPA-inducedK + I by approximately 40–50%, while PTX completely abolished it. This is the first direct evidence showing the activation of GIRK by LPA, and the involvement of G proteins of the Go family in rapid desensitization of LPA responses. [Copyright &y& Elsevier]K + - Published
- 2004
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9. Evolutionary analysis of rhodopsin and cone pigments: connecting the three-dimensional structure with spectral tuning and signal transfer
- Author
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Teller, David C., Stenkamp, Ronald E., and Palczewski, Krzysztof
- Subjects
RHODOPSIN ,G proteins ,PHOTORECEPTORS - Abstract
Extensive sequence data and structural sampling of expressed proteins from different species lead to the idea that entire molecules or specific domain folds belong to large superfamilies of proteins. A subset of G protein-coupled receptors, one of the largest families involved in cellular signaling, rod and cone opsins are involved in phototransduction in photoreceptor cells. Here, the evolutionary analysis of opsin sequences and structures predicts key residues involved in the transmission of the signal from the binding site of the chromophore to the cytoplasmic surface and residues that are involved in the spectral tuning of opsins to short wavelengths of light. [Copyright &y& Elsevier]
- Published
- 2003
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10. Constraints on the conformation of the cytoplasmic face of dark-adapted and light-excited rhodopsin inferred from antirhodopsin antibody imprints.
- Author
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Bailey, Brian W., Mumey, Brendan, Hargrave, Paul A., Arendt, Anatol, Ernst, Oliver P., Hofmann, Klaus Peter, Callis, Patrik R., Burritt, James B., Jesaitis, Algirdas J., and Dratz, Edward A.
- Abstract
Rhodopsin is the best-understood member of the large G protein-coupled receptor (GPCR) superfamily. The G-protein amplification cascade is triggered by poorly understood light-induced conformational changes in rhodopsin that are homologous to changes caused by agonists in other GPCRs. We have applied the 'antibody imprint' method to light-activated rhodopsin in native membranes by using nine monoclonal antibodies (mAbs) against aqueous faces of rhodopsin. Epitopes recognized by these mAbs were found by selection from random peptide libraries displayed on phage. A new computer algorithm, FINDMAP, was used to map the epitopes to discontinuous segments of rhodopsin that are distant in the primary sequence but are in close spatial proximity in the structure. The proximity of a segment of the N-terminal and the loop between helices VI and VIII found by FINDMAP is consistent with the X-ray structure of the dark-adapted rhodopsin. Epitopes to the cytoplasmic face segregated into two classes with different predicted spatial proximities of protein segments that correlate with different preferences of the antibodies for stabilizing the metarhodopsin I or metarhodopsin II conformations of light-excited rhodopsin. Epitopes of antibodies that stabilize metarhodopsin II indicate conformational changes from dark-adapted rhodopsin, including rearrangements of the C-terminal tail and altered exposure of the cytoplasmic end of helix VI, a portion of the C-3 loop, and helix VIII. As additional antibodies are subjected to antibody imprinting, this approach should provide increasingly detailed information on the conformation of light-excited rhodopsin and be applicable to structural studies of other challenging protein targets. [ABSTRACT FROM AUTHOR]
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- 2003
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11. Viral dsRNA activates mucin transcription in airway epithelial cells
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Londhe, Vedang, McNamara, Nancy, Lemjabbar, Hassan, and Basbaum, Carol
- Subjects
DOUBLE-stranded RNA ,RHINOVIRUSES ,RESPIRATORY infections ,MUCUS - Abstract
Double-stranded (ds) RNA is a biologically active component of many viruses including rhinoviruses infecting the upper respiratory tract. Mucus production is a common symptom of such infections. Here, we show that mucin, the glycoprotein subunit of mucus gels, is transcriptionally upregulated in an NF-κB- and p38-dependent manner when homogeneous cultures of epithelial cells are exposed to dsRNA. Furthermore, upstream of p38 in this system, dsRNA stimulates the extracellular release of ATP and activation of cell surface ATP receptors, which are G protein-coupled. This results in the stimulation of phospholipase C and protein kinase C. These findings suggest that ATP receptor antagonists could be used to modulate mucus production induced by virus. [Copyright &y& Elsevier]
- Published
- 2003
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12. Proteolytic cleavage of the EMR2 receptor requires both the extracellular stalk and the GPS motif
- Author
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Chang, Gin-Wen, Stacey, Martin, Kwakkenbos, Mark J., Hamann, Jörg, Gordon, Siamon, and Lin, Hsi-Hsien
- Subjects
PROTEINS ,AMINO acids ,PROTEOLYSIS - Abstract
EMR2 is a human myeloid-restricted member of the EGF-TM7 receptor family that contains a highly conserved
protein-coupled receptorG roteolysisp ite (GPS) in the membrane-proximal region. Here the post-translational proteolytic cleavage of EMR2 at GPS was investigated. We show the cleavage occurs at Leus 517 -Ser518 and is independent of the transmembrane domains. The non-covalent association of the resulting extracellular α-subunit and transmembrane β-subunit requires a minimum of eight amino acids in the β-subunit. The GPS motif is necessary, but not sufficient for receptor cleavage, which requires the entire extracellular stalk. Thus, an alternatively spliced EMR2 isoform with a truncated stalk fails to undergo proteolytic cleavage. Alternative splicing therefore provides a means to regulate GPS cleavage, producing receptors with two distinct structures. [Copyright &y& Elsevier]- Published
- 2003
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13. Control of Ras cycling by Ca2+
- Author
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Walker, Simon A., Cullen, Peter J., Taylor, James A., and Lockyer, Peter J.
- Subjects
GUANOSINE triphosphatase ,CYTOPLASM - Abstract
Ras GTPases are binary switches, cycling between an inactive GDP-bound form and an active GTP-bound form at the membrane. They transduce signals into the cytoplasm via effector pathways that regulate cell growth, differentiation and apoptosis. Ras activation is enhanced by guanine nucleotide exchange factors (GEFs); deactivation is accelerated by GTPase-activating proteins (GAPs). Recently, new roles for Ca
2+ and diacylglycerol (DAG) in the control of Ras cycling have emerged with the discovery of a series of novel GEFs and GAPs. These regulators of Ras cycling are likely to play a key role in the information processing of Ca2+ and DAG signals. [Copyright &y& Elsevier]- Published
- 2003
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14. Pharmacological analysis of a dopamine D2Short:Gαo fusion protein expressed in Sf9 cells
- Author
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Gazi, Lucien, Wurch, Thierry, Lopéz-Giménez, Juan F., Pauwels, Petrus J., and Strange, Philip G.
- Subjects
DOPAMINE ,PHARMACOLOGY - Abstract
A dopamine D
2Short receptor:Gαo fusion protein was expressed in Sf9 cells using the baculovirus expression system. [3 H]Spiperone bound to D2Short :Gαo with a pKd ≈10. Dopamine stimulated the binding of [35 S]guanosine-5′-O-(3-thio)triphosphate (GTPγS) to D2Short :Gαo expressed with Gβ1 γ2 (Emax >460%; pEC50 5.43±0.06). Most of the putative D2 antagonists behaved as inverse agonists (suppressing basal [35 S]GTPγS binding) at D2Short :Gαo /Gβ1 γ2 although (−)-sulpiride and ziprasidone were neutral antagonists. Competition of [3 H]spiperone binding by dopamine and 10,11-dihydroxy-N-n-propylnorapomorphine revealed two binding sites of different affinities, even in the presence of GTP (100 μM). The D2Short :Gαo fusion protein is therefore a good model for characterising D2 receptors. [Copyright &y& Elsevier]- Published
- 2003
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15. Mutation of an N-terminal acidic-rich region of p115-RhoGEF dissociates α13 binding and α13-promoted plasma membrane recruitment
- Author
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Bhattacharyya, Raja and Wedegaertner, Philip B.
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CARRIER proteins ,RAS proteins - Abstract
The Ras homology (Rho) guanine nucleotide exchange factor p115-RhoGEF couples the α
13 heterotrimeric guanine nucleotide binding protein (G protein) subunit to Rho GTPase. α13 binds to a regulator of G protein signaling (RGS) domain in p115-RhoGEF, but the mechanism of α13 activation of p115-RhoGEF is poorly understood. In this report, we demonstrate in cell-based assays that the acidic-rich N-terminus, adjacent to the RGS domain, is required for binding to activated α13 , and refine the importance of this region by showing that mutation of glutamic acids 27 and 29 in full-length p115-RhoGEF is sufficient to prevent interaction with activated α13 . However, α13 -interacting deficient N-terminal mutants of p115-RhoGEF retain α13 -dependent plasma membrane recruitment. Overall, these findings demonstrate a critical role for the N-terminal extension of p115-RhoGEF in mediating binding to α13 and dissociate two activities of p115-RhoGEF: binding to activated α13 and translocation to the PM in response to activated α13 . [Copyright &y& Elsevier]- Published
- 2003
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16. Platelet-derived growth factor (PDGF)-induced chemotaxis does not require the G protein-coupled receptor S1P1 in murine embryonic fibroblasts and vascular smooth muscle cells
- Author
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Kluk, Michael J., Colmont, Chantal, Wu, Ming-Tao, and Hla, Timothy
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SPHINGOSINE ,GROWTH factors ,POLYMERASE chain reaction - Abstract
Sphingosine 1-phosphate (S1P), a bioactive lipid mediator, signals via G protein-coupled receptors (GPCR). The prototypical S1P receptor, S1P
1 (also known as EDG-1), a Gi -linked receptor, is critical for vascular maturation during development. Recent work suggested that platelet-derived growth factor (PDGF)-induced cell migration required the S1P1 receptor, representing a novel mechanism for cross-talk between receptor tyrosine kinases and GPCRs. Since both S1P and PDGF are implicated in vascular smooth muscle cell (VSMC) pathobiology and development, we investigated this issue in rat VSMC and in embryonic fibroblasts derived from S1P1 null mice. Our data suggest that the S1P1 receptor is critical for S1P-induced, Gi -dependent migration but not for PDGF-BB-induced, receptor tyrosine kinase-dependent chemotaxis in VSMC. In addition, lack of S1P1 receptor in mouse embryonic fibroblasts did not significantly affect PDGF-induced cell migration. These data question the generality of the concept that S1P1 GPCR is a critical downstream component of PDGF-induced chemotaxis. [Copyright &y& Elsevier]- Published
- 2003
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17. Amphioxus homologs of Go-coupled rhodopsin and peropsin having 11-cis- and all-trans-retinals as their chromophores
- Author
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Koyanagi, Mitsumasa, Terakita, Akihisa, Kubokawa, Kaoru, and Shichida, Yoshinori
- Subjects
BIOLOGICAL pigments ,G proteins - Abstract
Because of low contents in the native organs and failure of the expression in cultured cells, the chromophore configurations of the pigments in Go-coupled opsin and peropsin groups in the opsin family are unknown. Here we have succeeded in expression of the amphioxus homologs of these groups in HEK293s cells and found that they can be regenerated with 11-cis- and all-trans-retinals, respectively. Light isomerized the chromophores of these opsins into the all-trans and 11-cis forms, respectively. The results strongly suggest that the physiological function of peropsin would be a retinal photoisomerase, while 11-cis configuration is necessary for the Go-coupled opsin groups. [Copyright &y& Elsevier]
- Published
- 2002
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18. Hetero-oligomerization of adenosine A1 receptors with P2Y1 receptors in rat brains
- Author
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Yoshioka, Kazuaki, Hosoda, Ritsuko, Kuroda, Yoichiro, and Nakata, Hiroyasu
- Subjects
CELL receptors ,ADENOSINE triphosphate - Abstract
Adenosine and ATP modulate cellular and tissue functions via specific P1 and P2 receptors, respectively. Although, in general, adenosine inhibits excitability and ATP functions as an excitatory transmitter in the central nervous system, little is known about the direct interaction between P1 and P2 receptors. We recently demonstrated that the G
i/o -coupled adenosine A1 receptor (A1 R) and Gq/11 -coupled P2Y1 receptor (P2Y1 R) form a heteromeric complex with a unique pharmacology in cotransfected HEK293T cells using the coimmunoprecipitation of differentially epitope-tagged forms of the receptor [Yoshioka et al. (2001) Proc. Natl. Acad. Sci. USA 98, 7617–7622], although it remained to be determined whether this hetero-oligomerization occurs in vivo. In the present study, we first demonstrated a high degree of colocalization of A1 R and P2Y1 R by double immunofluorescence experiments with confocal laser microscopy in rat cortex, hippocampus and cerebellum in addition to primary cultures of cortical neurons. Then, a direct association of A1 R with P2Y1 R was shown in coimmunoprecipitation studies using membrane extracts from these regions of rat brain. Together, these results suggest the widespread colocalization of A1 R and P2Y1 R in rat brain, and both receptors can exist in the same neuron, and therefore associate as hetero-oligomeric complexes in the rat brain. [Copyright &y& Elsevier]- Published
- 2002
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19. Mechanisms for 2-methoxyestradiol-induced apoptosis of prostate cancer cells
- Author
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Bu, Shizhong, Blaukat, Andree, Fu, Xin, Heldin, Nils-Erik, and Landström, Maréne
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PROSTATE cancer treatment ,APOPTOSIS - Abstract
Prostate and breast carcinomas are sex hormone-related carcinomas, which are known to be associated with an over-expression of the proto-oncogene Bcl-2. Here, we report that 2-methoxyestradiol (2-ME), an endogenous metabolite of estrogen that does not bind to nuclear estrogen receptors, effectively induces apoptosis in Bcl-2-expressing human prostate and breast carcinoma cells in vitro and in a rat prostate tumor model in vivo. In several cell lines derived from prostate, breast, liver and colorectal carcinomas, 2-ME treatment led to an activation of c-Jun N-terminal kinase (JNK) and phosphorylation of Bcl-2, which preceded the induction of apoptosis. In summary, our data suggest that 2-ME induces apoptosis in epithelial carcinomas by causing phosphorylation of JNK, which appears to be correlated with phosphorylation of Bcl-2. [Copyright &y& Elsevier]
- Published
- 2002
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20. The glucagon-like peptide-1 receptor binding site for the N-terminus of GLP-1 requires polarity at Asp198 rather than negative charge
- Author
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López de Maturana, Rakel and Donnelly, Dan
- Subjects
GLUCAGON-like peptide 1 ,G proteins - Abstract
The mutation of Asp198 to Asn in the receptor for glucagon-like peptide-1(7–36)amide (GLP-1) had no effect upon GLP-1 affinity whereas substitution with Ala greatly reduced affinity, demonstrating the importance of polarity rather than negative charge at Asp198. However, the Asp198-Ala mutation had less effect upon the affinity of Exendin-4, a peptide agonist that has been shown previously not to require its N-terminus for high affinity. Moreover, the affinity of a truncated GLP-1 analogue lacking the first eight residues was not affected by the Asp198-Ala mutation, demonstrating that Asp198 is required for maintaining the binding site of the N-terminal region of GLP-1. [Copyright &y& Elsevier]
- Published
- 2002
21. Agonist-promoted heteromeric oligomerization between adenosine A1 and P2Y1 receptors in living cells
- Author
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Yoshioka, Kazuaki, Saitoh, Osamu, and Nakata, Hiroyasu
- Subjects
OLIGOMERS ,ADENOSINES ,BIOLUMINESCENCE ,ENERGY transfer - Abstract
We have explored the process of oligomerization of G protein-coupled purinergic receptors, adenosine A
1 receptor (A1 R) and P2Y1 receptor (P2Y1 R), in intact HEK293T cells by means of modified bioluminescence resonance energy transfer technology (BRET2 ) that offers greatly improved separation of the emission spectra of the donor and acceptor moieties compared to traditional BRET. This approach identified both constitutive and agonist-promoted heteromeric oligomerization between Myc-tagged P2Y1 R fused to a donor, Renilla luciferase (Myc-P2Y1 R-Rluc) and HA-tagged A1 R fused to an acceptor, a different form of green fluorescent protein (HA-A1 R-GFP2 ). The BRET2 signal increased in a time-dependent manner in the cells expressing HA-A1 R-GFP2 /Myc-P2Y1 R-Rluc upon addition of agonists for both receptors, which could be inhibited by pretreatment with the P2Y1 R antagonist MRS2179. A high degree of HA-A1 R-GFP2 and Myc-P2Y1 R-Rluc co-localization in the co-transfected HEK293T cells was also observed by confocal laser microscopy. These results indicate that A1 R and P2Y1 R can form constitutive hetero-oligomers in living cells and this process is promoted by the simultaneous activation of both receptors. [Copyright &y& Elsevier]- Published
- 2002
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22. Lysophosphatidic acid (LPA) receptors are activated differentially by biological fluids: possible role of LPA-binding proteins in activation of LPA receptors
- Author
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Hama, Kotaro, Bandoh, Koji, Kakehi, Yoshiyuki, Aoki, Junken, and Arai, Hiroyuki
- Subjects
LYSOPHOSPHOLIPIDS ,SERUM albumin ,CALCIUM ions ,RATS - Abstract
Lysophosphatidic acid (LPA) exerts multiple biological functions through G protein-coupled receptors (EDG2/LPA
1 , EDG4/LPA2 , and EDG7/LPA3 ) and is present in serum where it is associated with albumin. In this study we examined LPA activity in various biological fluids by measuring the LPA-induced increase in the intracellular concentration of calcium ion in three types of Sf9 insect cells, each expressing one of the LPA receptors. Using this system, we found that EDG2 and EDG4, but not EDG7, were activated strongly by addition of incubated plasma. By contrast, LPA detected in seminal plasma, which contains a low concentration of albumin, selectively activated EDG7. After LPA in these samples was extracted and reconstituted, it activated all three receptors. We also found that serum albumin readily inhibits the activation of EDG7 but not the activation of EDG2 or EDG4. In addition, plasma from Nagase analbuminemic rats but not plasma from control Sprague–Dawley rats was found to strongly activate EDG7, although the plasma of these two types of rats contained equal amounts of LPA and activated both EDG2 and EDG4. The present study shows that serum albumin can negatively regulate EDG7 but not EDG2 or EDG4, and suggests that protein factors are present in seminal plasma and deliver LPA efficiently to EDG7 but not to EDG2 or EDG4. [Copyright &y& Elsevier]- Published
- 2002
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23. Human substance P receptor undergoes agonist-dependent phosphorylation by G protein-coupled receptor kinase 5 in vitro
- Author
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Warabi, Kengo, Richardson, Mark D., Barry, William T., Yamaguchi, Keisuke, Roush, Eric D., Nishimura, Kinya, and Kwatra, Madan M.
- Subjects
G proteins ,PROTEIN kinases - Abstract
G protein-coupled receptor kinases (GRKs) phosphorylate agonist-occupied G protein-coupled receptors, leading to receptor desensitization. Seven GRKs, designated GRK1 through 7, have been characterized. GRK5 is negatively regulated by protein kinase C. We investigated whether human substance P receptor (hSPR) is a substrate of GRK5. We report that membrane-bound hSPR is phosphorylated by purified GRK5, and that both the rate and extent of phosphorylation increase dramatically in the presence of substance P. The phosphorylation has a high stoichiometry (20±4 mol phosphate/mol hSPR) and a low K
m (1.7±0.1 nM). These data provide the first evidence that hSPR is a substrate of GRK5. [Copyright &y& Elsevier]- Published
- 2002
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24. Identification of G protein-coupled receptor genes from the human genome sequence
- Author
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Takeda, Shigeki, Kadowaki, Shiro, Haga, Tatsuya, Takaesu, Hirotomo, and Mitaku, Shigeki
- Subjects
G proteins ,HUMAN genome ,LIGANDS (Biochemistry) - Abstract
We have identified novel G protein-coupled receptors (GPCRs) with no introns in the coding region from the human genome sequence: 322 olfactory receptors; 22 taste receptors; 128 registered GPCRs for endogenous ligands; 50 novel GPCR candidates homologous to registered GPCRs for endogenous ligands; and 59 novel GPCR candidates not homologous to registered GPCRs. The total number of GPCRs with and without introns in the human genome was estimated to be approximately 950, of which 500 are odorant or taste receptors and 450 are receptors for endogenous ligands. [Copyright &y& Elsevier]
- Published
- 2002
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25. A conserved Asn in TM7 of the thyrotropin receptor is a common requirement for activation by both mutations and its natural agonist
- Author
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Claeysen, Sylvie, Govaerts, Cédric, Lefort, Anne, Van Sande, Jacqueline, Costagliola, Sabine, Pardo, Leonardo, and Vassart, Gilbert
- Subjects
THYROTROPIN ,GLYCOPROTEIN hormones - Abstract
The wide spectrum of naturally occurring mutations able to activate the thyrotropin (TSH) receptor provides a useful tool to approach the structure of the active state(s) of the glycoprotein hormone receptors. Here we show that the side-chain of the highly conserved N7.49 (Asn 674) in TM7 is mandatory for activation of the TSH receptor, not only by TSH, but also by a panel of eight natural and two artificial activating mutations. Basal activity levels of the mutants were significantly decreased by suppression of the side-chain of N7.49 (N7.49A double mutants). In addition, comparative effects of the N7.49A substitution on the ten mutants demonstrate that basal activity and agonist- or mutation-stimulated activity might involve different structural changes. [Copyright &y& Elsevier]
- Published
- 2002
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26. β-Arrestin is involved in the desensitization but not in the internalization of the somatostatin receptor 2A expressed in CHO cells
- Author
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Brasselet, Sabrina, Guillen, Stéphanie, Vincent, Jean-Pierre, and Mazella, Jean
- Subjects
SOMATOSTATIN ,RECEPTOR-ligand complexes - Abstract
The interaction of β-arrestin-1 with the somatostatin receptor type 2A (sst2A) was monitored using both biochemical and confocal imaging approaches. We show that, using transient transfection of either β-arrestin-1 or its dominant negative Δ-arrestin-1 in CHO cells stably transfected with the sst2A, β-arrestin-1 is colocalized with the receptor in endosomal vesicles after somatostatin-induced sequestration. However, this interaction leads to a role of β-arrestin-1 in the desensitization of the sst2A rather than in the internalization process of the receptor–ligand complex. [Copyright &y& Elsevier]
- Published
- 2002
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27. Mass spectrometric analysis of the kinetics of in vivo rhodopsin phosphorylation.
- Author
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Lee, Kimberly A., Craven, Kimberley B., Niemi, Gregory A., and Hurley, James B.
- Abstract
On stimulation, rhodopsin, the light-sensing protein in the rod cells of the retina, is phosphorylated at several sites on its C terminus as the first step in deactivation. We have developed a mass spectrometry-based method to quantify the kinetics of phosphorylation at each site in vivo. After exposing either a freshly dissected mouse retina or the eye of an anesthetized mouse to a flash of light, phosphorylation and dephosphorylation reactions are terminated by rapidly homogenizing the retina or enucleated eye in 8 M urea. The C-terminal peptide containing all known phosphorylation sites is cleaved from rhodopsin, partially purified by ultracentrifugation, and analyzed by liquid chromatography coupled with mass spectrometry (LCMS). The mass spectrometer responds linearly to the peptide from 10 fmole to 100 pmole. The relative sensitivity to peptides with zero to five phosphates was determined using purified phosphopeptide standards. High pressure liquid chromatography (HPLC) coupled with tandem mass spectrometry (LCMS/MS) was used to distinguish the three primary sites of phosphorylation, Ser 334, Ser 338, and Ser 343. Peptides monophosphorylated on Ser 334 were separable from those monophosphorylated on Ser 338 and Ser 343 by reversed-phase HPLC. Although peptides monophosphorylated at Ser 338 and Ser 343 normally coelute, the relative amounts of each species in the single peak could be determined by monitoring the ratio of specific daughter ions characteristic of each peptide. Doubly phosphorylated rhodopsin peptides with different sites of phosphorylation also were distinguished by LCMS/MS. The sensitivity of these methods was evaluated by using them to measure rhodopsin phosphorylation stimulated either by light flashes or by continuous illumination over a range of intensities. [ABSTRACT FROM AUTHOR]
- Published
- 2002
- Full Text
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28. Functional and protein chemical characterization of the N-terminal domain of the rat corticotropin-releasing factor receptor 1.
- Author
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Hofmann, Bernhard A., Sydow, Sabine, Jahn, Olaf, Van Werven, Lars, Liepold, Thomas, Eckart, Klaus, and Spiess, Joachim
- Abstract
Rat corticotropin-releasing factor receptor 1 (rCRFR1) was produced either in transfected HEK 293 cells as a complex glycosylated protein or in the presence of the mannosidase I inhibitor kifunensine as a high mannose glycosylated protein. The altered glycosylation did not influence the biological function of rCRFR1 as demonstrated by competitive binding of rat urocortin (rUcn) or human/rat corticotropin-releasing factor (h/rCRF) and agonist-induced cAMP accumulation. The low production rate of the N-terminal domain of rCRFR1 (rCRFR1-NT) by transfected HEK 293 cells, was increased by a factor of 100 in the presence of kifunensine. The product, rCRFR1-NT-Kif, bound rUcn specifically (K
D = 27 nM) and astressin (KI = 60 nM). This affinity was 10-fold lower than the affinity of full length rCRFR1. However, it was sufficiently high for rCRFR1-NT-Kif to serve as a model for the N-terminal domain of rCRFR1. With protein fragmentation, Edman degradation, and mass spectrometric analysis, evidence was found for the signal peptide cleavage site C-terminally to Thr23 and three disulfide bridges between precursor residues 30 and 54, 44 and 87, and 68 and 102. Of all putative N-glycosylation sites in positions 32, 38, 45, 78, 90, and 98, all Asn residues except for Asn32 were glycosylated to a significant extent. No O-glycosylation was observed. [ABSTRACT FROM AUTHOR]- Published
- 2001
- Full Text
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