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The third intracellular loop (IL3) of G protein-coupled receptors (GPCRs) is an important contact domain between GPCRs and their G proteins. Previously, the IL3 of Ste2p, a Saccharomyces cerevisiae GPCR, was suggested to undergo a conformational change upon activation as detected by differential protease susceptibility in the presence and absence of ligand. In this study using disulfide cross-linking experiments we show that the Ste2p cytoplasmic ends of helix 5 (TM5) and helix 6 (TM6) that flank the amino and carboxyl sides of IL3 undergo conformational changes upon ligand binding, whereas the center of the IL3 loop does not. Single Cys substitution of residues in the middle of IL3 led to receptors that formed high levels of cross-linked Ste2p, whereas Cys substitution at the interface of IL3 and the contiguous cytoplasmic ends of TM5 and TM6 resulted in minimal disulfide-mediated cross-linked receptor. The alternating pattern of residues involved in cross-linking suggested the presence of a 3(10) helix in the middle of IL3. Agonist (WHWLQLKPGQPNleY) induced Ste2p activation reduced cross-linking mediated by Cys substitutions at the cytoplasmic ends of TM5 and TM6 but not by residues in the middle of IL3. Thus, the cytoplasmic ends of TM5 and TM6 undergo conformational change upon ligand binding. An α-factor antagonist (des-Trp, des-His-α-factor) did not influence disulfide-mediated Ste2p cross-linking, suggesting that the interaction of the N-terminus of α-factor with Ste2p is critical for inducing conformational changes at TM5 and TM6. We propose that the changes in conformation revealed for residues at the ends of TM5 and TM6 are affected by the presence of G protein but not G protein activation. This study provides new information about role of specific residues of a GPCR in signal transduction and how peptide ligand binding activates the receptor. 相似文献
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The evolutionary origin of the pinnipeds (seals, sea lions, and walruses)
is still uncertain. Most authors support a hypothesis of a monophyletic
origin of the pinnipeds from a caniform carnivore. A minority view suggests
a diphyletic origin with true seals being related to the mustelids (otters
and ferrets). The phylogenetic relationships of the walrus to other
pinniped and carnivore families are also still particularly problematic.
Here we examined the relative support for mono- and diphyletic hypotheses
using DNA sequence data from the mitochondrial small subunit (12S) rRNA and
cytochrome b genes. We first analyzed a small group of taxa representing
the three pinniped families (Phocidae, Otariidae, and Odobenidae) and
caniform carnivore families thought to be related to them. We inferred
phylogenetic reconstructions from DNA sequence data using standard
parsimony and neighbor-joining algorithms for phylogenetic inference as
well as a new method called spectral analysis (Hendy and Penny) in which
phylogenetic information is displayed independently of any selected tree.
We identified and compensated for potential sources of error known to lead
to selection of incorrect phylogenetic trees. These include sampling error,
unequal evolutionary rates on lineages, unequal nucleotide composition
among lineages, unequal rates of change at different sites, and
inappropriate tree selection criteria. To correct for these errors, we
performed additional transformations of the observed substitution patterns
in the sequence data, applied more stringent structural constraints to the
analyses, and included several additional taxa to help resolve long,
unbranched lineages in the tree. We find that there is strong support for a
monophyletic origin of the pinnipeds from within the caniform carnivores,
close to the bear/raccoon/panda radiation. Evidence for a diphyletic origin
was very weak and can be partially attributed to unequal nucleotide
compositions among the taxa analyzed. Subsequently, there is slightly more
evidence for grouping the walrus with the eared seals versus the true
seals. A more conservative interpretation, however, is that the walrus is
an early, but not the first, independent divergence from the common
pinniped ancestor.
相似文献
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Umanah GK Huang L Ding FX Arshava B Farley AR Link AJ Naider F Becker JM 《The Journal of biological chemistry》2010,285(50):39425-39436
Fundamental knowledge about how G protein-coupled receptors and their ligands interact is important for understanding receptor-ligand binding and the development of new drug discovery strategies. We have used cross-linking and tandem mass spectrometry analyses to investigate the interaction of the N terminus of the Saccharomyces cerevisiae tridecapeptide pheromone, α-factor (WHWLQLKPGQPMY), and Ste2p, its cognate G protein-coupled receptor. The Trp(1) residue of α-factor was replaced by 3,4-dihydroxyphenylalanine (DOPA) for periodate-mediated chemical cross-linking, and biotin was conjugated to Lys(7) for detection purposes to create the peptide [DOPA(1),Lys(7)(BioACA),Nle(12)]α-factor, called Bio-DOPA(1)-α-factor. This ligand analog was a potent agonist and bound to Ste2p with ~65 nanomolar affinity. Immunoblot analysis of purified Ste2p samples that were treated with Bio-DOPA(1)-α-factor showed that the peptide analog cross-linked efficiently to Ste2p. The cross-linking was inhibited by the presence of either native α-factor or an α-factor antagonist. MALDI-TOF and immunoblot analyses revealed that Bio-DOPA(1)-α-factor cross-linked to a fragment of Ste2p encompassing residues Ser(251)-Met(294). Fragmentation of the cross-linked fragment and Ste2p using tandem mass spectrometry pinpointed the cross-link point of the DOPA(1) of the α-factor analog to the Ste2p Lys(269) side chain near the extracellular surface of the TM6-TM7 bundle. This conclusion was confirmed by a greatly diminished cross-linking of Bio-DOPA(1)-α-factor into a Ste2p(K269A) mutant. Based on these and previously obtained binding contact data, a mechanism of α-factor binding to Ste2p is proposed. The model for bound α-factor shows how ligand binding leads to conformational changes resulting in receptor activation of the signal transduction pathway. 相似文献
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Ste2p is the G protein-coupled receptor (GPCR) for the tridecapeptide pheromone alpha factor of Saccharomyces cerevisiae. This receptor-pheromone pair has been used extensively as a paradigm for investigating GPCR structure and function. Expression in yeast harboring a cognate tRNA/aminoacyl-tRNA synthetase pair specifically evolved to incorporate p-benzoyl- l-phenylalanine (Bpa) in response to the amber codon allowed the biosynthesis of Bpa-substituted Ste2p in its native cell. We replaced natural amino acid residues in Ste2p with Bpa by engineering amber TAG stop codons into STE2 encoded on a plasmid. Several of the expressed Bpa-substituted Ste2p receptors exhibited high-affinity ligand binding, and incorporation of Bpa into Ste2p influenced biological activity as measured by growth arrest of whole cells in response to alpha factor. We found that, at concentrations of 0.1-0.5 mM, a dipeptide containing Bpa could be used to enhance delivery of Bpa into the cell, while at 2 mM, both dipeptide and Bpa were equally effective. The application of a peptide delivery system for unnatural amino acids will extend the use of the unnatural amino acid replacement methodology to amino acids that are impermeable to yeast. Incorporation of Bpa into Ste2p was verified by mass spectrometric analysis, and two Bpa-Ste2p mutants were able to selectively capture alpha factor into the ligand-binding site after photoactivation. To our knowledge, this is the first experimental evidence documenting an unnatural amino acid replacement in a GPCR expressed in its native environment and the use of a mutated receptor to photocapture a peptide ligand. 相似文献
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Stuart A Suttie Alan GK Li Martha Quinn Kenneth GM Park 《World journal of surgical oncology》2007,5(1):1-9
Background
Caveolin-1 is thought to have an important impact on both signal transduction and mediation of intracellular processes. Furthermore, it has been suggested that Caveolin-1 may contribute to certain steps of carcinogenesis in various types of cancer. We examined the potential clinical relevance of Caveolin-1 in normal, benign and malignant breast tissue specimens.Methods
Using tissue microarray (TMA) technology cases of invasive breast cancer, DCIS, benign breast disease (i.e. fibroadenoma, sclerosing adenosis, ductal hyperplasia and radial scar) and normal breast tissue were evaluated for Caveolin-1 expression. Immunohistochemical staining with an anti-Caveolin-1-antibody was performed. Staining intensity was quantified semiquantitatively. In invasive lesions staining results were correlated with clinical and pathological data.Results
No Caveolin-1 expression was observed in epithelial cells of normal breast tissue (n = 5), benign breast disease (n = 295) and DCIS (n = 108). However, Caveolin-1 expression was found in 32 of 109 cases of invasive breast carcinomas (29.4%). Caveolin-1 expression in invasive breast cancer could neither be correlated with survival parameters such as overall or disease-free survival nor with established clinical and pathological markers.Conclusion
In this study we demonstrated expression of Caveolin-1 in one third of invasive breast cancers. A significant increase in Caveolin-1 expression was observed comparing invasive breast cancer to both benign breast tissue and non-invasive breast cancer. Since inhibitors of Caveolin-1 signalling are available, targeting Caveolin-1 in breast cancer may represent a potential option for future breast cancer treatment. 相似文献9.
Yu‐Chan Chen George K E Umanah Noah Dephoure Shaida A Andrabi Steven P Gygi Ted M Dawson Valina L Dawson Jared Rutter 《The EMBO journal》2014,33(14):1548-1564
The majority of ER-targeted tail-anchored (TA) proteins are inserted into membranes by the Guided Entry of Tail-anchored protein (GET) system. Disruption of this system causes a subset of TA proteins to mislocalize to mitochondria. We show that the AAA+ ATPase Msp1 limits the accumulation of mislocalized TA proteins on mitochondria. Deletion of MSP1 causes the Pex15 and Gos1 TA proteins to accumulate on mitochondria when the GET system is impaired. Likely as a result of failing to extract mislocalized TA proteins, yeast with combined mutation of the MSP1 gene and the GET system exhibit strong synergistic growth defects and severe mitochondrial damage, including loss of mitochondrial DNA and protein and aberrant mitochondrial morphology. Like yeast Msp1, human ATAD1 limits the mitochondrial mislocalization of PEX26 and GOS28, orthologs of Pex15 and Gos1, respectively. GOS28 protein level is also increased in ATAD1−/− mouse tissues. Therefore, we propose that yeast Msp1 and mammalian ATAD1 are conserved members of the mitochondrial protein quality control system that might promote the extraction and degradation of mislocalized TA proteins to maintain mitochondrial integrity. 相似文献
10.
NaCl-preferring NZB/B1NJ mice and NaCl-avoiding CBA/J mice have similar amiloride inhibition of chorda tympani responses to NaCl 总被引:1,自引:1,他引:0
Integrated chorda tympani nerve responses to NaCl were studied in two mouse
strains, an NaCl-preferring NZB/B1NJ and an NaCl-avoiding CBA/J. The NaCl
responses of both strains had similar magnitude and were suppressed by
amiloride to a similar extent. This suggests that peripheral gustatory
responsiveness to NaCl is not the only mechanism underlying mouse strain
variation in NaCl acceptance.
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