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81.
Louise Valentin-Hansen Minyoung Park Thomas Huber Amy Grunbeck Saranga Naganathan Thue W. Schwartz Thomas P. Sakmar 《The Journal of biological chemistry》2014,289(26):18045-18054
Substance P (SP) is a neuropeptide that mediates numerous physiological responses, including transmission of pain and inflammation through the neurokinin-1 (NK1) receptor, a G protein-coupled receptor. Previous mutagenesis studies and photoaffinity labeling using ligand analogues suggested that the binding site for SP includes multiple domains in the N-terminal (Nt) segment and the second extracellular loop (ECLII) of NK1. To map precisely the NK1 residues that interact with SP, we applied a novel receptor-based targeted photocross-linking approach. We used amber codon suppression to introduce the photoreactive unnatural amino acid p-benzoyl-l-phenylalanine (BzF) at 11 selected individual positions in the Nt tail (residues 11–21) and 23 positions in the ECLII (residues 170(C-10)–193(C+13)) of NK1. The 34 NK1 variants were expressed in mammalian HEK293 cells and retained the ability to interact with a fluorescently labeled SP analog. Notably, 10 of the receptor variants with BzF in the Nt tail and 4 of those with BzF in ECLII cross-linked efficiently to SP, indicating that these 14 sites are juxtaposed to SP in the ligand-bound receptor. These results show that two distinct regions of the NK1 receptor possess multiple determinants for SP binding and demonstrate the utility of genetically encoded photocross-linking to map complex multitopic binding sites on G protein-coupled receptors in a cell-based assay format. 相似文献
82.
Heptahelical G-protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors couple to heterotrimeric G proteins to relay extracellular signals to intracellular signaling networks, but the molecular mechanism underlying guanosine 5′-diphosphate (GDP) release by the G protein α-subunit is not well understood. Amino acid substitutions in the conserved α5 helix of Gi, which extends from the C-terminal region to the nucleotide-binding pocket, cause dramatic increases in basal (receptor-independent) GDP release rates. For example, mutant Gαi1-T329A shows an 18-fold increase in basal GDP release rate and, when expressed in culture, it causes a significant decrease in forskolin-stimulated cAMP accumulation. The crystal structure of Gαi1-T329A·GDP shows substantial conformational rearrangement of the switch I region and additional striking alterations of side chains lining the catalytic pocket that disrupt the Mg+2 coordination sphere and dislodge bound Mg+2. We propose a “sequential release” mechanism whereby a transient conformational change in the α5 helix alters switch I to induce GDP release. Interestingly, this mechanistic model for heterotrimeric G protein activation is similar to that suggested for the activation of the plant small G protein Rop4 by RopGEF8. 相似文献
83.
A nuclear gene for higher level phylogenetics: phosphoenolpyruvate carboxykinase tracks mesozoic-age divergences within Lepidoptera (Insecta) 总被引:3,自引:0,他引:3
The sequence of phosphoenolpyruvate carboxykinase (PEPCK) has been
previously identified as a promising candidate for reconstructing
Mesozoic-age divergences (Friedlander, Regier, and Mitter 1992, 1994). To
test this hypothesis more rigorously, 597 nucleotides of aligned PEPCK
coding sequence (approximately 30% of the coding region) were generated
from 18 species representing Mesozoic-age lineages of moths (Insecta:
Lepidoptera) and outgroup taxa. Relationships among basal Lepidoptera are
well established by morphological analysis, providing a strong test for the
utility of a gene which has not previously been used in systematics.
Parsimony and other phylogenetic analyses were conducted on nucleotides by
codon positions (nt1, nt2, nt3) separately and in combination, and on amino
acids, for comparison to the test phylogeny. The highest concordance was
achieved with nt1 + nt2, for which one of two most-parsimonious trees was
identical to the test phylogeny, and with all nucleotides when nt3 was
down-weighted sevenfold or higher, for which a single most-parsimonious
tree identical to the test phylogeny resulted. Substitutions in nt3
approached saturation in many, but not all, pairwise comparisons and their
exclusion or severe downweighting greatly increased the degree of
concordance with the test phylogeny. Neighbor-joining analysis confirms
this finding. The utility of PEPCK for phylogenetics is demonstrated over a
time span for which few other suitable genes are currently available.
相似文献
84.
TP Souza-Junior L Loren?o-Lima D Ganini CV Vardaris TG Polotow MP Barros 《Biology of sport / Institute of Sport》2014,31(4):271-276
Reactive oxygen species are produced during anaerobic exercise mostly by Fe ions released into plasma and endothelial/muscle xanthine oxidase activation that generates uric acid (UA) as the endpoint metabolite. Paradoxically, UA is considered a major antioxidant by virtue of being able to chelate pro-oxidative iron ions. This work aimed to evaluate the relationship between UA and plasma markers of oxidative stress following the exhaustive Wingate test. Plasma samples of 17 male undergraduate students were collected before, 5 and 60 min after maximal anaerobic effort for the measurement of total iron, haem iron, UA, ferric-reducing antioxidant activity in plasma (FRAP), and malondialdehyde (MDA, biomarker of lipoperoxidation). Iron and FRAP showed similar kinetics in plasma, demonstrating an adequate pro-/antioxidant balance immediately after exercise and during the recovery period (5–60 min). Slight variations of haem iron concentrations did not support a relevant contribution of rhabdomyolysis or haemolysis for iron overload following exercise. UA concentration did not vary immediately after exercise but rather increased 29% during the recovery period. Unaltered MDA levels were concomitantly measured. We propose that delayed UA accumulation in plasma is an auxiliary antioxidant response to post-exercise (iron-mediated) oxidative stress, and the high correlation between total UA and FRAP in plasma (R-Square = 0.636; p = 0.00582) supports this hypothesis. 相似文献
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T P Sakmar 《Biophysical journal》1999,77(3):1189-1191