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Results: 11

1.
FIGURE 9.

FIGURE 9. From: Spatial Approximations between Residues 6 and 12 in the Amino-terminal Region of Glucagon-like Peptide 1 and Its Receptor.

Identification of the receptor residue labeled with the Bpa6 probe. Left, a diagram illustrating the sequence of the CNBr fragment from the wild-type GLP1 receptor used for radiochemical sequencing. Right, a representative radioactivity elution profile of Edman degradation sequencing of the fragment resulting from CNBr cleavage of the wild-type receptor (Tyr205–Met233). A peak in radioactivity occurred in elution cycle 1, representing labeling of the receptor Tyr205 residue by the Bpa6 probe.

Quan Chen, et al. J Biol Chem. 2010 August 6;285(32):24508-24518.
2.
FIGURE 6.

FIGURE 6. From: Spatial Approximations between Residues 6 and 12 in the Amino-terminal Region of Glucagon-like Peptide 1 and Its Receptor.

Identification of the receptor residue labeled with the Bpa12 probe. Left, a diagram of the CNBr fragment (Leu144–Met204) resulting from cleavage of the F143M receptor mutant. Right, representative radioactivity elution profile of Edman degradation sequencing of this fragment covalently attached with the Bpa12 probe. A peak eluted in radioactivity appeared in cycle 2, representing labeling of the receptor Tyr145 residue by the Bpa12 probe. Data are representative of three independent experiments.

Quan Chen, et al. J Biol Chem. 2010 August 6;285(32):24508-24518.
3.
FIGURE 8.

FIGURE 8. From: Spatial Approximations between Residues 6 and 12 in the Amino-terminal Region of Glucagon-like Peptide 1 and Its Receptor.

Sequential CNBr and Skatole cleavage of the Bpa6 probe-labeled GLP1 receptor. Left, a diagram illustrating the predicted site resulting from Skatole cleavage of the candidate Tyr205–Met233 fragment of the wild-time receptor. Right, the non-glycosylated Mr = 6,500 band resulting from CNBr cleavage of the labeled wild-type GLP1 receptor shifted to Mr = 4,500 after further Skatole cleavage. This is most consistent with labeling of the ECL1 with the Bpa6 probe, representing the receptor region between residues Tyr205 and Met233. Based on the migration, the site of labeling with the Bpa6 probe was likely within the segment between Tyr205 and Trp214.

Quan Chen, et al. J Biol Chem. 2010 August 6;285(32):24508-24518.
4.
FIGURE 4.

FIGURE 4. From: Spatial Approximations between Residues 6 and 12 in the Amino-terminal Region of Glucagon-like Peptide 1 and Its Receptor.

Further localization of the domain of labeling with the Bpa12 probe by Lys-C cleavage. Left, a diagram illustrating the predicted sites from Lys-C cleavage of the wild-type GLP1 receptor. Right, Lys-C cleavage of the wild-type receptor yielded a band migrating at molecular weight of approximately 11,000 that did not shift further after deglycosylation with PNGase F. This indicated that the Bpa12 probe labeled the segment between Arg131 and Lys197 of the GLP1 receptor spanning the amino-terminal domain, TM1 and TM2 domains, and the first intracellular loop (bold circles). Data are representative of three independent experiments.

Quan Chen, et al. J Biol Chem. 2010 August 6;285(32):24508-24518.
5.
FIGURE 5.

FIGURE 5. From: Spatial Approximations between Residues 6 and 12 in the Amino-terminal Region of Glucagon-like Peptide 1 and Its Receptor.

CNBr cleavage of the F143M GLP1 receptor mutant labeled with the Bpa12 probe. Left, a diagram illustrating the theoretical fragments resulting from CNBr cleavage of the F143M GLP1 receptor mutant, along with the masses of protein cores of these fragments. Right, CNBr cleavage of the labeled wild-type receptor resulted in a band migrating at molecular weight of approximately 50,000 and shifted to 11,000 in the F143M mutant receptor. This provides the definitive identification of the fragment between Leu144 and Met204 (black circles) as the domain of labeling by the Bpa12 probe. Data are representative of three independent experiments.

Quan Chen, et al. J Biol Chem. 2010 August 6;285(32):24508-24518.
6.
FIGURE 7.

FIGURE 7. From: Spatial Approximations between Residues 6 and 12 in the Amino-terminal Region of Glucagon-like Peptide 1 and Its Receptor.

CNBr cleavage of the Bpa6 probe-labeled GLP1 receptor. Left, a diagram illustrating the theoretical sites of CNBr cleavage of the GLP1 receptor. Right, CNBr cleavage of the labeled GLP1 receptor yielded a fragment migrating at molecular weight of approximately 6,500 that did not shift further after deglycosylation with PNGase F. The best candidates representing this are the fragments including the first (Tyr205–Met233, highlighted in black circles) and third (Asp372–Met397, highlighted in gray circles) extracellular loops. Data are representative of at least three independent experiments.

Quan Chen, et al. J Biol Chem. 2010 August 6;285(32):24508-24518.
7.
FIGURE 3.

FIGURE 3. From: Spatial Approximations between Residues 6 and 12 in the Amino-terminal Region of Glucagon-like Peptide 1 and Its Receptor.

CNBr cleavage of the Bpa12 probe-labeled GLP1 receptor. Left, a diagram illustrating the theoretical fragments of the GLP1 receptor from CNBr cleavage. Right, a representative autoradiograph of a 10% NuPAGE gel used to separate the products of CNBr cleavage of the GLP1 receptor labeled with the Bpa12 probe. CNBr cleavage of the labeled GLP1 receptor yielded a fragment migrating at about Mr = 50,000 that shifted to approximately 24,000 after deglycosylation with PNGase F, indicating the site of labeling being within the large glycosylated Val18–Met204 fragment that includes the amino-terminal domain, TM1 and TM2, and the first intracellular loop of the GLP1 receptor (black circles). Data are representative of three independent experiments.

Quan Chen, et al. J Biol Chem. 2010 August 6;285(32):24508-24518.
8.
FIGURE 10.

FIGURE 10. From: Spatial Approximations between Residues 6 and 12 in the Amino-terminal Region of Glucagon-like Peptide 1 and Its Receptor.

Characterization of the Y145A GLP1 receptor mutant. Shown in the left panel are competition-binding curves of increasing concentrations of GLP1 to displace the binding of radioligand 125I-GLP1 to COS-1 cells expressing wild-type (WT) and Y145A GLP1 receptors. Values illustrated represent saturable binding as percentages of maximal binding observed in the absence of the competing peptide. They are expressed as the mean ± S.E. of duplicate values from a minimum of three independent experiments. Shown in the right panel are intracellular cAMP responses to increasing concentrations of GLP1 in these cells. Data points represent the mean ± S.E. of three independent experiments performed in duplicate, normalized relative to the maximal response to GLP1.

Quan Chen, et al. J Biol Chem. 2010 August 6;285(32):24508-24518.
9.
FIGURE 2.

FIGURE 2. From: Spatial Approximations between Residues 6 and 12 in the Amino-terminal Region of Glucagon-like Peptide 1 and Its Receptor.

Photoaffinity labeling of the GLP1 receptor. Left, representative autoradiographs of 10% SDS-PAGE gels used to separate the products of affinity labeling of CHO-GLP1R membranes with Bpa6 (bottom) or Bpa12 (top) probes in the presence of increasing concentrations of competing unlabeled GLP1 (from 0 to 1 μm). Shown also is the labeling of the non-receptor-bearing CHO cell membranes in the absence of competitor. The labeled GLP1 receptor with each probe migrated at molecular weight of approximately 66,000 that shifted to approximately 42,000 after deglycosylation by PNGase F as shown. Right, densitometric analysis of the competition for receptor labeling with Bpa6 (bottom) or Bpa12 (top) probes, performed in three independent experiments (mean ± S.E.).

Quan Chen, et al. J Biol Chem. 2010 August 6;285(32):24508-24518.
10.
FIGURE 1.

FIGURE 1. From: Spatial Approximations between Residues 6 and 12 in the Amino-terminal Region of Glucagon-like Peptide 1 and Its Receptor.

Probe characterization. Left, competition-binding curves for increasing concentrations of GLP1, the Bpa6 or Bpa12 probes to displace the binding of the radioligand 125I-GLP1 to CHO-GLP1R membranes. Values represent percentages of the maximal saturable binding observed in the absence of competitor and expressed as mean ± S.E. of duplicate data from three independent experiments. Right, intracellular cAMP responses in CHO-GLP1R cells to increasing concentrations of GLP1 and the Bpa6 and Bpa12 probes. Values are expressed as mean ± S.E. of data from three independent experiments performed in duplicate, with data normalized relative to the maximal response to GLP1. Absolute basal (5.1 ± 1.3 pmol/million cells) and maximal (194 ± 38 pmol/million cells) cAMP levels were similar for all three peptides.

Quan Chen, et al. J Biol Chem. 2010 August 6;285(32):24508-24518.
11.
FIGURE 11.

FIGURE 11. From: Spatial Approximations between Residues 6 and 12 in the Amino-terminal Region of Glucagon-like Peptide 1 and Its Receptor.

Graphic illustration of the feasibility of docking GLP1 with the two major regions of the GLP1 receptor, the amino terminus and the helical bundle core domain. Shown are homology models of docking GLP1 with the amino terminus (orange) and the helical bundle core domain (gray) of the GLP1 receptor. It should be noted that the region between residues 133 and 144 linking the amino terminus with the top of transmembrane segment was not included in this illustration because its structure was not well defined in the published crystal structures. The GLP1 peptide is colored blue to red from its amino terminus to its carboxyl terminus. Residues within GLP1 representing positions of photolabile moieties for affinity labeling (His7, Phe12, Ala24, and Gly35) are shown in green. The receptor residue, Glu125, labeled by the probe at position 35 was colored in cyan, whereas the receptor residue labeled by the probe at position 24, Glu133, is not illustrated, because it is within an unconstrained region of the receptor. The two receptor residues photoaffinity labeled by the probes at positions 12 and 6 in the current study, Tyr145 and Tyr205, respectively, are highlighted by Corey-Pauling-Koltun representation. The green-dotted lines link the sites of photoactivation with sites of covalent labeling.

Quan Chen, et al. J Biol Chem. 2010 August 6;285(32):24508-24518.

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