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1.
Figure 1

Figure 1. AC2 peptide design. From: Identification of new G?? interaction sites in adenylyl cyclase 2.

(A) Schematic of the AC2 transmembrane domains and cystosolic (C1 and C2) domains is shown. Transmembrane domains are shown in grey, C1 domain in yellow and C2 domain in red. Peptides are various colors, as indicated by the list on the right. (B) Table summarizing peptide sequences assayed in this study. The sequences are color coded according to the schematic in A and numbered according to the AC2 sequence (Rat; UniProt accession number P26769).

Aislyn D.W. Boran, et al. Cell Signal. ;23(9):1489-1495.
2.
Figure 5

Figure 5. Modulation of AC2 activation by Gβγ by various AC2 peptides. From: Identification of new G?? interaction sites in adenylyl cyclase 2.

Plots of AC2 activity under various conditions are shown. AC2 activity was measured using a radiometric assay where [αP32]-ATP is converted to [αP32]-cAMP. The activity is expressed in units of pmol of cAMP generated per milligram of membrane protein per minute (pmol/mg/min). (A) AC2 was stimulated with Gαs alone, Gαs + Gβγ or Gαs + Gβγ with increasing concentrations of the C1a or C1b peptides. (B) AC2 was stimulated with Gαs alone or Gαs with increasing concentrations of the C1a or C1b peptides. (C) AC2 basal activity was measured with increasing concentrations of the C1a or C1b peptides. Open symbols represent Gαs + Gβγ + various peptides, ▼ Gαs alone, ● Gαs + Gβγ and * basal.

Aislyn D.W. Boran, et al. Cell Signal. ;23(9):1489-1495.
3.
Figure 6

Figure 6. Modulation of AC2 activation by Gβγ by AC2 PFAHL C1b peptides. From: Identification of new G?? interaction sites in adenylyl cyclase 2.

Plots of the AC2 activity under various conditions are shown. AC2 activity was measured using a commercially available cAMP Lance™ assay kit. The activity is expressed in units of pmol of cAMP generated per milligram of membrane protein per minute (pmol/mg/min). (A) AC2 was stimulated with Gαs alone, Gαs + Gβγ or Gαs + Gβγ with increasing concentrations of the PFAHL C1b peptides. (B) AC2 was stimulated with Gαs alone or Gαs with increasing concentrations of the PFAHL C1b peptides. (C) AC2 basal activity was measured with increasing concentrations of the PFAHL C1b peptides. Open symbols represent Gαs + Gβγ + various peptides, ▼ Gαs alone, ● Gαs + Gβγ and * basal.

Aislyn D.W. Boran, et al. Cell Signal. ;23(9):1489-1495.
4.
Figure 3

Figure 3. Biacore binding isotherms for interaction between Gβγ and immobilized AC2 C1b PFAHL region peptides. From: Identification of new G?? interaction sites in adenylyl cyclase 2.

Biacore data is shown for binding between Gβγ and various AC2 C1b PFAHL region peptides: AC2 C1b 493-509 (A), 495-514 (B) and 515-541 (C). The peptides were biotinylated and immobilized and Gβγ was flowed over each chip at 12.5, 25, 50, 100 and 200 nM and at a flow rate of 20 μL/min. The residual is an indication of efficiency of the data fitting the binding isotherm.

Aislyn D.W. Boran, et al. Cell Signal. ;23(9):1489-1495.
5.
Figure 2

Figure 2. Biacore binding isotherms for interaction between Gβγ and immobilized AC2 C1a peptides and Gβγ. From: Identification of new G?? interaction sites in adenylyl cyclase 2.

Biacore data is shown for binding between Gβγ and (A) the AC2 C1a (339-360) peptide and (B) the scrambled form of the AC2 C1a (339-360) peptide. In order to scramble the 339-360 sequence, several residues were swapped to those with opposite charges and several alanine substitutions were made. The peptides were biotinylated and immobilized and Gβγ was flowed over each chip at 12.5, 25, 50, 100 and 200 nM and at a flow rate of 20 μL/min. The residual is an indication of efficiency of the data fitting to the binding isotherm.

Aislyn D.W. Boran, et al. Cell Signal. ;23(9):1489-1495.
6.
Figure 4

Figure 4. Biacore binding isotherms for interaction between Gβγ and immobilized AC2 C1b and C2 peptides. From: Identification of new G?? interaction sites in adenylyl cyclase 2.

Biacore data is shown for binding between Gβγ and various AC2 peptides: AC2 C1b 554-571 (A), AC2 C1b 578-602 (B), a scrambled form of the AC2 C1b 578-602 (C) and the QEHA peptide (AC2 C2 956-982) (D). The AC2 C1b 578-602 sequence was scrambled by substituting charged amino acids with amino acids containing the opposite charge. The peptides were biotinylated and immobilized and Gβγ was flowed over each chip at 12.5, 25, 50, 100 and 200 nM and at a flow rate of 20 μL/min. The residual is an indication of efficiency of the data fitting the binding isotherm.

Aislyn D.W. Boran, et al. Cell Signal. ;23(9):1489-1495.
7.
Figure 7

Figure 7. Homology of regions within the C1 and C2 domains of AC isoforms and structural analysis of AC activation. From: Identification of new G?? interaction sites in adenylyl cyclase 2.

Sequence alignments of a segment from the C1a region of AC2 (A) and the C1b region of AC2 (B). The score represents the percent homology for each region between each AC and AC2 as generated by alignments using EMBL-EBI ClustalW software. Regions of high homology are highlighted in grey and the sequence ranges covered by the AC2 peptides in this study are depicted at the top of the sequence alignments, color coded as in Figure 1. AC rat sequences were used when available otherwise, the AC mouse sequence was used. AC accession numbers and species (accession number/ AC isoform/ species) were as follows: P26769/ ADCY2/ RAT, P26770/ ADCY4/ RAT, P51829/ ADCY7/ MOUSE, Q04400/ ADCY5/ RAT, Q03343/ ADCY6/ RAT, O88444/ ADCY1/ MOUSE, P40146/ ADCY8/ RAT, P21932/ ADCY3/ RAT, and P51830/ ADCY9/ MOUSE. (C) Cartoon rendering of AC-Gαs structure where AC5 C1 is shown in green, AC2 C2 is shown in red and Gαs is shown in blue. The AC2 C2 peptide sequence (QEHA 956-982) is highlighted in cyan, AC2 C2 KF loop (927-933) is highlighted in purple and the AC5 C1a peptide region (339-360) is highlighted in orange. The Switch I region of Gαs is highlighted in pink and the Switch II is in yellow. Dashed lines with the color corresponding to the respective AC region or Gαs region represent linkages to the membrane for which the amino acid sequences have been omitted from the structure. The structural view on the right is rotated approximately 45° with respect to the view on the left and Gαs was omitted for clarity. The protein renderings were made using PyMol [35] and the coordinates from the Protein Data Bank file (accession number 1AZS).

Aislyn D.W. Boran, et al. Cell Signal. ;23(9):1489-1495.

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