Journal: Proceedings of the National Academy of Sciences of the United States of America
Article Title: CAP1 binds and activates adenylyl cyclase in mammalian cells
doi: 10.1073/pnas.2024576118
Figure Lengend Snippet: CAP1-cyclase interaction involves CAP1’s N-terminal coiled-coil domain. (A) CAP1 (2 to 41) contains the C1–C2 binding domain. Immobilized GST or GST-N-CAP deletion proteins were incubated with 1 μg purified His-C1a and His-C2, and binding was assessed by Western blots with an anti–His-tag antibody. (B) MST analysis of in vitro RED-Tris-NTA labeled His-C1a (Left, AC7; 100 nM) or His-C2 (Right, AC7; 100 nM) with purified GST-CAP1 (2 to 41) or GST alone as titrants. (C) Peptide array–based mapping. Peptides covering the CAP1 (2 to 41) sequence (15-mers, Δ3 amino acid shift) were spotted on membranes, incubated with purified GST-IC1IIC2, and binding was revealed by anti–GST-HRP antibody. (D) Alanine mutagenesis (peptide array format) identifies the involvement of the RLE motifs in binding to GST-IC1IIC2. (E) Leucine residues in CAP1 (2 to 41) RLE motifs are required for binding C1 and C2. Immobilized GST and GST-CAP (2 to 41) RLE mutants were incubated with purified His-C1a (AC7; 1 μg) and His-C2 (AC7; 1 μg), and binding was assessed by Western blots with an anti-His antibody. (F) Leucine residues in both RLE submotifs (L11S and L18S) are required for the interaction of full-length CAP1 with the soluble IC1a-IIC2a (HA-C1C2) fusion construct. Lysates from cells cotransfected with Xpress-tagged WT or mutant CAP1 and with HA-C1C2 were analyzed by HA-immunoprecipitation coupled to anti-Xpress blotting. All immunoblots and peptide arrays are representative of three independent experiments. MST data are expressed as mean ± SEM (n = 3). Significance tested was performed using a two-tailed Student's t test against GST control group (****P < 0.001).
Article Snippet: Labeling of His-N-CAP1 proteins with NT647-Maleimide and NT647-NHS, or His-C1a and His-C2 with RED-Tris-NTA second generation fluorescent dyes followed the protocols provided by NanoTemper.
Techniques: Binding Assay, Incubation, Purification, Western Blot, In Vitro, Labeling, Peptide Microarray, Sequencing, Mutagenesis, Construct, Immunoprecipitation, Two Tailed Test
