Fig 1.
Isolating MAS Ct interacting proteins by pull down assays.
(A) MAS Ct comprising the last 25 amino acids with an N-terminal biotin was synthesized as bait for the pull down assay. The PDZ binding motif (-E-T-V-V) in MAS is underlined while the cysteine to serine mutation in the peptide is underlined. (B) The major steps in the pull down assay are shown in a flow chart. (C) Western blot analysis of various fractions (input, unbound, washes and elutions) from pull down experiments of HEK293, HL-1 and cardiac tissue lysates. Presence of PDZ protein is detected by pan-MAGUK antibody. Below the western blots, Ponceau S stained membrane portions of corresponding regions (75 kDa to 150 kDa) from the blots are shown as a loading control.
Fig 2.
Identification of proteins from pull down assay by MS.
(A) Protein gels for MS analysis. The elution fractions from resin alone control ('–') and Ct bound resin ('+') were resolved on SDS-PAGE gels and the entire lanes were subjected for MS analysis to identify the proteins. (B) Venn-diagram of PDZ proteins identified to be specifically pulled down by MAS Ct in three different sample lysates. The total numbers of PDZ proteins identified are given in parenthesis. All PDZ hits are almost exclusively present in '+' samples. List of non-PDZ proteins identified to interact with Ct are given in Table 2.
Table 1.
List of PDZ protein hits with their synonyms, structural and functional domains.
Table 2.
List of non-PDZ proteins identified in the pull-down assays with different samples.
Fig 3.
Ingenuity Pathway Analysis (IPA) of protein hits.
List of IPA predicted (A and D) top 10 signaling networks, (B and E) top 10 function categories and (C and F) disease categories involving protein hits from (A, B and C) HEK293 and (B, E and F) HL-1 and Human cardiac tissue samples. In panels A, B, D and E a p-value of less than 0.05 (or -log(p-value) < 1.3) is considered statistically significant. In panels C and F, the numbers of protein hits that are involved in a particular disease or functional category are expressed as a percentage of total protein hits that were included in the analysis and shown as a bar graph which is truncated to show categories with greater than 30% representation.
Fig 4.
Summary of ΔPDZ-MAS expression and signaling compared to WT.
(A) Surface expression of ΔPDZ-MAS to WT evaluated using enzyme linked immunosorbent assay (ELISA). (B) The bar graph showing the constitutive/basal IP1 levels (in the absence of any ligand treatment) in ΔPDZ-MAS relative to the WT after normalizing for cell surface expression. The ligand dose-responses of ΔPDZ-MAS in (C) IP1 and (D) calcium functional assays compared to WT. (E) The calcium flux kinetics (t1/2) upon treatment with NPFF and AR-agonist compared to WT. The EC50 or IC50 values are also shown in panels C and D while t1/2 values are shown in panel E. The relative EC50, IC50 and t1/2 values as compared to WT (fold increase) are shown in parentheses. Data for WT and the experimental methodology for ELISA, IP1 and calcium functional assays have been reported previously [23]; Values are mean±SEM from at least two independent experiments; Statistical significance (t-test)—*p<0.05.
Fig 5.
Interaction of MAS and PDZ proteins in HEK293 cells.
Western blots of Cytoplasmic (Cy), detergent insoluble membrane (iM) and detergent soluble (sM) fractions from (A) Vector alone and WT-MAS and (B) WT-MAS and ΔPDZ-MAS transfected HEK293 cells. The western blots were initially probed with anti-c-myc antibody and then stripped and re-probed multiple times with different primary antibodies. The data for WT-MAS in panels (A) and (B) are from two independent experiments. (C) The pan-MAGUK signal in the western blots in panels (A) and (B) was as quantified using the Odyssey® Infrared Imaging System (LI-COR Biosciences, Lincoln, NE) and the ratio of pan-MAGUK band intensities of iM to sM fractions is shown. (D) The sM and iM fractions from WT-MAS and ΔPDZ-MAS were resolved on SDS-PAGE gels and the regions between ~75kDa to 150kDa (boxed regions) corresponding to the regions positive for pan-MAGUK signals (see panel B) were subjected for MS analysis to identify the PDZ proteins that were differentially enriched in different fractions.
Table 3.
List of PDZ proteins identified to be significantly present in the membrane fractions of WT and ΔPDZ from areas highlighted by boxes on the protein gel in Fig 5B.
Fig 6.
Hypothetical ‘MAS-signalosome’ model.
The figure shows hypothetical assembly of PDZ and non-PDZ proteins leading to the formation of a signalosome. The ‘MAS-signalosome’ can potentially initiate novel signaling pathways or modulate different signaling or post-signaling events by MAS or other receptors.