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. Author manuscript; available in PMC: 2016 May 1.

Published in final edited form as: Cell Signal. 2015 Feb 12;27(5):908–922. doi: 10.1016/j.cellsig.2015.02.003

(A) Schematic presentation of human PDE4 isoforms. As a note, rat long isoform PDE4A5 is the homolog of human PDE4A1, and rat super-short isoform PDE4A1 is the homolog of human PDE4A4. (B) UCR1C and UCR2 alignment in human PDE4 long isoforms. Arrow indicates the critical residues responsible for UCR1-UCR2 interaction. As a note, UCR1C and UCR2 are 100% conserved among different long isoforms from the same genes. Thus, representative long isoforms for each PDE4 genes (PDE4D3, PDE4B3, PDE4A1 and PDE4C2) were used for alignment. (C) Activation mode of PDE4 long isoforms: UCR1 binds UCR2 via electrostatic interactions between residues Arg98, and Arg101 of UCR1C and residues Glu146, Glu147, Asp149 of UCR2N in human PDE4D3. UCRs form a regulatory module, where UCR2 itself exerts an autoinhibitory effect on PDE activity. Phosphorylation as well as interaction of UCRs with lipids and proteins [PA/PS, XAP2, DISC1 and SH3 domains (Lyn and Fyn)] modulates PDE4 activity. Thus, we hypothesize that UCR1C (amino acids 81-136, in gray) binds and modulates PDE4 long isoforms. (D) Interaction of UCR1 and UCR1C with PDE4D3. HEK293T cells were co-transfected with VSV-PDE4D3 and FLAG-UCR1 expression constructs. Forty-eight hours later, cell lysates were harvested for immunoprecipitation (IP) with FLAG antibody. Ponceau S staining shows the successful IP (middle panels). UCR1C binding to PDE4D3 was determined by measuring the amount of VSV-PDE4D3 associated with the immunoprecipitants by Western blot. Both UCR1 (amino acids 17-136) and UCR1C bind PDE4D3. Double mutation of R98A and R101A (UCR1C R/A) abolished interaction. (E) UCR1C, but not UCR1C R/A, enhances activity of PDE4D3. Lysates from 2C were used for in vitro cAMP-PDE assay. Western blot analysis was performed to ensure equal expression of PDE4D3 and expressed of UCR1 mutants and control. The specific activity of PDE4D3 was calculated by normalizing the cAMP-PDE activity to PDE4D3 levels. (F) UCR1C activates long, but not short, PDE4A isoforms. HEK293T cells were co-transfected with GFP-tagged rat PDE4A isoforms and FLAG-UCR1C constructs. The long isoform GFP-PDE4A5 (homolog of human long isoform PDE4A1) and super short-isoform GFP-PDE4A1 (homolog of human super-short isoform PDE4A4) were immunoprecipitated then subjected to in vitro PDE assays and Western analysis. Specific activity was calculated from the cAMP-PDE assay normalized to GFP-PDE4A levels. Expression of UCR1C and its vector control were also shown for similar expression. InStat was used to perform for statistical analysis. A p-value < 0.05 was significant (*) while a p-value > 0.05 was not significant (n.s.). (G–H) UCR1C activates long PDE4B and PDE4C isoforms. HEK293T cells were co-transfected with FLAG-UCR1C constructs and V5-tagged human PDE4B3 (G) or VSV-tagged human PDE4C2 isoforms (H) respectively. The transfected lysates were used for in vitro PDE assays and Western analysis. Specific activity was calculated from the cAMP-PDE assay normalized to PDE4B3 or PDE4C2 respectively. Data represent the mean± s.e.m from 2 independent experiments.

(A) Schematic presentation of human PDE4 isoforms. As a note, rat long isoform PDE4A5 is the homolog of human PDE4A1, and rat super-short isoform PDE4A1 is the homolog of human PDE4A4. (B) UCR1C and UCR2 alignment in human PDE4 long isoforms. Arrow indicates the critical residues responsible for UCR1-UCR2 interaction. As a note, UCR1C and UCR2 are 100% conserved among different long isoforms from the same genes. Thus, representative long isoforms for each PDE4 genes (PDE4D3, PDE4B3, PDE4A1 and PDE4C2) were used for alignment. (C) Activation mode of PDE4 long isoforms: UCR1 binds UCR2 via electrostatic interactions between residues Arg98, and Arg101 of UCR1C and residues Glu146, Glu147, Asp149 of UCR2N in human PDE4D3. UCRs form a regulatory module, where UCR2 itself exerts an autoinhibitory effect on PDE activity. Phosphorylation as well as interaction of UCRs with lipids and proteins [PA/PS, XAP2, DISC1 and SH3 domains (Lyn and Fyn)] modulates PDE4 activity. Thus, we hypothesize that UCR1C (amino acids 81-136, in gray) binds and modulates PDE4 long isoforms. (D) Interaction of UCR1 and UCR1C with PDE4D3. HEK293T cells were co-transfected with VSV-PDE4D3 and FLAG-UCR1 expression constructs. Forty-eight hours later, cell lysates were harvested for immunoprecipitation (IP) with FLAG antibody. Ponceau S staining shows the successful IP (middle panels). UCR1C binding to PDE4D3 was determined by measuring the amount of VSV-PDE4D3 associated with the immunoprecipitants by Western blot. Both UCR1 (amino acids 17-136) and UCR1C bind PDE4D3. Double mutation of R98A and R101A (UCR1C R/A) abolished interaction. (E) UCR1C, but not UCR1C R/A, enhances activity of PDE4D3. Lysates from 2C were used for in vitro cAMP-PDE assay. Western blot analysis was performed to ensure equal expression of PDE4D3 and expressed of UCR1 mutants and control. The specific activity of PDE4D3 was calculated by normalizing the cAMP-PDE activity to PDE4D3 levels. (F) UCR1C activates long, but not short, PDE4A isoforms. HEK293T cells were co-transfected with GFP-tagged rat PDE4A isoforms and FLAG-UCR1C constructs. The long isoform GFP-PDE4A5 (homolog of human long isoform PDE4A1) and super short-isoform GFP-PDE4A1 (homolog of human super-short isoform PDE4A4) were immunoprecipitated then subjected to in vitro PDE assays and Western analysis. Specific activity was calculated from the cAMP-PDE assay normalized to GFP-PDE4A levels. Expression of UCR1C and its vector control were also shown for similar expression. InStat was used to perform for statistical analysis. A p-value < 0.05 was significant (*) while a p-value > 0.05 was not significant (n.s.). (G–H) UCR1C activates long PDE4B and PDE4C isoforms. HEK293T cells were co-transfected with FLAG-UCR1C constructs and V5-tagged human PDE4B3 (G) or VSV-tagged human PDE4C2 isoforms (H) respectively. The transfected lysates were used for in vitro PDE assays and Western analysis. Specific activity was calculated from the cAMP-PDE assay normalized to PDE4B3 or PDE4C2 respectively. Data represent the mean± s.e.m from 2 independent experiments.