Fic GEFs [66]. Cells 2021, ten, x FOR PEER Review of 14 Nevertheless, the CBD of RAPGEF2/RAPGEF6 doesn’t contain conserved residues6important for cyclic nucleotide binding [67] and just isn’t responsive to cAMP or other nucleotides [68].Figure 3. Phylogenetic analyses in the CBD of PKA, PKG and EPAC1, EPAC2, RAPGEF two and six. (a) Unrooted cladogram of Figure three. Phylogenetic analyses in the CBD of PKA, PKG (b) Rooted phylogram of two and six. (a) Unrooted cladogram CBD of PKA, PKG and EPAC1, EPAC2, RAPGEF 2 and six.and EPAC1, EPAC2, RAPGEFchordate CBD of EPAC1. (c) Rooted of CBD of PKA, PKG and EPAC1, bars: 0.01 represents 1 (b) Rooted phylogram of phylogram of chordate EPAC2. ScaleEPAC2, RAPGEF 2 and 6. aa substitution per 100.chordate CBD of EPAC1. (c) Rootedphylogram of chordate EPAC2. Scale bars: 0.01 represents 1 aa substitution per 100.Cells 2021, 10,six ofA BLAST search using the GEF domain of EPAC1 and EPAC2 led to the identification of 897 sequences across the RAPGEF family members from non-repetitive species (Supplementary data 3). An unrooted cladogram of GEF domain of RAPGEF was generated with MSA (Figure 4a). EPAC GEF phylogeny nevertheless followed the general trend of animal taxonomy as shown within the full-length EPAC tree (Figure 2a) with the constraints in the larger RAPGEF families. EPAC1 and EPAC2 GEFs have been extra closely clustered with every single other amongst all RAPGEF members of your loved ones. It appeared that the GEF domain of RAPGEFs is originated from RAPGEF1, which contained species which can be far more primitive. GEF domain Cells 2021, 10, x FOR PEER Assessment RAPGEF2 and RAPGEF6 type a separate group, leaving EPAC1, EPAC2 and RAPGEF5 7 of 14 of clustered within a Balovaptan medchemexpress somewhat closely connected group.Figure four. Phylogenetic analyses from the GEF of RAPGEF1-6. (a) Unrooted cladogram in the GEF RAPGEF1-6. (b) Rooted Figure 4. Phylogenetic analyses of the GEF of RAPGEF1-6. (a) Unrooted cladogram with the GEF ofof RAPGEF1-6. (b) Rooted phylogram with the mammalian GEF of EPAC1. (c) Rooted phylogram of the mammalian GEF of EPAC2. Scale bars: 0.01 phylogram with the mammalian GEF of EPAC1. (c) Rooted phylogram with the mammalian GEF of EPAC2. Scale bars: 0.01 represents 1 aa substitution per 100. represents 1 aa substitution per 100.three.three. Identification of Isoform-Specific Sequence Motifs Certainly one of our objectives should be to search for distinctive sequence signatures which can differentiate the two EPAC isoforms. Ideally, such a sequence motif could be hugely conserved inside its personal isoform among all species, but absent in the other isoform. To attain this purpose, we aligned sequences for both EPAC isoforms in all species, and at each amino acid position determined (1) regardless of whether the aligned human residue for EPAC1 and EPAC2 was theCells 2021, ten,7 ofWe could clearly observe that EPAC1 GEF originates at a later root than the origins of EPAC2 GEF in primitive species, parallel to chordate EPAC2 GEF sequences. Rooted phylograms of mammalian EPAC1 and EPAC2 GEF, drawn towards the exact same scale, showed that EPAC1 GEF are more divergent than EPAC2 counterparts (Figure 4b,c). We compared the sequence identity of GEFs once more amongst humans and zebrafish, and we located that EPAC2 GEFs have a sequence identity of 83.6 , although EPAC1 GEFs have an identity of 66.three . As anticipated, the mammalian EPAC1 GEF tree featured exactly the same taxonomy groups (Figure 4b), as in comparison to the tree derived in the full-length EPAC1 sequence (Figure 2b). On the other hand, the mammalian EPAC2 GEF tree (Figure 4c) contained the Etomoxir Purity & Documentation marsupial taxa, a group evolut.