P1R3A (GM): residues R63VSF; PPP1R3B (GL): residues R62VSF; PPP1R3C (R5/PTG): R84VVF; numbering refers to the human counterpart of each and every protein, as outlined by Uniprot]. We demonstrate that in R6 this motif is crucial to preserve the ability from the protein to induce glycogen synthesis. We also present data indicating that R6 contains a area (W267DNND) involved in binding to glycogenic substrates (GS and GP) (Fig 7). This motif is well conserved in other glycogen targeting subunits [PPP1R3A (GM): residues W219SNNN; PPP1R3B (GL): residues W222DSNR; PPP1R3C (R5/PTG): residues W246DNND] but, to our understanding, only within the case of R6 (this work) and in the case of murine R5/PTG [11] and rabbit GM [10], the functionality of this motif in glycogen synthesis has been evaluated. Consistent with its binding properties, this domain is well exposed to the solvent in both the model we present for R6 (Fig 1B) and within the case of GL (pdb accession code: 2EEF). Our work indicates that R6 includes an added region regulating binding to PP1 glycogenic substrates, which is crucial for the glycogenic activity from the targeting subunit: R256VHF (Fig 7). Although not conserved, a comparable area is present in R5/PTG (K231IEF) and GL (R207MEF) (Fig 1A), raising the possibility that it could form part of an extended location of speak to with PP1 glycogenic substrates that would comprise in 
R6 from R256VHF towards the W267DNND area (Fig 1B). Alternatively, and because the hydrophobic residues (Val and Phe) within the R256VHF domain (mutated inside the R6-RAHA type which has impaired interaction with glycogenic substrates) are buried and not exposed for the solvent in the structural model we present (Fig 1B) and also in the 10205015 case of GL (pdb accession code: 2EEF), they could participate in intramolecular contacts, so when they are changed to Ala residues a conformational modification in this region could come about, affecting the binding properties in the W267DNND motif that is definitely nearby (Fig 1B). In any case, and in agreement using a earlier report [11], our benefits indicate that binding of R6 to PP1c and PP1 glycogenic substrates are independent processes, even though impairment of any of them outcomes inside the very same loss of functionality in glycogen homeostasis. We also report a novel functional domain in R6 involved in binding to 14-3-3 proteins (RARS74LP) (Fig 7). This domain is absent in other glycogen targeting subunits as GL or R5/ PTG (Fig 1A); in actual fact, we had been not able to detect any interaction of those glycogenic subunits with 14-3-3 proteins by yeast two-hybrid (not shown). Mutation inside the vital Ser74 residue of R6 disrupts binding to 14-3-3 proteins, despite the fact that it still allows the binding to PP1c and to PP1 glycogenic substrates. As distinctive mass spectrometry analyses indicate that Ser74 is 5,7-Dihydroxy-4′-methoxyflavone structure phosphorylated in vivo ([32], [33], [34], [35]), binding of R6 to 14-3-3 proteins could safeguard this web site from dephosphorylation and avoid the fast degradation on the protein by the lysosomal pathway. It’s surprising that a form that cannot be phosphorylated at Ser74 (and therefore it can’t bind 14-3-3 proteins) presented hyper-glycogenic properties. In our hands, the expression of even very low amounts in the R6-S74A protein induced the production of high levels of glycogen. Possibly this R6-S74A protein has far better dephosphorylation kinetics against glycogenic enzymes or, alternatively, it could influence other unknown regulators of glycogen synthesis. The fact that the R6-S74A kind is quite unst
