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Molecular Recognition of the Protein Phosphatase 1 Glycogen Targeting Subunit by Glycogen Phosphorylase

2008; Elsevier BV; Volume: 283; Issue: 14 Linguagem: Inglês

10.1074/jbc.m706612200

1083-351X

Alexander Pautsch, N. Stadler, Oliver Wissdorf, Elke Langkopf, Werner Moreth, Rüdiger Streicher,

Glycosylation and Glycoproteins Research

Disrupting the interaction between glycogen phosphorylase and the glycogen targeting subunit (GL) of protein phosphatase 1 is emerging as a novel target for the treatment of type 2 diabetes. To elucidate the molecular basis of binding, we have determined the crystal structure of liver phosphorylase bound to a GL-derived peptide. The structure reveals the C terminus of GL binding in a hydrophobically collapsed conformation to the allosteric regulator-binding site at the phosphorylase dimer interface. GL mimics interactions that are otherwise employed by the activator AMP. Functional studies show that GL binds tighter than AMP and confirm that the C-terminal Tyr-Tyr motif is the major determinant for GL binding potency. Our study validates the GL-phosphorylase interface as a novel target for small molecule interaction. Disrupting the interaction between glycogen phosphorylase and the glycogen targeting subunit (GL) of protein phosphatase 1 is emerging as a novel target for the treatment of type 2 diabetes. To elucidate the molecular basis of binding, we have determined the crystal structure of liver phosphorylase bound to a GL-derived peptide. The structure reveals the C terminus of GL binding in a hydrophobically collapsed conformation to the allosteric regulator-binding site at the phosphorylase dimer interface. GL mimics interactions that are otherwise employed by the activator AMP. Functional studies show that GL binds tighter than AMP and confirm that the C-terminal Tyr-Tyr motif is the major determinant for GL binding potency. Our study validates the GL-phosphorylase interface as a novel target for small molecule interaction. Diabetes is one of the major public health problems. Approximately 194 million people worldwide, or 5.1%, in the age group 20–79 were estimated to have diabetes in 2003. This estimate is expected to increase to some 333 million, or 6.3% of the adult population, by 2025 (1Gan D.E. 3rd Ed. Diabetes Atlas. International Diabetes Federation, Brussels, Belgium2007Google Scholar). Type 2 diabetes, the most common form of diabetes is characterized by defects in insulin secretion, insulin resistance, and elevated hepatic glucose production. Both increased gluconeogenesis and increased glycogenolysis contribute to excessive hepatic glucose output despite hyperglycemia (2DeFronzo R.A. Ann. Intern. Med. 1999; 131: 281-303Crossref PubMed Scopus (1030) Google Scholar). Several novel pharmacological strategies are aiming to treat hyperglycemia by normalizing or increasing depleted glycogen stores (3Cohen P. Nat. Rev. Mol. Cell Biol. 2006; 7: 867-873Crossref PubMed Scopus (179) Google Scholar). For example, drug discovery has focused on competitive as well as allosteric inhibition of glycogen phosphorylase activity (4Henke B.R. Sparks S.M. Mini. Rev. Med. Chem. 2006; 6: 845-857Crossref PubMed Scopus (88) Google Scholar). Glycogen phosphorylase (GP) 2The abbreviations used are: GPglycogen phosphorylaseGLglycogen targeting subunit of PP1PP1protein phosphatase 1SPAscintillation proximity assayhlGPhuman liver GPBES2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid. is an important allosteric enzyme in carbohydrate metabolism that catalyzes phosphorolysis of an α-1,4-glycosidic bond of glycogen to glucose-1-phosphate. In humans there are three GP isoforms (liver, muscle, and brain GP), which are named after the tissues where they are predominantly expressed. Glycogen phosphorylase is a homodimer that cycles between two conformations: active (R) and inactive (T) state. Phosphorylation of Ser14 by phosphorylase kinase and active site as well as allosteric binders modulate the equilibrium between both states (5Johnson L.N. FASEB J. 1992; 6: 2274-2282Crossref PubMed Scopus (261) Google Scholar), but the isozymes differ in their responsiveness to regulatory mechanisms. In the liver, phosphorylation is the major regulator of GP activation. Conversion of unphosphorylated liver GPb to phosphorylated GPa fully activates the enzyme. AMP stimulates liver GPb by 10–20%, whereas it does not further activate GPa (6Kobayashi M. Soman G. Graves D.J. J. Biol. Chem. 1982; 257: 14041-14047Abstract Full Text PDF PubMed Google Scholar). In contrast, AMP activates the unphosphorylated muscle isoform to 80% of the maximal activity and increases the activity of phosphorylated muscle GPa by a further 10%. Crystallographic studies have shown endogenous and synthetic modulators bound to four major sites (see Fig. 1a): active site (7Rath V.L. Ammirati M. LeMotte P.K. Fennell K.F. Mansour M.N. Danley D. sE. Hynes T.R. Schulte G.K. Wasilko D.J. Pandit J. Mol. Cell. 2000; 6: 139-148Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), purine site (8Ekstrom J.L. Pauly T.A. Carty M.D. Soeller W.C. Culp J. Danley D.E. Hoover D.J. Treadway J.L. Gibbs E.M. Fletterick R.J. Day Y.S. Myszka D.G. Rath V.L. Chem. Biol. 2002; 9: 915-924Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), central cavity (9Rath V.L. Ammirati M. Danley D.E. Ekstrom J.L. Gibbs E.M. Hynes T.R. Mathiowetz A.M. McPherson R.K. Olson T.V. Treadway J.L. Hoover D.J. Chem. Biol. 2000; 7: 677-682Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), and allosteric AMP site (10Klabunde T. Wendt K.U. Kadereit D. Brachvogel V. Burger H.J. Herling A.W. Oikonomakos N.G. Kosmopoulou M.N. Schmoll D. Sarubbi E. von R.E. Schonafinger K. Defossa E. J. Med. Chem. 2005; 48: 6178-6193Crossref PubMed Scopus (66) Google Scholar, 11Barford D. Hu S.H. Johnson L.N. J. Mol. Biol. 1991; 218: 233-260Crossref PubMed Scopus (222) Google Scholar). glycogen phosphorylase glycogen targeting subunit of PP1 protein phosphatase 1 scintillation proximity assay human liver GP 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid. Important for glycogen metabolism is the strong reciprocal control between GPa and glycogen synthase activity. Activation of glycogen synthase via its phosphatase (protein phosphatase 1 (PP1)) can be allosterically inhibited by binding of GPa (12Alemany S. Cohen P. FEBS Lett. 1986; 198: 194-202Crossref PubMed Scopus (69) Google Scholar) to GL (13Moorhead G. MacKintosh C. Morrice N. Cohen P. FEBS Lett. 1995; 362: 101-105Crossref PubMed Scopus (82) Google Scholar), a glycogen targeting subunit of PP1. PP1 in turn suppresses GP and phosphorylase kinase activities through dephosphorylation. Glycogen targeting subunits bind to PP1, modulate its activity toward substrates, localize it to specific cellular sites, and are proposed to function as a scaffold for the assembly and regulation of glycogen metabolizing enzymes. There is an increasing number of glycogen-targeting subunits. So far, seven family members of glycogen-targeting subunits are described in humans: GM (PPP1R3A) (14Chen Y.H. Hansen L. Chen M.X. Bjorbaek C. Vestergaard H. Hansen T. Cohen P.T. Pedersen O. Diabetes. 1994; 43: 1234-1241Crossref PubMed Scopus (59) Google Scholar), GL (PPP1R3B) (15Doherty M.J. Moorhead G. Morrice N. Cohen P. Cohen P.T. FEBS Lett. 1995; 375: 294-298Crossref PubMed Scopus (140) Google Scholar), R5/PTG (PPP1R3C) (16Doherty M.J. Young P.R. Cohen P.T. FEBS Lett. 1996; 399: 339-343Crossref PubMed Scopus (91) Google Scholar, 17Printen J.A. Brady M.J. Saltiel A.R. Science. 1997; 275: 1475-1478Crossref PubMed Scopus (243) Google Scholar), R6 (PPP1R3D) (18Armstrong C.G. Browne G.J. Cohen P. Cohen P.T. FEBS Lett. 1997; 418: 210-214Crossref PubMed Scopus (85) Google Scholar), PPP1R3E (19Munro S. Ceulemans H. Bollen M. Diplexcito J. Cohen P.T. FEBS J. 2005; 272: 1478-1489Crossref PubMed Scopus (57) Google Scholar), and PPP1R3F and PPP1R3G (20Ceulemans H. Stalmans W. Bollen M. Bioessays. 2002; 24: 371-381Crossref PubMed Scopus (127) Google Scholar). Mutational analysis of the rat liver targeting subunit GL has identified three separate regions that are responsible for binding to PP1 (residues 59–94), glycogen (residues 94–257), and phosphorylase a (residues 269–284 at the GL C terminus) (21Armstrong C.G. Doherty M.J. Cohen P.T. Biochem. J. 1998; 336: 699-704Crossref PubMed Scopus (80) Google Scholar). The GL C-terminal region is unique in GL and absent in other glycogen targeting subunits but is conserved between rodent and human. Pharmacological inhibition of the interaction of phosphorylase a with GL could provide a novel mechanism to lower blood glucose levels by inducing the dephosphorylation and activation of glycogen synthase (3Cohen P. Nat. Rev. Mol. Cell Biol. 2006; 7: 867-873Crossref PubMed Scopus (179) Google Scholar). Here we show that the GL C-terminal region structurally and functionally mimics AMP binding to human liver glycogen phosphorylase (hlGP). Using x-ray crystallography we identify the C terminus of GL bound in the allosteric regulator site of hlGP. Using functional assays we map binding contributions of the GL peptide and show that it activates phosphorylase b, in vitro. Protein Production—Human liver glycogen phosphorylase was prepared as described (7Rath V.L. Ammirati M. LeMotte P.K. Fennell K.F. Mansour M.N. Danley D. sE. Hynes T.R. Schulte G.K. Wasilko D.J. Pandit J. Mol. Cell. 2000; 6: 139-148Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Briefly, full-length hlGPa was expressed in insect cells and purified using copper-chelating, anion exchange, and size exclusion chromatography. hlGPb was expressed in Escherichia coli and purified analogously. Crystallization and Structure Determination—Crystals were obtained at 20 °C in 1 + 1 μl hanging drops from hlGPa concentrated to 8 mg/ml in 20 mm BES, pH 6.7, 1 mm EDTA, 0.5 mm dithiothreitol, 50 mm glucose, 0.5 mm AMP over a reservoir of 0.1 m Tris, pH 8.5, 7–8% (w/v) polyethylene glycol 8000. Macroseeding improved crystal size and reproducibility. The GL complex was obtained by soaking crystals for 24 h in reservoir supplemented with 1 mm GL-Cterm peptide (see below). For cryoprotection the mother liquor was incrementally exchanged to 0.1 m Tris, pH 8.5, 20% glycerol, 20% polyethylene glycol 8000, 1 mm GL-Cterm peptide. The crystals were then flash frozen in a 100 K nitrogen stream. The diffraction data were collected on the PX-1 beamline at the SLS (Villigen, CH) and processed with XDS (22Kabsch W. J. Appl. Crystallogr. 1993; 26: 795-800Crossref Scopus (3243) Google Scholar) (see Table 1). The complex structure was solved using difference Fourier methods with the coordinates of hlGPa-AMP (Protein Data Bank accession code 1FA9) as a template. Following rigid body refinement the (Fo – Fc)αcalc maps included easily interpretable electron density for the bound ligand. Restrained refinement was performed with REFMAC (23Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar) and BUSTER (Global Phasing Ltd.) iterated with model building in COOT (24Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23628) Google Scholar). The final model has been completed to residues 4–838 of hlGPa, residues 281–284 of GL, and 154 water molecules. 98.5% of residues are in the most favored and additionally allowed regions of the Ramachandran plot, and 0.3% are in disallowed regions. Several regions were not well defined in the final electron density and are omitted from the final model (residues 162–165, 251–262, 282–285, and 317–322). The final model statistics are listed in Table 1. Surface calculations were made with the EBI PISA server. The figures were prepared using PyMOL (DeLano Scientific LLC).TABLE 1Data collection and refinement statisticsData setaThe values in parantheses are for the highest resolution shell.hlGPa-GLSpace groupP3121Cell dimensions a, c (Å)124.0, 125.7Resolution (Å)20-2.56 (2.71-2.56)Observed reflections145576Unique reflections35387Completeness (%)98.6 (99.9)Rsym (%)bRsym = ∑hkl∑i|Ii — 〈I〉|/∑hkl|∑iIi.7.5 (47.1)〈I/σ(I)〉12.5 (3.1)Rwork/RfreecRwork = ∑hkl∥Fobs|— k|Fcalc∥/∑hkl||Fobs|, and Rfree was calculated using 5% of data excluded from refinement.20.3/27.6No. of refined atoms6734Average B-factor (Å2)64.9Root mean square deviation Bond length (Å)0.008 Bond angles (Å)1.12a The values in parantheses are for the highest resolution shell.b Rsym = ∑hkl∑i|Ii — 〈I〉|/∑hkl|∑iIi.c Rwork = ∑hkl∥Fobs|— k|Fcalc∥/∑hkl||Fobs|, and Rfree was calculated using 5% of data excluded from refinement. Open table in a new tab Coordinates—The coordinates and structure factors have been deposited in the Protein Data Bank (Protein Data Bank accession code 2QLL). Peptides—Synthetic peptides were purchased from Thermo-Electron (Ulm, Germany). The peptides were delivered as 5-mg lyophilized aliquots and resuspended in dimethyl sulfoxide before use. The sequence of the 16mer GL C-terminal peptide is NH2-PEWPSYLGYEKLGPYY-COOH (Gl-Cterm). A biotin-labeled 17-mer GL-peptide, biotin-FPEW-PSYLGYEKLGPYY-COOH (GL probe) was synthesized by Interactiva (Ulm, Germany). SPA Assay for GL/hlGPa Interaction—A SPA was used to measure the interaction between 33P-radiolabeled hlGPa and a biotin-labeled GL-peptide (GL probe). A one-step phosphorylation reaction by phosphorylase kinase (Sigma) transformed hlGPb into 33P-radiolabeled hlGPa. The incubation reaction contained test compound, 5 μgof 33P-hlGPa, 50 pmol of GL probe, 0.4 mg SPA-Beads (streptavidin-SPA beads; Amersham Biosciences, RPNQ 0007) in test buffer (50 mm Tris, 5 mm EDTA, pH 7.5)/well in a 384-well format. Scintillation was measured after overnight incubation at room temperature. Triplicate determinations were made in all of the binding experiments. rmGPb Activation Assay—Activation of enzymatic activity of rabbit muscle glycogen phosphorylase b (rmGPb from Sigma) was measured in direction of glycogen degradation by coupling glucose-1-phosphate production to NADP consumption (25Serrano F.S.J. Lopez J.L.S. Martin O.G. Int. J. Biochem. Cell Biol. 1995; 27: 911-916Crossref PubMed Scopus (5) Google Scholar). Activation experiments were performed as triplicate determinations. All of the reagents and enzymes were purchased from Sigma. Structure of GL Bound to hlGPa—To identify the molecular basis of GL binding, we diffused a peptide comprising the last 16 residues of GL (Gl-Cterm, 269GL-284GL; numbering refers to the sequence of rat GL) into crystals of hlGPa and determined the crystal structure of the resulting hlGPa-GL complex (Fig. 1 and Table 1). Only the terminal four amino acids (residues 281GL–284GL, Gly-Pro-Tyr-Tyr) were visible in difference electron density maps (Fig. 2A), whereas the remainder of the peptide was disordered. The GL-binding site is located at the subunit interface and overlaps with the binding site for the allosteric regulator AMP (Fig. 2C). The binding site is ∼14 Å from the Ser14 phosphorylation site, 32 Å from the catalytic site, and 25 Å from the central cavity where the allosteric inhibitor CP-403700 binds (9Rath V.L. Ammirati M. Danley D.E. Ekstrom J.L. Gibbs E.M. Hynes T.R. Mathiowetz A.M. McPherson R.K. Olson T.V. Treadway J.L. Hoover D.J. Chem. Biol. 2000; 7: 677-682Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). The allosteric site is lined by helices α2 and α8, and a short strand, β7, and is closed by the cap′ region (residues 36′-47′; the prime symbol refers to residues from the second chain of the homodimer) from the other subunit. GL binds in an U-shaped, hydrophobically collapsed conformation where it exploits numerous polar and hydrophobic contacts to hlGPa (Fig. 2, A and B). The GL peptide protrudes deep into the pocket, and 78% (542 Å2) of its total solvent-accessible surface becomes buried upon binding. The terminal carboxylate group mimics the AMP phosphate by addressing an arginine-rich region that also comprises the binding site for the allosteric effector phosphate. The Tyr284GL side chain points into the ribose binding region, where it forms hydrophobic contacts to Trp67, Gln71, Tyr75, and Val45′ and a strong hydrogen bond to Asp42′. Tyr283GL protrudes into a region that is not contacted by AMP. Its phenol group is incorporated in a hydrogen bonding network with Asp306 and Arg242. We further observe an edge-to-face interaction with Phe196 and stacking of the phenol ring against the Arg309 guanidinium group. The pyrrolidine of Pro282GL is packed through hydrophobic stacking interactions between the two subunits: the phenolic side chain of Tyr75 from subunit A and two residues from the subunit B cap region (carbonyl oxygen of Asn44′ and CG2 of Val45′). In this it partially mimics the stacking interactions of the AMP adenine between Tyr75 and Asn44′ (Fig. 2C). With Gly281GL, the GL peptide leaves the pocket and protrudes into the solvent. The last notable interaction is a van der Waals' contact of its carbonyl oxygen to Cβ of Ala313. Conformational Changes—Prior to soaking with GL, hlGPa was crystallized in the active, AMP-bound conformation (7Rath V.L. Ammirati M. LeMotte P.K. Fennell K.F. Mansour M.N. Danley D. sE. Hynes T.R. Schulte G.K. Wasilko D.J. Pandit J. Mol. Cell. 2000; 6: 139-148Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), and on the subunit level there are no large conformational changes upon GL binding. The backbone atoms of hlGP-GL and hlGP-AMP align with an root mean square deviation of 0.48 Å. However, the side chains of three residues undergo local reorganization to accommodate the Tyr283GL phenol (Phe196 and Arg309) or adapt to the terminal carboxylic acid (Tyr155). Moreover, there is a subtle change in quaternary structure as the two subunits rotate ∼2° toward each other (not shown). Within the allosteric pocket this translates to a 0.7-Å shift of the cap′ loop (Fig. 2C) to improve contacts to Pro282GL. Phosphorylase a is a potent allosteric inhibitor of the PP1-GL complex (12Alemany S. Cohen P. FEBS Lett. 1986; 198: 194-202Crossref PubMed Scopus (69) Google Scholar, 21Armstrong C.G. Doherty M.J. Cohen P.T. Biochem. J. 1998; 336: 699-704Crossref PubMed Scopus (80) Google Scholar), whereas the inactive phosphorylase b is not (12Alemany S. Cohen P. FEBS Lett. 1986; 198: 194-202Crossref PubMed Scopus (69) Google Scholar). To address the molecular basis of this finding we compared hlGPa-GL to a complex with the inhibitor N-acetyl-β-d-glucopyranosylamine (hlGPa-GlcNac), which defines the inactive (T state) conformation (7Rath V.L. Ammirati M. LeMotte P.K. Fennell K.F. Mansour M.N. Danley D. sE. Hynes T.R. Schulte G.K. Wasilko D.J. Pandit J. Mol. Cell. 2000; 6: 139-148Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Upon inhibitor binding the subunits rotate "outward" by ∼7°, and the dimer interface (including the allosteric site) is remodeled (Fig. 2D). Consequently, several interactions that stabilize the GL complex are lost (for example: cap′ interactions, stacking with Tyr75, hydrogen bond to Tyr155) or would lead to steric conflicts (for example: Arg309, Phe196, and Asp306). Functional Analysis of GL Recognition by Glycogen Phosphorylase—After solving the complex structure, we sought to elucidate the determinants that are crucial for conferring binding potency. We developed a SPA to measure the competitive binding of a biotinylated GL peptide to 33P-labeled hlGPa. The dissociation constant (KD) for the interaction of the GL peptide with hlGP was determined from a saturation binding curve (Fig. 3A) to be 145 nm. Next, we measured the competitive displacement of the GL probe through several peptide variants as well as AMP. Representative binding curves for the displacement of the GL probe by Gl-Cterm and AMP are shown in Fig. 3B. The apparent IC50 values of the different peptides (Table 2) allow an assessment of the relative importance of individual amino acids for binding. We find that the hexa- to tetrapeptides inhibit the hlGPa-GL interaction in a similar range as the complete Gl-Cterm. This is in agreement with the structure, because residues 269GL–280GL are not ordered and therefore are not supposed to contribute strongly to binding. An approximately 8-fold drop in affinity is observed once truncation includes Gly281GL, and no binding could be detected when only the two terminal tyrosines were probed. Likewise, mutation of either tyrosine to alanine or truncation of Tyr284GL was deleterious for binding. Again, this is in good agreement to the numerous contacts these residues involve in the structure. AMP is not a good competitor and inhibits the hlGPa-GL interaction only with an IC50 of 21 μm, roughly 10 times weaker than Gl-Cterm.TABLE 2Functional analysis of ligand bindingLigandGL displacement (IC50)aThe values are the means of three independent measurements unless noted otherwise. Standard deviations are shown as ±S.D. Activation of rmGPb through GL-Cterm was performed as a single experiment only.rmGPb activationaThe values are the means of three independent measurements unless noted otherwise. Standard deviations are shown as ±S.D. Activation of rmGPb through GL-Cterm was performed as a single experiment only.μm% control/30 μm ligandGL-CtermbThe values are the means of two independent measurements.2.3 ± 0.8104KLGPYY5.8 ± 1.721 ± 5.2LGPYY5.4 ± 1.023 ± 8.3GPYY4.1 ± 1.129 ± 7.2PYY18.7 ± 4.13 ± 0.0YYbThe values are the means of two independent measurements.>300GPYAbThe values are the means of two independent measurements.>300GPAYbThe values are the means of two independent measurements.>300GPYbThe values are the means of two independent measurements.>300AMPbThe values are the means of two independent measurements.21.0 ± 3.9100a The values are the means of three independent measurements unless noted otherwise. Standard deviations are shown as ±S.D. Activation of rmGPb through GL-Cterm was performed as a single experiment only.b The values are the means of two independent measurements. Open table in a new tab We then tested the ability of GL derivatives to activate the inactive rabbit muscle phosphorylase b (rmGPb; Table 2) and compared it with AMP-induced activation (6Kobayashi M. Soman G. Graves D.J. J. Biol. Chem. 1982; 257: 14041-14047Abstract Full Text PDF PubMed Google Scholar). Interestingly, GL peptides are able to activate rmGPb. Activation through Gl-Cterm matches that of AMP at 30 μm, which represents a maximally active concentration. Also the three shorter, tightly binding peptides still activate rmGPb between 20 and 30%. GL as an AMP Mimic—In vitro binding studies have shown that AMP is able to inhibit the specific interaction between phosphorylase a and recombinant GL protein (15Doherty M.J. Moorhead G. Morrice N. Cohen P. Cohen P.T. FEBS Lett. 1995; 375: 294-298Crossref PubMed Scopus (140) Google Scholar). The structural analysis of the GPa-GL complex revealed that the very C terminus of GL binds in an AMP-competitive fashion, mimicking many of the nucleotides interactions. Additional contacts (mostly provided by Tyr283GL) result in a 10-fold tighter binding compared with AMP. The improved potency might be necessary to counterbalance the high cellular AMP levels. Phosphorylase itself can accommodate GL smoothly, without undergoing any larger conformational changes. A structural comparison between active and inactive states of hlGP explains why the latter cannot directly accommodate GL and consequently is not effective as an inhibitor of PP1 (12Alemany S. Cohen P. FEBS Lett. 1986; 198: 194-202Crossref PubMed Scopus (69) Google Scholar). Nevertheless, GL peptides are able to activate muscle GPb. Because GL cannot directly bind to GPb in its inactive (T state) conformation, we propose that the peptide acts analogous to AMP (5Johnson L.N. FASEB J. 1992; 6: 2274-2282Crossref PubMed Scopus (261) Google Scholar) and shifts the equilibrium between active and inactive conformations toward the R state. The hlGP-GL Interface as a Drug Target—Inhibiting the interaction of phosphorylase a and GL has the potential to block the allosteric inhibition of the PP1/GL activity on glycogen synthase by phosphorylase a. It has been hypothesized that a stimulation of the glycogen synthase pathway via pharmacological dissociation of phosphorylase a from GL could help to normalize hyperglycemia (3Cohen P. Nat. Rev. Mol. Cell Biol. 2006; 7: 867-873Crossref PubMed Scopus (179) Google Scholar). We identified the AMP site as a high affinity binding site for GL. Our data indicate that it might be feasible to antagonize binding of GL protein to GPa with small molecules. Because in humans GL is expressed in liver and muscle cells, glycogen synthesis might be activated in both tissues. Increasing GL activity by overexpression was found in cultured primary human myotubes (26Montori-Grau M. Guitart M. Lerin C. Andreu A.L. Newgard C.B. Garcia-Martinez C. Gomez-Foix A.M. Biochem. J. 2007; 405: 107-113Crossref PubMed Scopus (20) Google Scholar) as well as in primary hepatocytes (27Gasa R. Jensen P.B. Berman H.K. Brady M.J. Paoli-Roach A.A. Newgard C.B. J. Biol. Chem. 2000; 275: 26396-26403Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) to stimulate glycogen synthase activity and to exhibit a high glycogenic effect. The effects of increasing activity of different glycogen targeting subunits by hepatic overexpression have also been studied in animal models (28Gasa R. Clark C. Yang R. Paoli-Roach A.A. Newgard C.B. J. Biol. Chem. 2002; 277: 1524-1530Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 29Yang R. Newgard C.B. J. Biol. Chem. 2003; 278: 23418-23425Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Despite the profound effects of hepatic GL overexpression on glycogen stores in the livers of these animal models, no or only modest and transient effects on hyperglycemia are reported. This is in contrast to the improved glycemic situation following the expression of GMΔC, a truncated version of the muscle-targeting subunit in diabetic rats. The difference was explained by a larger increment in hepatic glycogen storage under oral glucose tolerance test with GMΔC overexpression than with GL overexpression. Higher glycogen stores even in the fasted state following GL overexpression were discussed as indicative for reduced glycogenolytic sensitivity. Therefore, the potential in vivo profile of an inhibitor of GL binding to GP with respect to effects on glycogenic and glycogenolytic pathways is hard to predict. It is clear that our small peptides might not exhibit the ideal profile, because they behave like AMP with respect to rmGPb activation. Increasing GP activity might counterbalance the stimulation of glycogen synthesis, thus neutralizing potential beneficial effects. Therefore, drug development strategies should aim for compounds lacking GP activating properties. We thank Katja Mück for initial support in protein purification; Petra Wieland and Rene Schiller for assistance with the biochemical assays; Stefan Hörer, Herbert Nar, and Stefan Kauschke for discussions; and Clemens Schulze-Briese and the beamline staff of SLS-PX1 for support during data collection.