title: Functional analysis of the nucleotide binding domain of membrane-associated guanylate kinases.
abstract: Membrane-associated guanylate kinases (MAGUKs) regulate cellular adhesion and signal transduction at sites of cell-cell contact. MAGUKs are composed of modular protein-protein interaction motifs including L27, PDZ, Src homology (SH) 3, and guanylate kinase domains that aggregate adhesion molecules and receptors. Genetic analyses reveal that lethal mutations of MAGUKs often occur in the guanylate kinase domain, indicating a critical role for this domain. Here, we explored whether GMP binding to the guanylate kinase domain regulates MAGUK function. Surprisingly, and in contrast to previously published studies, we failed to detect GMP binding to the MAGUKs postsynaptic density-95 (PSD-95) and CASK. Two amino acid residues in the GMP binding pocket that differ between MAGUKs and authentic guanylate kinase explain this lack of binding, as swapping these residues largely prevent GMP binding to yeast guanylate kinase. Conversely, these mutations restore GMP binding but not catalytic activity to PSD-95. Protein ligands for the PSD-95 guanylate kinase domain, guanylate kinase-associated protein (GKAP) and MAP1A, appear not to interact with the canonical GMP binding pocket, and GMP binding does not influence the intramolecular SH3/guanylate kinase (GK) interaction within PSD-95. These studies indicate that MAGUK proteins have lost affinity for GMP but may have retained the guanylate kinase structure to accommodate a related regulatory ligand.
authors.affiliation: Department of Physiology and Program in Neuroscience, University of California at San Francisco, San Francisco, California 94143-0444, USA.
authors.forename: Olav
authors.initials: O
authors.lastname: Olsen
authors.forename: David S
authors.initials: DS
authors.lastname: Bredt
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D047388:Casein Kinases; D000076122:Disks Large Homolog 4 Protein; D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006157:Guanosine Monophosphate / Q000494:pharmacology; D051528:Guanylate Kinases; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008024:Ligands; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000074407:SAP90-PSD95 Associated Proteins; D017386:Sequence Homology, Amino Acid; D014162:Transfection
chemical_list: D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008164:Luminescent Proteins; C490546:Map1a protein, rat; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; C501451:Mpp2 protein, rat; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D000074407:SAP90-PSD95 Associated Proteins; C033652:postsynaptic density proteins; D049452:Green Fluorescent Proteins; D006157:Guanosine Monophosphate; D005982:Glutathione Transferase; D011494:Protein Kinases; C106868:CASK kinases; D047388:Casein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D009703:Nucleoside-Phosphate Kinase; D051528:Guanylate Kinases