Selective Binding of Synapse-associated Protein 97 to GluR-A α-Amino-5-hydroxy-3-methyl-4-isoxazole Propionate Receptor Subunit Is Determined by a Novel Sequence Motif

Abstract

A family of four closely related PDZ domain-containing membrane-associated guanylate kinase homologues (MAGUKs) is involved in the regulation of the amount and functional state of ionotropic glutamate receptors in excitatory synapses. To understand the mechanisms that determine the specificity of these interactions, we examined the structural basis of the highly selective association between the ionotropic GluR subunit GluR-A and synapse-associated protein 97 (SAP97). The C terminus of GluR-A bound to the PDZ domains of SAP97, but not to those of three related MAGUKs, PSD-93, PSD-95, and SAP102. Experiments with single PDZ domains indicated that the strongest contribution was by the second PDZ domain. Unexpectedly, mutation analysis of the GluR-A C terminus revealed that a tripeptide sequence SSG at position ؊9 to ؊11 plays an essential role in this binding, in addition to a C-terminal type I PDZ binding motif (leucine at C terminus and threonine at the ؊2 position). Analysis of the in vitro MAGUK-binding properties of a GluR-D mutant with a one-residue deletion at the C terminus provides further support for the view that an SSG sequence located N-terminally from a type I PDZ binding motif can mediate selective binding to SAP97 and suggest the existence of a novel variation of the PDZ domain-peptide interaction.Synapse-associated protein 97 (SAP97) 1 and the closely related SAP90/PSD-95, SAP102, and PSD-93/chapsyn-110 form a family of membrane-associated guanylate kinase homologues (MAGUKs), characterized by the presence of three PDZ domains, an SH3 domain, and a C-terminal guanylate kinase homologous domain (1-5). Interactions of the PDZ domains of MAGUK proteins with type I C-terminal binding motifs present in ionotropic glutamate receptor subunits have been implicated in the regulation of the organization and functional activity of glutamatergic synapses (for review, see Refs. 6 and 7). Direct physical association of these MAGUK proteins with the subunits of N-methyl-D-aspartate (NMDA) (4, 8 -10), kainate (11), and ␣-amino-5-hydroxy-3-methyl-4-isoxazole propionate (AMPA)-selective glutamate receptors (12) has been demonstrated, but the regulation and detailed physiological functions of these interactions are still unclear.Interaction between the AMPA receptor subunit GluR-A (GluR1) and SAP97 is particularly interesting (12). First, an interaction between the C terminus of GluR-A and a type I PDZ domain protein has been implicated in several models of activitydependent regulation of synaptic strength (13). Second, it is to date the only PDZ domain interaction of AMPA receptors that does not involve the GluR-B subunit and, therefore, may be relevant for the synaptic organization of calcium-permeable AMPA receptors, which lack this subunit (14). Third, in contrast to the majority of synaptic MAGUKs, which generally share binding partners, GluR-A appears to bind only to SAP97. However, the true selectivity and its underlying mechanisms have yet to be defined. In the present study, we show that GluR-A binds to the...

Protein Trafficking and Anchoring Complexes Revealed by Proteomic Analysis of Inward Rectifier Potassium Channel (Kir2.x)-associated Proteins

“…The C-terminal PDZ binding motifs of these channels, as well as nearby upstream amino acids, probably contribute to their specific association with different protein complexes. Supporting this idea, the peptide sequence SSG, which lies upstream of the PDZ binding motif in the GluR1 AMPA receptor, confers selective binding to SAP97 (77). Interestingly, Kir2.2 also contains this sequence in roughly the same upstream location.…”

Postmortem Brain: An Underutilized Substrate for Studying Severe Mental Illness

“…These domains are specialized, regulatable motifs that facilitate colocalization of proteins in specialized microdomains such as the PSD. One of the PDZ domains of SAP97 interacts with the PDZ domain on the C terminus of the AMPA receptor GluA1 subunit in GluA1/GluA2 hetero-oligomers (Cai et al, 2002;Leonard et al, 1998).…”

Direct binding of the human homologue of the Drosophila disc large tumor suppressor gene to seven-pass transmembrane proteins, tumor endothelial marker 5 (TEM5), and a novel TEM5-like protein

“…Taken together, our present findings suggest that hDlg is recruited to the plasma membrane by TEM5 and scaffolds it in endothelial cells during tumor angiogenesis and neoangiogenesis. It is known that hDlg scaffolds some receptors and ion channels in neurons, T lymphocytes, and cardiac myocytes (Kim et al, 1995;Kim and Sheng, 1996;Hanada et al, 1997;Leonard et al, 1998;Murata et al, 2001;Cai et al, 2002), but the function of hDlg in endothelial cells has not yet been reported. This is the first identification of the transmembrane protein, which binds to hDlg, in endothelial cells.…”

“…Thus, hDlg, like Dlg, is likely to be involved in the negative regulation of cell proliferation. Furthermore, hDlg directly binds alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors and Shaker-type K þ channels via its PDZ domain and scaffolds them at synapses (Kim et al, 1995;Kim and Sheng, 1996;Leonard et al, 1998;Cai et al, 2002). hDlg binds Shaker-type K þ channels in T lymphocytes and cardiac myocytes (Hanada et al, 1997;Murata et al, 2001).…”