Structural Basis of the Na+/H+ Exchanger Regulatory Factor PDZ1 Interaction with the Carboxyl-terminal Region of the Cystic Fibrosis Transmembrane Conductance Regulator

104

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...

Section: Discussionmentioning

confidence: 99%

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

Cai

Coleman

Niemi

et al. 2002

104

“…9). Rather, we found that RACK1 and NHERF1, a scaffold protein that binds four carboxyl-terminal amino acids (DTRL) of CFTR through either of its PDZ (PDZ1 and PDZ2) domains (29,31,37,38), coimmunoprecipitate from Calu-3 cells and are binding partners (Fig. 8).…”

Section: Discussionmentioning

confidence: 99%

Protein Kinase Cε-dependent Regulation of Cystic Fibrosis Transmembrane Regulator Involves Binding to a Receptor for Activated C Kinase (RACK1) and RACK1 Binding to Na+/H+ Exchange Regulatory Factor

Liedtke¹,

Yun²,

Kyle³

et al. 2002

106

Protein kinase C (PKC) regulation of cystic fibrosis transmembrane regulator (CFTR) chloride function has been demonstrated in several cell lines, including Calu-3 cells that express native, wild-type CFTR. We demonstrated previously that PKC⑀ was required for cAMP-dependent CFTR function. The goal of this study was to determine whether PKC⑀ interacts directly with CFTR. Using overlay assay, immunoprecipitation, pulldown and binding assays, we show that PKC⑀ does not bind to CFTR, but does bind to a receptor for activated C kinase (RACK1), a 37-kDa scaffold protein, and that RACK1 binds to Na

“…5A) (19,44,45). More recently, structures have been published for PDZ domains from ␣-syntrophin (22) and the H ϩ /Na ϩ exchange regulatory factor (HNERF PDZ1) (48,49), in which specific hydrophobic or hydrogen bond interactions are observed to the Ϫ1 position side chain (Fig. 5).…”

Section: Discussionmentioning

confidence: 99%

Origins of PDZ Domain Ligand Specificity

Skelton¹,

Koehler²,

Zobel³

et al. 2003

141

The LAP (leucine-rich repeat and PDZ-containing) family of proteins play a role in maintaining epithelial and neuronal cell size, and mutation of these proteins can have oncogenic consequences. The LAP protein Erbin has been implicated previously in a number of cellular activities by virtue of its PDZ domain-dependent association with the C termini of both ERB-B2 and the p120-catenins. The present work describes the NMR structure of Erbin PDZ in complex with a high affinity peptide ligand and includes a comprehensive energetic analysis of both the ligand and PDZ domain side chains responsible for binding. C-terminal phage display has been used to identify preferred ligands, whereas binding affinity measurements provide precise details of the energetic importance of each ligand side chain to binding. Alanine and homolog scanning mutagenesis (in a combinatorial phage display format) identifies Erbin side chains that make energetically important contacts with the ligand. The structure of a phage-optimized peptide (Ac-TGW ؊4 ETW ؊1 V; IC 50 ‫؍‬ ϳ0.15 M) in complex with Erbin PDZ provides a structural context to understand the binding energetics. In particular, the very favorable interactions with Trp ؊1 are not Erbin side chain-mediated (and therefore may be generally applicable to many PDZ domains), whereas the ␤2-␤3 loop provides a binding site for the Trp ؊4 side chain (specific to Erbin because it has an unusually long loop). These results contribute to a growing appreciation for the importance of at least five ligand C-terminal side chains in determining PDZ domain binding energy and highlight the mechanisms of ligand discrimination among the several hundred PDZ domains present in the human genome.