In this investigation, we report identification and characterization of a 95 kDa postsynaptic density protein (PSD-95)/discs-large/ZO-1 (PDZ) domain-containing protein termed tamalin, also recently named GRP1-associated scaffold protein (GRASP), that interacts with group 1 metabotropic glutamate receptors (mGluRs). The yeast two-hybrid system and in vitro pull-down assays indicated that the PDZ domain-containing, amino-terminal half of tamalin directly binds to the class I PDZ-binding motif of group 1 mGluRs. The C-terminal half of tamalin also bound to cytohesins, the members of guanine nucleotide exchange factors (GEFs) specific for the ADP-ribosylation factor (ARF) family of small GTP-binding proteins. Tamalin mRNA is expressed predominantly in the telencephalic region and highly overlaps with the expression of group 1 mGluR mRNAs. Both tamalin and cytohesin-2 were enriched and codistributed with mGluR1a in postsynaptic membrane fractions. Importantly, recombinant and native mGluR1a/tamalin/cytohesin-2 complexes were coimmunoprecipitated from transfected COS-7 cells and rat brain tissue, respectively. Transfection of tamalin and mutant tamalin lacking a cytohesin-binding domain caused an increase and decrease in cell-surface expression of mGluR1a in COS-7 cells, respectively. Furthermore, adenovirus-mediated expression of tamalin and dominant-negative tamalin facilitated and reduced the neuritic distribution of endogenous mGluR5 in cultured hippocampal neurons, respectively. The results indicate that tamalin plays a key role in the association of group 1 mGluRs with the ARF-specific GEF proteins and contributes to intracellular trafficking and the macromolecular organization of group 1 mGluRs at synapses.

Tamalin is a scaffold protein that comprises multiple protein-interacting domains, including a 95-kDa postsynaptic density protein (PSD-95)/discs-large/ZO-1 (PDZ) domain, a leucine-zipper region, and a carboxyl-terminal PDZ binding motif. Tamalin forms a complex with metabotropic glutamate receptors and guanine nucleotide exchange factor cytohesins and promotes intracellular trafficking and cell surface expression of group 1 metabotropic glutamate receptors. In the present study, using several different approaches we have shown that tamalin interacts with multiple neuronal proteins through its distinct protein-binding domains. The PDZ domain of tamalin binds to the PDZ binding motifs of SAP90/PSD-95-associated protein and tamalin itself, whereas the PDZ binding motif of tamalin is capable of interacting with the PDZ domain of S-SCAM. In addition, tamalin forms a complex with PSD-95 and Mint2/ X11␤/X11L by mechanisms different from the PDZ-mediated interaction. Tamalin has the ability to assemble with these proteins in vivo; their protein complex with tamalin was verified by coimmunoprecipitation of rat brain lysates. Interestingly, the distinct protein-interacting domains of tamalin are evolutionarily conserved, and mRNA expression is developmentally up-regulated at the postnatal period. The results indicate that tamalin exists as a key element that forms a protein complex with multiple postsynaptic and protein-trafficking scaffold proteins.Multimolecular protein assembly through protein-protein interaction is important as a general mechanism for diverse cellular functions in neuronal and other cells (reviewed in Refs. 1-5). Molecular assembly of protein complexes is built around one or more central scaffold proteins that contain multiple domains for protein-protein interaction. The 95-kDa postsynaptic density protein (PSD-95) 1 /discs-large/ZO-1 (PDZ) domain is a key protein-binding domain comprised of ϳ90 amino acid residues and interacts with a PDZ binding motif with the consensus sequences S/TXV/I/L (X is any amino acid) (6, 7). In neurons, postsynaptic PDZ domain-containing scaffold proteins, PSD-95 and S-SCAM, interact with a number of membrane and cytoplasmic proteins, including NMDA receptors (7-9) and SAP90/PSD-95-associated proteins (SAPAPs) (also called guanylate kinase-associated proteins/human Discs Large-associated proteins) (10 -12). PSD-95 and S-SCAM are localized at the PSD of excitatory synapses and play an important role in functional assembly of a postsynaptic macromolecular complex. The PDZ domain-containing scaffold proteins are also important in subcellular trafficking of their partner proteins (reviewed in Refs. 1, 4, and 13). LIN-2, LIN-7, and LIN-10 of Caenorhabditis elegans are all PDZ domain-containing proteins and are important for the proper localization of the LET-23 receptor tyrosine kinase (13). LIN-2 is the homolog of mammalian scaffold protein CASK, which was simultaneously discovered as a protein interacting with the cell surface protein neurexin (14, 15). The mammalian homolo...

Hyperpolarization-activated cation currents, termed I h , are non-uniformly distributed along dendritic arbors with current density increasing with increasing distance from the soma. The nonuniform distribution of I h currents contributes to normalization of location-dependent variability in temporal integration of synaptic input, but the molecular basis for the graded HCN distribution remains to be investigated. The hyperpolarization-activated, cyclic nucleotide-gated cation channels (

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