We have isolated, from a rat brain cDNA library, a clone corresponding to a 2779-bp cDNA encoding a novel splice form of the glutamate receptor interacting protein-1 (GRIP1). We call this 696-amino acid splice form GRIP1c 4-7 to differentiate it from longer splice forms of GRIP1a/b containing seven PDZ domains. The four PDZ domains of GRIP1c 4-7 are identical to PDZ domains 4 -7 of GRIP1a/b. GRIP1c 4-7 also contains 35 amino acids at the N terminus and 12 amino acids at the C terminus that are different from GRIP1a/b. In transfected HEK293 cells, a majority of GRIP1c 4-7 was associated with the plasma membrane. GRIP1c 4-7 interacted with GluR2/3 subunits of the ␣-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor. In low density hippocampal cultures, GRIP1c 4-7 clusters colocalized with GABAergic (where GABA is ␥-aminobutyric acid) and glutamatergic synapses, although a higher percentage of GRIP1c 4-7 clusters colocalized with ␥-aminobutyric acid, type A, receptor (GABA A R) clusters than with ␣-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor clusters. Transfection of hippocampal neurons with hemagglutinin-tagged GRIP1c 4-7 showed that it could target to the postsynaptic complex of GABAergic synapses colocalizing with GABA A R clusters. GRIP1c 4-7-specific antibodies, which did not recognize previously described splice forms of GRIP1, recognized a 75-kDa protein that is enriched in a postsynaptic density fraction isolated from rat brain. EM immunocytochemistry experiments showed that in intact brain GRIP1c 4-7 concentrates at postsynaptic complexes of both type I glutamatergic and type II GABAergic synapses although it is also presynaptically localized. These results indicate that GRIP1c 4-7 plays a role not only in glutamatergic synapses but also in GABAergic synapses.

We cloned two novel alternatively-spliced mRNA isoforms of glutamate receptor interacting protein 1 (GRIP1) which we named GRIP1d and GRIP1e 4-7. GRIP1d is a 135 kDa, 7-PDZ-domain variant of GRIP1, containing the 12 amino acid C-terminus originally described for the 4-PDZ-domain GRIP1c 4-7. GRIP1e 4-7 is a 75 kDa 4-PDZ-domain variant of GRIP1, containing the 12 amino acid C-terminus originally described for the 7-PDZ-domain GRIP1a/b. Northern blots indicated that GRIP1d mRNA is 5.1 kb long and abundant in brain. An antibody to the C-terminus of the 75 kDa GRIP1c 4-7 also recognized an abundant 135 kDa protein, consistent with the predicted size of GRIP1d. Similarly, an antibody to the C-terminus of the 135 kDa GRIP1a/b also recognized a low abundance 75 kDa protein, consistent with the predicted size of GRIP1e 4-7. Immunocytochemistry of hippocampal cultures and intact brain using these antibodies showed that (i) these isoforms are present in both GABAergic and glutamatergic synapses, and (ii) the isoforms co-localize in individual synapses. While GRIP1a/b isoforms are abundant in interneurons and highly concentrated in GABAergic presynaptic terminals, the isoforms recognized by the antibody to the C-terminus common to GRIP1c 4-7 and GRIP1d are much less abundant in interneurons and preferentially concentrate at the postsynaptic complex. Keywords: electron microscopy, GABAergic synapse, glutamate receptor interacting protein 1, immunocytochemistry, PDZ domain, postsynaptic density. The glutamate receptor interacting protein (GRIP) family comprises highly homologous multiple PDZ domain-containing proteins that are encoded by the GRIP1 (Dong et al. 1997(Dong et al. , 1999 and GRIP2 (Bruckner et al. 1999;Dong et al. 1999;Wyszynski et al. 1999) GRIP-/-mouse mutants have shown that GRIP1 is required for normal cell-matrix interactions during embryonic development (Bladt et al. 2002;Takamiya et al. 2004). GRIP1 also plays an important role in dendrite morphogenesis (Hoogenraad et al. 2005), and we have recently reported that GRIP1 is present at GABAergic synapses and in the intact brain (Charych et al. 2004a;Li et al. 2005a). In addition, GRIP1 has been shown to interact with the GABA A R interacting protein GABARAP via PDZ domains 4-6 (Kittler et al. 2004).GRIP1 exhibits a high degree of molecular heterogeneity resulting from alternative splicing (Fig. 1). Alternate 18 and 19 amino acid N-terminal peptides were shown in mouse to give rise to GRIP1a and GRIP1b, respectively, such that GRIP1b can be palmitoylated and GRIP1a cannot (Yamazaki et al. 2001). Consequently, GRIP1b is associated with membranes whereas GRIP1a is localized to the cytoplasm (Yamazaki et al. 2001). Alternative splicing of GRIP1 also gives rise to a 90 kDa short form of GRIP1, called DIP2 (DLX