Primary Structure of GKAP
A yeast two-hybrid screen of a rat brain cDNA library using PSD-95 GK domain as bait yielded five independent overlapping cDNA clones of the same gene, which we termed GKAP (Fig. A). A BLAST search of the GenBank database did not reveal homology to any known polypeptides, except for a human EST (expressed sequence tag) clone. The EST clone (accession number Z45015) was obtained, fully sequenced, and identified as a human homologue of GKAP (hGKAP, 98% identity at the amino acid level to rat GKAP; see Fig. ). The coding sequence of hGKAP predicted a polypeptide of 627 residues and ~70 kD molecular weight. No significant stretch of hydrophobic residues was found by hydrophobicity analysis suggesting that GKAP is not a transmembrane protein.
Figure 1 Primary structure of GKAP. (A) Rat brain cDNA clones isolated from the yeast two-hybrid screen using PSD-95 GK domain as bait are shown (black lines) aligned below a schematic of the domain organization of GKAP protein (drawn to scale). Numbers in parentheses (more ...)
GKAP clones contained four sequence variations (type A-D) at the NH2-terminal end, presumably due to differential splicing (Fig. A). An in-frame upstream stop codon was present in the 5′ end (type D) of the hGKAP EST clone, and so a putative translation initiation site was assigned to the next methionine, which was in a good Kozak consensus. By 5′-RACE, we were able to isolate the same type D 5′ end with an in-frame upstream stop codon from rat brain cDNA. None of the other 5′ variants of rat GKAP clones (type A, B, and C) isolated by the yeast two-hybrid screen had in-frame upstream stop codons. This is expected from the nature of yeast two- hybrid screening since positive clones are expressed as COOH-terminal fusion proteins. However, expression of the GKAP clone 2.18 (containing the type A insertion) in heterologous cells produced GKAP protein with a size identical to GKAP proteins found in rat brain (see below), allowing us to assign a putative ATG codon in the ‘A' insertion (Fig. B).
In addition to probable splice variation at the NH2-terminus, insertions of a different sequence were found in the middle and at the COOH terminus of GKAP, presumably also due to alternative splicing (Fig. A). Both kinds of middle insertion (1 and 2) and COOH-terminal variants (X and Y) were detected by PCR in both human and rat cDNA libraries. 3′ termination codons were present in both X and Y COOH-terminal variants of GKAP clones, allowing us to designate the COOH terminus of these proteins. The sequence of some of these insertions are of interest. For instance, the type ‘2' middle and the type ‘X' COOH-terminal insertions of the protein (Fig. B) both contain proline-rich potential SH3 domain-binding motifs.
All the cDNAs isolated by the two-hybrid screen overlapped in the NH2-terminal half of GKAP, suggesting that this region mediates the binding to the GK domain of PSD-95 (Fig. A). Five 14–amino acid (14 aa) repeats were noted in the NH2-terminal region. A stretch of ~100 amino acids near the COOH terminus of GKAP (termed the GH1 domain) showed significant sequence similarity (33–35% identity at the amino acid level) to a region of a C. elegans and a human open reading frame of unknown function (GenBank accession # U00058 and D13633, respectively).
GKAP Interacts Specifically with Members of the PSD-95 Family
In addition to PSD-95, the GK domain is present in SAP97, chapsyn-110/PSD-93, and SAP102 as well as in more distantly related MAGUK proteins (ZO-1, ZO-2, p55, CASK, dlg2, etc.). The binding specificity of GKAP for various different GK domains was tested by yeast twohybrid assay (Fig. A
). GKAP interacts strongly with the GK domains of PSD-95, SAP97, and chapsyn-110, but not with that from the tight junction protein, ZO-1 (Willott et al., 1993
), indicating that GKAP binding may be specific for members of the PSD-95 subfamily of MAGUKs. Indeed, the amino acid sequence identity between the GK domains of chapsyns is 70–75%, whereas PSD-95 and ZO-1 GK domains share only ~30% identity.
Figure 2 GKAP interacts specifically with GK domains from members of the PSD-95 family. (A) GK domains from PSD-95, SAP97, chapsyn-110, and ZO-1 were tested for their binding to GKAP in the yeast two-hybrid assay, based on induction of yeast reporter genes (more ...)
To further test the specificity of the GK-GKAP interaction, we performed a reverse yeast two-hybrid screen using the entire GKAP clone 2.18 as bait. The screen yielded a total of ten independent GKAP-interacting clones, nine of which were cDNA fragments containing GK domains of one of the four known chapsyns: PSD-95, SAP97, chapsyn-110, and SAP102 (Fig. B).
Domains Mediating Interaction between the GK and GKAP
Minimal sequence requirements for GK-GKAP binding were determined using deletions of the GK domain of PSD-95 (Fig. A), and of the NH2-terminal region of GKAP (Fig. B). Small deletions into either the NH2- or COOH-terminal side of the GK domain resulted in the loss of interaction (Fig. A), suggesting that the entire GK domain is required for binding to GKAP. The last 13 amino acids of PSD-95 COOH-terminal to the GK domain were not required for GKAP binding.
Figure 3 Minimal domains required for binding between PSD-95 GK domain and GKAP. (A) Various deletion variants of the GK domain of PSD-95 are shown as black lines aligned below the COOH-terminal region of PSD-95 in which the GK domain is represented by hatched (more ...)
On the GKAP side, we tested whether the 14 aa repeats found near the NH2 terminus are involved in GK binding (Fig. B). In the yeast two-hybrid assay, fragments of GKAP incorporating the first repeat (aa 28-58) or the second repeat (aa 64-102) showed a weak but significant interaction with the GK domain. Constructs that included two or more of the 14 aa repeats showed much stronger binding. Moreover, essentially non-overlapping constructs containing the first and second repeats (aa 46-102) or containing the third and fourth repeats (aa 96-104), were each able to interact strongly with the PSD-95 GK domain. Since the intervening sequences between the repeats are quite dissimilar, the above results are consistent with the idea that the 14 aa repeats of GKAP may independently contribute to the overall binding affinity for GK. However, a 1,000-fold excess of a peptide (SPKPSPKVAARRESYLKATQ) corresponding to the second 14 aa repeat (underlined) was unable to inhibit GK-GKAP binding in solution binding or in filter binding assays (data not shown). This negative result could mean that these 14 aa repeats are not directly important in GK-GKAP interaction. Alternatively, the structural context of the 14 aa repeats is critical, i.e., the correct “folding” of the 14 aa repeats is dependent on surrounding sequences that were missing in the synthetic peptide. Thirdly, cooperative binding of the 5 repeats of GKAP may result in such a strong avidity that competition with a single 14 aa repeat is inadequate, even at high relative concentrations. Finally, although the 5 repeats are similar, they are not identical in sequence, so their binding specificities may be slightly different; in this case, a single peptide may not be able to compete the overall GKAP binding efficiently. These reasons are not mutually exclusive.
Direct Interaction between the GK Domain and GKAP
To show direct biochemical association between the GK domain and GKAP, overlay filter binding assays were performed. GKAP fusion protein bound specifically to the GK domains of PSD-95, SAP97, and chapsyn-110 (Fig. A), but not to negative controls including GST alone and the PDZ1-2 and SH3 domains of PSD-95.
Figure 4 Direct GK-GKAP binding in overlay filter binding assays. (A) GST-fusion proteins containing no insert (GST), or different regions of PSD-95 (PDZ1-2, SH3, or the GK domain), or GK domains of SAP97 and chapsyn-110, were separated by SDSPAGE, transferred (more ...)
The filter binding assay was also performed in a reverse orientation as further confirmation of the interaction. The GK domains from PSD-95, SAP97, and chapsyn-110 specifically bound to the NH2-terminal half of GKAP (containing the 14 aa repeats) but not to the COOH-terminal region of GKAP (containing the GH1 domain) (Fig. B). Thus the NH2-terminal region of GKAP binds directly to the GK domains of PSD-95 family proteins, in agreement with two-hybrid analysis.
GKAP Is Enriched in the Postsynaptic Density
GKAP antibodies (termed GKAP2.1) were raised against the NH2-terminal two-thirds of GKAP, using as immunogen a H6 fusion protein of GKAP clone 2.1. Affinity-purified GKAP2.1 antibodies specifically recognized two prominent bands of ~95 kD and ~130 kD on immunoblots of rat brain membranes (Fig. A). The 95-kD brain band comigrated exactly with GKAP expressed in COS-7 cells transfected with presumptive full-length GKAP cDNA (clone 2.18; Fig. A). The nature of the ~130-kD band is less clear, but both the 95-kD and the 130-kD bands almost certainly represent GKAP proteins because the identical bands were also recognized by an independent antibody raised against a non-overlapping COOH-terminal part of GKAP (aa 446-666 of clone 2.18) (data not shown). The 130-kD immunoreactive polypeptide in rat brain may be the result of GKAP variants that have a longer NH2-terminal extension than represented in our cDNA clones. As noted above, some of the GKAP cDNAs do not have upstream stop codons, so they may be incomplete open reading frames. Alternatively, the 130-kD band might reflect posttranslational modifications of GKAP that do not occur in COS-7 cells. Whatever the case, the 95-kD and 130kD bands codistribute and cofractionate very similarly (Fig. ), and coimmunoprecipitate with PSD-95 in rat brain (see below), providing further evidence that they both represent GKAP proteins.
Figure 5 Expression pattern of GKAP protein in rat brain. (A) Specificity of GKAP antibodies and differential regional expression of GKAP in rat brain. Whole cell extracts of untransfected COS-7 cells (Untrans.), or of COS cells transfected with GKAP cDNA, (more ...)
In the rat brain, GKAP is widely distributed in membrane preparations from different regions of the brain but shows no detectable expression in liver (Fig. A). GKAP proteins are predominantly associated with the membrane rather than the soluble fractions of rat brain (Fig. B). And like PSD-95, an abundant postsynaptic density (PSD) protein, GKAP is highly enriched in PSD fractions, where it is resistant to Triton and sarkosyl detergent extraction (Fig. B). The biochemical cofractionation of PSD-95 and GKAP is consistent with these proteins being associated at postsynaptic sites in rat brain.
In Vivo Association between PSD-95 and GKAP
Colocalization of GKAP and PSD-95 in neurons is a prerequisite for association in vivo. In double immunofluorescence studies, we found GKAP immunoreactivity to colocalize strikingly with PSD-95 in discrete puncta along the dendrites of cultured hippocampal neurons (Fig. ). The punctate localizations of PSD-95 and GKAP are at presumptive synaptic sites, since they are apposed to presynaptic markers such as SV2 and synaptophysin (data not shown). The colocalization of PSD-95 and GKAP indicates they are both synaptic proteins but does not prove that they interact directly in vivo.
Figure 6 Colocalization of GKAP and PSD-95 in cultured hippocampal neurons. Double immunofluorescence labeling of neurons with rabbit anti-GKAP (GKAP2.1) antibodies (green, top panel), and with guinea pig anti-PSD-95 antibodies (red, bottom panel). Middle (more ...)
To demonstrate biochemical association of PSD-95 and GKAP in a cellular context, we have performed coimmunoprecipitation of PSD-95 and GKAP from transfected heterologous cells as well as from rat brain. Since PSD-95 can also interact with Shaker-type K+
channels via its PDZ domains (Kim et al., 1995
), we tried coimmunoprecipitation of PSD-95, GKAP, and Shaker-type subunit Kv1.4 from COS cells triply transfected with all three genes (Fig. A
). Antibodies specific for each of the three proteins were able to immunoprecipitate the other two proteins in addition to their cognate antigen, while none of these proteins were immunoprecipitated by control NR2B antibodies. The finding that anti-Kv1.4 antibodies can precipitate GKAP, and that GKAP antibodies can bring down Kv1.4, is of special significance, since it implies the formation of a ternary complex containing the K+
channel and GKAP linked together by PSD-95. In confirmation of this, when the wild-type PSD-95 was replaced by a mutant lacking only the GK domain (PSD-95ΔGK) in the triple transfection, GKAP could no longer be coimmunoprecipitated by Kv1.4 or PSD-95 antibodies, while the interaction between PSD-95ΔGK and Kv1.4 was maintained (Fig. A
). Thus, there is no direct association of Kv1.4 and GKAP. These results are consistent with the formation of a ternary complex in which PSD-95 binds to the K+
channel via its PDZ domains and to GKAP via its GK domain.
Figure 7 Coimmunoprecipitation of PSD95 and GKAP from cotransfected COS cells and from rat brain. (A) Extracts from COS-7 cells triply transfected with either Kv1.4 + PSD-95 + GKAP (left), or with Kv1.4 + PSD-95ΔGK + (more ...)
To demonstrate existence of a protein complex containing PSD-95 and GKAP in the rat brain, coimmunoprecipitation was performed from solubilized brain membranes. As noted above (Fig. ), neither PSD-95 nor GKAP are extracted by mild detergents. Nevertheless, following a SDS/Triton extraction protocol recently developed by Huganir and colleagues (Müller et al., 1996
) for coimmunoprecipitation of postsynaptic density proteins, GKAP was efficiently solubilized and could be coimmunoprecipitated with PSD-95 by PSD-95 antibodies (Fig. B
GKAP Is Recruited into Ion Channel/PSD-95 Clusters
We have previously shown that PSD-95 and its relatives have the remarkable property of clustering Shaker K+
channels and NMDA receptors in heterologous cells. To test whether GKAP is recruited into ion channel/PSD-95 clusters, we coexpressed GKAP with Kv1.4 and PSD-95 in COS-7 cells (Fig. ). When Kv1.4 and GKAP are cotransfected in the absence of PSD-95, both Kv1.4 and GKAP proteins are diffusely distributed in a reticular pattern in the cell (Fig. , a
). In triply (Kv1.4 + PSD-95 + GKAP) transfected cells, plaque-like clusters of Kv1.4 are formed, and GKAP immunoreactivity colocalized exactly with Kv1.4 in these clusters (Fig. , c
). In separate experiments, PSD-95 also coclusters with Kv1.4 in these triply transfected cells (data not shown) in agreement with earlier findings (Kim et al., 1995
). Thus, GKAP recruitment to Kv1.4/PSD-95 coclusters presumably reflects binding of GKAP to PSD-95. In support of this conclusion, when PSD-95 is replaced with mutant PSD-95ΔGK in the same triple transfection, the colocalization of GKAP in Kv1.4 clusters is lost. GKAP is now diffusely distributed in the cell, while the clustering of KV1.4 is maintained (Fig. , e
). These results indicate that GKAP recruitment to Kv1.4/PSD-95 clusters depends on the GK domain of PSD-95. Interestingly, clustering of Kv1.4 by PSD-95 does not require the GK domain (Fig. e
). Similarly, GKAP was also recruited to clusters formed by PSD-95 and NMDA receptor subunit NR2B suggesting that PSD-95 can bring GKAP in close proximity to NMDA receptor channels (data not shown). Taken together with the coimmunoprecipitation data (Fig. A
), these results indicate that by virtue of its multimodular protein binding domains, PSD-95 can nucleate a macroscopic protein cluster containing both ion channels and GKAP.
Figure 8 Recruitment of GKAP into Kv1.4/PSD-95 coclusters in COS cells, studied by double immunofluorescence labeling. In cells cotransfected with Kv1.4 and GKAP, (a and b), both Kv1.4 (a, red) and GKAP (b, green) are mainly distributed in a diffuse intracellular (more ...)