Association of the type 1 inositol (1,4,5)-trisphosphate ... of the type 1 inositol (1,4,5)-trisphosphate receptor with 4.1N protein in neurons Anton Maximov,1 Tie-Shan Tang,1 and

Association of the type 1 inositol (1,4,5)-trisphosphate receptorwith 4.1N protein in neurons

Anton Maximov,1 Tie-Shan Tang,1 and Ilya Bezprozvanny*Department of Physiology, UT Southwestern Medical Center at Dallas, Dallas, TX 75390, USA

Received 25 June 2002; revised 1 November 2002; accepted 6 November 2002

Abstract

The type 1 inositol (1,4,5)-trisphosphate receptor (InsP3R1) is an intracellular calcium (Ca2�) release channel that plays an importantrole in neuronal function. In yeast two-hybrid screen of rat brain cDNA library with the InsP3R1 carboxy-terminal bait we isolated multipleclones of neuronal cytoskeletal protein 4.1N. We mapped the 4.1N-interaction site to a short fragment (50 amino acids) within thecarboxy-terminal tail of the InsP3R1 and the InsP3R1-interaction site to the carboxy-terminal domain (CTD) of 4.1N. We established thatInsP3R1 carboxy-terminal binds selectively to the CTD� alternatively spliced form of the 4.1N protein. In biochemical experiments wedemonstrated that 4.1N and InsP3R1 specifically associate in vitro. We showed that both 4.1N and InsP3R1 were enriched in synapticlocations and immunoprecipitated the 4.1N–InsP3R1 complex from rat brain synaptosomes. In biochemical experiments we demonstrateda possibility of InsP3R1–4.1N–CASK–syndecan-2 quaternary complex formation. From our findings we hypothesize that InsP3R1–4.1Nassociation may play a role in InsP3R1 localization or Ca2� signaling in neurons.© 2003 Elsevier Science (USA). All rights reserved.

Introduction

The inositol (1,4,5)-trisphosphate receptor (InsP3R) is anintracellular calcium (Ca2�) release channel that plays animportant role in Ca2� signaling in neurons (Berridge,1998). Three isoforms of InsP3R have been identified (Fu-ruichi et al., 1994). The type 1 receptor (InsP3R1) is apredominant neuronal isoform. Mice lacking InsP3R1 dis-play severe ataxic behavior (Matsumoto et al., 1996), andmice with spontaneous mutation in the InsP3R1 gene expe-rience convulsions and ataxia (Street et al., 1997), suggest-ing a major role of the InsP3R1 in neuronal function. Neu-ronal Ca2� signaling is a highly compartmentalized process,and subcellular localization of Ca2� channels plays an im-portant role in their function. High-power Ca2� imagingrevealed highly localized Ca2� release from InsP3R located

in postsynaptic spine apparatus of cerebellar Purkinje neu-rons (Finch and Augustine, 1998; Miyata et al., 2000; Take-chi et al., 1998). What molecular mechanisms are respon-sible for the InsP3R1 targeting to postsynaptic spines?Amino-terminal region of the InsP3R1 binds to the mGluR-associated adaptor protein Homer (Tu et al., 1998), andHomer–Shank interaction is involved in InsP3R1 targetingto postsynaptic spines in hippocampal neurons (Sala et al.,2001). Are there additional InsP3R1 binding partners thatmay be involved in subcellular localization of InsP3R1 inneurons? To identify novel InsP3R1 binding partners weperformed an yeast two-hybrid screen of the rat brain cDNAlibrary with the bait corresponding to the InsP3R1 carboxy-terminal cytosolic tail and isolated multiple clones of 4.1Nprotein.

4.1N protein (Walensky et al., 1999; Yamakawa et al.,1999) is a brain-specific member of the 4.1 protein family ofcytoskeletal proteins (Hoover and Bryant, 2000). Geneticknockout of 4.1R protein resulted in neurobehavioral phe-notype (Walensky et al., 1998b), suggesting an importantrole of 4.1 proteins in brain function. The main knownfunction of 4.1 proteins in cells is to link plasma membraneproteins, such as glycophorin C in red blood cells (Marfatia

* Corresponding author. Department of Physiology, K4.112, UTSouthwestern Medical Center at Dallas, 5323 Harry Hines Blvd.,Dallas, TX 75390-9040, USA. Fax: �1-214-648-2974.

E-mail address: [email protected] (I. Bez-prozvanny).

1 Contributed equally.

R

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Molecular and Cellular Neuroscience 22 (2003) 271–283 www.elsevier.com/locate/ymcne

1044-7431/03/$ – see front matter © 2003 Elsevier Science (USA). All rights reserved.doi:10.1016/S1044-7431(02)00027-1

et al., 1995) with the spectrin–actin cytoskeleton. Twohighly conserved domains, the 4.1-ezrin-radixin-moesin(FERM) domain and the spectrin/actin binding domain(SABD) are involved in interactions of 4.1 family of pro-teins with the plasma membrane proteins and with thespectrin–actin cytoskeleton, respectively (Hoover and Bry-ant, 2000). The association of 4.1-FERM domain with car-boxy-terminal tail of syndecans (Cohen et al., 1998) andneurexins (Biederer and Sudhof, 2001) has been reported.4.1-FERM domain is also involved in interactions with themembers of the MAGUK family p55 (Marfatia et al., 1995)and CASK (Biederer and Sudhof, 2001; Cohen et al., 1998).The functions of the third domain conserved in 4.1 family(carboxy-terminal domain, CTD) is less well understood.Recently, it has been shown that 4.1G-CTD domain bindsFKBP13 (Walensky et al., 1998a), that 4.1N-CTD and4.1R-CTD domains bind nuclear mitotic apparatus protein(NuMA) (Mattagajasingh et al., 1999; Ye et al., 1999), andthat 4.1N-CTD and 4.1G-CTD domains bind GluR1 car-boxy-terminal region (Shen et al., 2000). Thus, most likely4.1-CTD domain serves to recruit additional signaling part-ners.

We mapped the 4.1N-interaction site to a short (50-amino acid) fragment within the cytosolic tail of the

InsP3R1. We established that the 4.1N-CTD domain is bothnecessary and sufficient for binding to the InsP3R1 carboxy-terminal. In biochemical experiments we demonstrated that4.1N and InsP3R1 associate in vitro and can be coimmuno-precipitated from the brain synaptosomes. Furthermore, wedemonstrated a possibility of InsP3R1–4.1N–CASK–synde-can-2 complex formation. From our findings we hypothe-size that InsP3R1–4.1N association may play a role inInsP3R1 localization or Ca2� signaling in neurons.

Results

InsP3R1 carboxy-terminal specifically binds 4.1N proteinin yeast two-hybrid assay

To search for novel InsP3R1 neuronal binding partnerswe aimed to identify proteins that bind to the carboxy-terminal cytosolic region of the InsP3R1. The carboxy-tailregion of the rat InsP3R1 in pLexN vector (IC bait, Fig. 1A)was used in yeast two-hybrid screen of the rat brain cDNAlibrary. Of 16 isolated prey clones, 2 clones (clones 7 and14, see Fig. 2A diagram) represented carboxy-terminal frag-ments of the rat 4.1N protein (Walensky et al., 1999; Ya-

Fig. 1. The 4.1N-binding domain in the InsP3R1 carboxy-terminal. (A) The strength of interaction with clone 7 (r4.1N-U3- CTD�), determined for each ICbait from �-galactosidase activity, is presented in percentages relative to IC6/clone 7 pair (mean�SE, n�3). The minimal fragment of rat InsP3R1carboxy-termini (F2627–R2676), essential for binding to rat 4.1N protein, is shaded. Predicated secondary structure elements of IC sequence are shown onthe top. The type 1 InsP3R-specific sequence is shown as a hatched box. IC6-2 and IC6-3 are fragments of rat InsP3R2 and InsP3R3. (B) Sequence alignmentof the minimal 4.1N-binding motif in rat InsP3R1 (Mignery et al., 1990) (F2627–R2676, Accession No. P29994) with the corresponding regions of ratInsP3R2 (Sudhof et al., 1991) (F2579–R2628, Accession No. P29995) and rat InsP3R3 (Blondel et al., 1993) (F2554–R2603, Accession No. Q63269).

272 A. Maximov et al. / Molecular and Cellular Neuroscience 22 (2003) 271–283

makawa et al., 1999). The third clone (clone 8) corre-sponded to the full-length rat 4.1N protein. In contrast, theproteins encoded by the other remaining 13 clones occurredonly once. When tested in liquid yeast two-hybrid assaywith IC bait, 2 of the clones corresponding to the 4.1Nprotein (clones 7 and 8) gave the strongest signal whencompared to most other preys (data not shown). Thus, inthis study we focused on 4.1N protein as a potentiallyrelevant InsP3R1-binding partner.

In the first series of experiments we used a semiquanti-tative liquid yeast two-hybird assay to identify minimaldomains involved in interaction between the InsP3R1 car-boxy-termini and the 4.1N protein. Carboxy-terminal re-gions of InsP3R1, InsP3R2, and InsP3R3 isoforms are highlyconserved with the exemption of the short sequence at thevery carboxy-termini (hatched box on Fig. 1A). TheIC_G2736X mutant, which lacked the sequence unique forthe InsP3R1, still interacted with the clone 7 (r4.1N-U3-CTD�) (Fig. 1A). To systematically map 4.1N-interactingdomain, a number of InsP3R1 carboxy-terminal fragmentswere cloned into pLexN vector, and the strength of inter-

action between generated baits and clone 7 (r4.1N-U3-CTD�) was measured. Using PSIPRED server (McGuffinet al., 2000), we predicted that the InsP3R1 IC region(D2590-A2749) consists of six �-helices (�1-�6) and two�-strands (�1 and �2) connected via loop regions (Fig. 1A).From analysis of the carboxy-terminal (IC5-IC8) and theamino-terminal (IC3, IC4, IC9-IC11) deletion mutants of ICbait, we determined that the minimal region required forinteraction with the 4.1N protein corresponds to the mid-dle 50-amino acid-long portion of IC bait (F2627–R2676,shaded), which includes predicted �2,3 helices, �2 strand,loop, and �4 helix (Fig. 1A).

The alignment of the minimal 4.1N-interacting region inInsP3R1 (F2627-R2676) with the corresponding regions ofInsP3R2 (F2579–R2628) and InsP3R3 (F2554-R2603)shows high degree of conservation with few nonconservedsubstitutions (Fig. 1B). The IC6 bait (aa D2589–R2676 ofInsP3R1) includes the minimal 4.1N-interacting domainwithin InsP3R1 sequence (Fig. 1). To further determine thespecificity of 4.1N binding, we generated IC6-2 and IC6-3baits, corresponding to the analogous fragments of rat

Fig. 2. InsP3R1 carboxy-terminal binds to the 4.1N-CTD� domain. (A) The strength of interaction between 4.1 prey constructs and IC6 bait (a.a.D2590–R2676) is normalized to clone7/IC6 interaction (mean�SE, n�3). FERM, SABD, U3, and CTD domains are indicated on domain structure of 4.1Nprotein (Hoover and Bryant, 2000). h4.1N, human 4.1N protein (hKIAA0338) (Nagase et al., 1997); r4.1N, rat 4.1N protein (Yamakawa et al., 1999); CTD�,spliced out form of CTD domain. (B) Alignment of rat 4.1N-CTD domain (Yamakawa et al., 1999) (S1423-S1551, Accession No. BAA76625) with thecorresponding region of mouse 4.1G (Walensky et al., 1998a) (V861–E988, Accession No. AAC40083), mouse 4.1B (Parra et al., 2000) (G801–D929,Accession No. AAD38048), and mouse 4.1R (Huang et al., 1993) (1733–E858, Accession No. L00919) proteins. The sequence spliced-out in 4.1N-CTD�isoform is shown in Italics and marked by a bracket.

273A. Maximov et al. / Molecular and Cellular Neuroscience 22 (2003) 271–283

InsP3R2 and rat InsP3R3 proteins. We did not observe anyinteraction between IC6-2 and IC6-3 baits and clone 7(r4.1N-U3-CTD�) in liquid yeast two-hybrid assay (Fig.1A). Thus, despite high degree of sequence conservationbetween InsP3R isoforms in the 4.1N-interacting region(Fig. 1B), only InsP3R1 carboxy-terminal binds to 4.1N inyeast two-hybrid assay.

In the next series of experiments we used a similarapproach to map the InsP3R1-interaction domain on the4.1N protein sequence. The strength of interaction between4.1N fragments in the prey vector and the IC6 bait (D2589–R2676 of InsP3R1) was compared in the liquid yeast two-hybird assay. The domain structure of 4.1N protein is typ-ical for the 4.1 protein family (Fig. 2A) (Hoover and Bryant,2000). Clone 7, which contains the U3 domain and the CTDdomain, interacted with the IC6 bait as strongly as thefull-length 4.1N protein (clone 8) (Fig. 2A). Thus, theFERM and SABD domains of the 4.1N protein are notrequired for interaction with the InsP3R1 carboxy-terminal.Binding of clone 14, composed of a truncated U3 domainand a CTD domain, was much weaker (Fig. 2A). Interest-ingly, clone 7 and clone 8 contained a CTD� spliced ver-sion of 4.1N-CTD domain, whereas clone 14 contained aCTD version (Fig. 2A and B). Thus, weak binding of clone14 may be attributed to truncation of the U3 domain or toalternative splicing of the CTD domain. To discriminatebetween these possibilities, we measured the strength ofinteraction between IC6 bait and preys corresponding to theisolated U3 domain, and to both splice variants of the CTDdomain. We determined that the U3 domain did not interactwith IC6 bait, and that CTD prey binds IC6 bait only weakly(Fig. 2A). In contrast, CTD� prey binds IC6 bait muchstronger, at the level comparable to clones 7 and 8 (Fig. 2A).Thus, we concluded that the 4.1N protein binds to theInsP3R carboxy-tail region via the CTD domain and thatalternative splicing of the CTD domain plays a regulatoryrole in the 4.1N interaction with the InsP3R1 carboxy-terminal.

All four members of 4.1 family are expressed in the brain(Parra et al., 1998; Parra et al., 2000; Walensky et al.,1998b; Walensky et al., 1999), but only 4.1N isoform wasisolated in our yeast two-hybrid screen of rat brain cDNAlibrary. The InsP3R1 binding motif is located within CTDdomain of 4.1N protein (Fig. 2A). The alignment of the rat4.1N-CTD domain with mouse 4.1G, 4.1B, and 4.1R CTDdomains shows that the amino-terminal portion of 4.1N-CTD domain contains a unique sequence (Fig. 2B). Incontrast, all remaining members of 4.1 family are conservedin the corresponding region (Fig. 2B). All four members of4.1 family show very high degree of conservation in thecarboxy-terminal portion of CTD domain (Fig. 2B). Inter-estingly, the sequence unique for 4.1N protein is spliced outin CTD� splice variant (Fig. 2B, shown in italics). To testthe specificity of InsP3R1 binding, we generated h4.1G-U3-CTD and h4.1G-CTD preys, corresponding to the analogousfragments of human 4.1G protein. No interactions was ob-

served between 4.1G preys and IC6 bait in liquid yeasttwo-hybrid assay (Fig. 2B), suggesting that InsP3R1 islikely to be specific for 4.1N isoform.

Association between recombinant InsP3R1and 4.1N proteins

To independently test for the specificity of InsP3R1-4.1Nassociation, we expressed full-length HA-tagged h4.1N andh4.1N� proteins in COS cells by transient transfection andperformed in vitro binding experiments with the full-lengthrecombinant InsP3R1, InsP3R3, and InsP3R2 expressed inSf9 cells by baculoviral infection. The recombinantInsP3R1, InsP3R2, and InsP3R3 were solubilized from Sf9cell-derived microsomes in CHAPS and mixed with lysatesprepared from HA-h4.1N-transfected COS cells. We foundthat in the presence of HA-h4.1N� a significant portion ofexpressed InsP3R1 was immunoprecipitated with anti-HAantibody (Fig. 3A). In complementary experiments, themixture of recombinant InsP3R1 and HA-h4.1N� or HA-h4.1N proteins was immunoprecipitated with polyclonalanti-InsP3R1 antibody and blotted with monoclonal anti-HAantibody. A significant fraction of expressed HA-h4.1N�and HA-h4.1N proteins was immunoprecipitated by anti-InsP3R1 antibody, but not by the preimmune serum (Fig.3B). In contrast to experiments with InsP3R1, anti-HA an-tibodies were not able to precipitate recombinant InsP3R3 orInsP3R2 from the binding reaction with HA-h4.1N� (Fig.3C and E). In agreement with this negative result, anti-InsP3R3 polyclonal antibodies precipitated InsP3R3 but notHA-h4.1N� proteins from the binding mixture (Fig. 3D).Thus, in agreement with the yeast two-hybrid analysis (Fig.1A) we concluded that 4.1N proteins bind strongly toInsP3R1 (Fig. 3A and B), but not at all or weakly to InsP3R2(Fig. 3E) or InsP3R3 (Fig. 3C and D).

InsP3R1 and 4.1N proteins bind in vitro (Fig. 3A and B).Can InsP3R1–4.1N complex be formed in cells? Expressionof full-length InsP3R1 in COS cells leads to formation ofER stacks with most of the expressed InsP3R1 trapped in theER (Takei et al., 1994). Thus, we resorted to experimentswith soluble InsP3R1 carboxy-terminal constructs fused togreen fluorescent protein (GFP) (GFP-IC4/IC6). We coex-pressed HA-h4.1N� and HA-h4.1N proteins with GFP-IC4and GFP-IC6 constructs in COS cells. Subcellular localiza-tion of HA-h4.1N proteins was visualized by immunostain-ing of permeabilized COS cells with anti-HA antibody andsecondary Rhodamine-conjugated anti-mouse antibody.Subcellular localization of GFP-IC4/IC6 was reported byGFP fluorescence. When transfected cells were imaged bylaser confocal microscopy, both HA-h4.1N� and HA-h4.1Nproteins were concentrated in proximity of the plasma mem-brane (Fig. 4, left column), confirming the functionality of4.1-FERM domain in COS cells. When cotransfected withHA-h4.1N� protein, GFP-IC6 fusion protein was also con-centrated near the plasma membrane (Fig. 4, top row). Incontrast, GFP-IC6 was diffuse and cytosolic in the presence


of h4.1N protein (Fig. 4, second row). Distribution of con-trol GFP-IC4 protein was diffuse and cytosolic in the pres-ence of either h4.1N-CTD splice variant (Fig. 4, third andforth rows). From these experiments we concluded thatHA-h4.1N� protein is able to recruit GFP-IC6 protein to theplasma membrane in COS cells. Formation of HA-h4.1N�/GFP-IC6 complex in cells is supported by high degree ofcolocalization between these two proteins (Fig. 4, top row,merged image).

InsP3R1 and 4.1N localize to synapses andform complex in vivo

Recombinant InsP3R1 and 4.1N associate in yeast two-hybrid and biochemical experiments (Figs. 1–4). DoInsP3R1 and 4.1N proteins colocalize in neurons or formcomplex in vivo? To address these questions we performed

quantitative analysis of subcellular distribution of InsP3R1,4.1N and 4.1G proteins in subcellular fractions from ratbrain. For these experiments we adapted the procedure fromJones and Matus (1974) and fractionated total rat brain, ratcortex, and rat cerebellum homogenates on sucrose densitygradients. The obtained fractions were analyzed by Westernblotting with anti-InsP3R1, anti-4.1N, and anti-4.1G anti-bodies. We previously described the InsP3R1 antibody(Kaznacheyeva et al., 1998). The anti-4.1N and anti-4.1Gantibodies were raised against peptides corresponding to themost carboxy-termini of 4.1N and 4.1G proteins coupled toKLH (see Experimental Methods). To test the specificity ofthe obtained antibodies, we used generated antisera to blotthe lysates from COS cells transfected with HA-r4.1N-U3-CTD� and HA-h4.1G-U3-CTD constructs. Obtained resultsconfirmed the relative specificty of 4.1N and 4.1G antibod-ies (Fig. 5A and B). It should be noted that the carboxy-

Fig. 3. Association between InsP3R and 4.1N recombinant proteins in vitro. (A, B) InsP3R1 binds HA-h4.1N. A mixture of lysates from RT1-infected Sf9 cells andHA-h4.1N or HA-h4.1N� transfected COS7 cells was analyzed by immunoprecipitation (IP) and Western blotting (WB) as indicated. Preimmune serum (P/S) wasused in control immunoprecipitation experiments (B). (C, D) InsP3R3 does not bind to HA-h4.1N�. A mixture of lysates from RT3-infected Sf9 cells andHA-h4.1N� transfected COS7 cells was analyzed by immunoprecipitation (IP) and Western blotting (WB) as indicated. To confirm the efficiency of immunopre-cipitation with polyclonal anti-InsP3R3 antibodies, the precipitated fractions were blotted with monoclonal anti-InsP3R3 antibodies (D). (E) InsP3R2 does not bindto HA-h4.1N�. A mixture of lysates from RT2-infected Sf9 cells and HA-h4.1N� transfected COS7 cells was precipitated by anti-HA monoclonal antibodies andblotted with anti-InsP3R2 polyclonal antibodies. The input lanes in (A–E) contain 1/50 of the lysates used in immunoprecipitation experiments.


termini of 4.1G, 4.1B, and 4.1R proteins have significantdegree of homology (Fig. 2B), and 4.1G antibody maycross-react with 4.1B and 4.1R proteins.

When compared to initial homogenate clarified by cen-trifugation (low-speed supernatant), the InsP3R1 and 4.1Nproteins were significantly enriched in synaptic plasmamembrane (SPM) fraction (Fig. 5C). The enrichment ofInsP3R1 in SPM was more pronounced in cerebellum andtotal brain samples (Fig. 5C, first and third row), but it wasalso significant in the cortex sample (Fig. 5C, second row).For subsequent analysis we focused on fractions from thetotal brain. In agreement with published immunolocaliza-

tion data (Walensky et al., 1999), 4.1N protein was enrichedin SPM fraction (Fig. 5C, forth row). In contrast, immuno-reactivity to 4.1G was uniformly distributed among differ-ent fractions (Fig. 5C, seventh row), suggesting that the4.1G isoform is not enriched in synaptic locations. Ourconclusions regarding 4.1N and 4.1G distribution are ingeneral agreement with recently published analysis of 4.1proteins present in postsynaptic density (PSD) fractions(Scott et al., 2001). As control for the fractionation proce-dure, we blotted obtained fractions with the antibodiesagainst postsynaptic marker PSD95. As expected, PSD95protein was significantly enriched in the SPM fraction (Fig.

Fig. 4. Localization of 4.1N and GFP-IC proteins coexpressed in COS7 cells. HA-h4.1N� (first and third rows) and HA-h4.1N (second and forth rows) werecoexpressed with GFP-IC6 (first and second rows) or GFP-IC4 (third and forth rows) in COS7 cells. Subcellular distribution of HA-h4.1N� and HA-h4.1Nproteins was visualized by anti-HA immunohistochemistry (first column), of GFP-IC6/IC4 proteins by GFP fluorescence (second column). Colocalization ofHA-4.1N� and GFP-IC6 proteins, but not other couples, is apparent on merged image (third column) and on the enlarged image (insert, first row).


5C, sixth row). The 4.1-binding partner CASK (Biedererand Sudhof, 2001; Cohen et al., 1998) was also enriched atSPM (Fig. 5C, fifth row) (Butz et al., 1998), albeit to a lesserdegree than 4.1N, InsP3R1, and PSD95 (Fig. 5C).

InsP3R1 and 4.1N are enriched in synaptic plasma mem-brane fractions (Fig. 5C). Do InsP3R1 and 4.1N form com-plexes in vivo? To test if InsP3R1 and 4.1N form complexesin the brain, we performed immunoprecipitation experi-ments with rat brain synaptosomes. In these experiments, P2fraction of total rat brain synaptosomes was solubilized inCHAPS and precipitated with anti-4.1N or anti-InsP3R1polyclonal antibodies. A [3H]InsP3 binding assay was usedto quantify the amount of InsP3R1 bound to the beads ineach sample as described under Experimental Methods. Wefound that the anti-4.1N antibodies precipitated approxi-mately 20% of specific [3H]InsP3 binding sites that could beprecipitated with anti-InsP3R1 antibody (Fig. 6). Inclusionof GST-IC6 fusion protein to the binding reaction resulted

in complete loss of signal observed with the anti-4.1Nantibody but had no effect on the signal observed with theanti-InsP3R1 antibody (Fig. 6). We reason that GST-IC6protein exerts its effect by competing native InsP3R1 fromthe complex with 4.1N protein. From these experiments weconcluded that a significant fraction of synaptic InsP3R1 isassociated with the 4.1N protein via the carboxy-terminaldomain.

InsP3R1–4.1N–CASK–syndecan-2 complex

4.1-FERM domain binds to syndecan and CASK pro-teins (Biederer and Sudhof, 2001; Cohen et al., 1998; Hsuehet al., 1998). Syndecans are the family of heparan sulfateproteoglycans that play an important role in cell adhesion,signaling, and cytoskeleton organization (Carey, 1997;Woods and Couchman, 1998). Both syndecan-2 (Syn2) andsyndecan-3 (Syn3) are expressed in the central nervous

Fig. 5. Subcellular fractionation of InsP3R1 and 4.1N in rat brain. (A, B) Control for specificity of anti-4.1N and anti-4.1G polyclonal antibodies. Lysatesfrom COS cells transfected with HA-h4.1G-U3-CTD (A) and HA-r4. 1N-U3-CTD� (B) constructs were analyzed by Western blotting with anti-HAmonoclonal antibodies, anti-4.1G and anti-4.1N polyclonal antibodies as indicated. (C) Subcellular fractions of rat brain, rat brain cortex, or cerebellum wereisolated on sucrose density gradient as described under Experimental Methods. The fractions were immunobloted with polyclonal antibodies against InsP3R1,4.1N, 4.1G, PSD-95, and monoclonal antibodies against CASK. 10 �g (cortex and total) or 2 �g (cerebellum) of protein was loaded on each lane.


system with Syn3 localized to axonal compartment andSyn2 enriched in postsynaptic locations (Hsueh and Sheng,1999; Hsueh et al., 1998). We reasoned that association ofInsP3R1 with 4.1N protein (Figs. 1–4) may lead to forma-tion of the InsP3R1–4.1N–CASK–Syn2 quaternary com-plex in postsynaptic locations (see Discussion).

The 4.1-FERM and CASK-PDZ binding motifs are lo-calized to carboxy-terminal cytosolic tail of syndecans(Carey, 1997; Woods and Couchman, 1998). To test thepossibility of InsP3R1–4.1N–CASK–Syn2 complex forma-tion, we performed affinity chromatography experimentswith the peptide corresponding to the carboxy-terminal tailof rat Syn2 (Sdc2). Sdc2-mut peptide that lacks 4.1-FERMand CASK-PDZ binding motifs was used in negative con-trol experiments. In the first series of experiments we testedassembly of recombinant InsP3R1, CASK, and HA-4.1Nproteins on Sdc2 peptides coupled to Sepharose beads (Sdc2beads). For these experiments InsP3R1 was expressed in Sf9cells, CASK and HA-4.1N proteins were expressed inHEK293 cells. The recombinant proteins were extracted in1% CHAPS, clarified by centrifugation, mixed, and incu-bated with Sdc2 beads. After extensive washes protein com-plexes attached to the beads were sequentially eluted with 1M NaCl and 1% SDS and analyzed by immunoblotting. Inagreement with previously published reports (Cohen et al.,1998; Hsueh et al., 1998) we observed specific binding ofCASK to Sdc2, but not to Sdc2-mut beads (Fig. 7A, firstrow). As expected from conservation in 4.1-FERM domainfunctionality, both HA-4.1N� and HA-4.1N specificallyassociated with Sdc2 beads (Fig. 7A, second and forthrows). In the presence of either HA-4.1N� or HA-4.1Nproteins InsP3R1 was associated with Sdc2 beads, but not

with Sdc2-mut beads (Fig. 7A, third and fifth row). Mostlikely because of indirect nature of InsP3R1-Sdc2 associa-tion, the fraction of InsP3R1 bound to Sdc2 beads (whennormalized to input) was about twofold lower than forCASK and 4.1N.

The experiments shown on Fig. 7A suggest that theInsP3R1–4.1N–CASK–Syn2 complex can form in vitro.Can this complex form at synaptic locations in vivo? Toanswer this question, we purified P2 fraction from rat cortexand rat cerebellum homogenates (Jones and Matus, 1974),solubilized obtained material in 1% CHAPS, and incubatedwith Sdc2 and Sdc2-mut Sepharose beads. Following bind-ing reaction, the beads were washed extensively and pro-teins attached to the beads were sequentially eluted with 1

Fig. 7. Association of InsP3R1 with CASK–4.1N–syndecan complexes.(A) Extracts of HEK293 cells expressing recombinant CASK and HA-4.1Nproteins were combined with extracts of Sf9 cells expressing InsP3R1 andincubated with Sepharose beads coupled to the peptide corresponding tothe carboxy-terminal tail of rat syndecan-2 (Sdc2) or to the mutated peptide(Sdc2-mut). Proteins specifically associated with the beads were sequen-tially eluted with 1M NaCl and 1% SDS and analyzed by immunoblottingwith antibodies against CASK, HA, and InsP3R1 as indicated. (B) Extractsof rat brain cortex or cerebellum synaptosomes were incubated with Sdc2and Sdc2-mut beads and attached proteins were sequentially eluted with1M NaCl and 1% SDS. Eluted fractions were analyzed by immunoblottingwith antibodies against CASK, 4.1N, and InsP3R1 as indicated.

Fig. 6. InsP3R1-4.1N complex in synaptic membrane fraction. P2 fractionof rat brain synaptosomes was extracted in 1% CHAPS and immunopre-cipitated with antibodies against InsP3R1 or r4.1N as described underExperimental Methods. The amount of precipitated InsP3Rs was quantifiedby [3H]InsP3 binding. Nonspecific signal obtained in parallel immunopre-cipitation experiments with the corresponding preimmune serum was sub-tracted. The effect of GST-IC6 (3 �M) addition is shown. GST-IC6 proteinis not recognized by anti-InsP3R1 antibodies (data not shown).


M NaCl and 1% SDS. The eluted fractions were analyzedby immunoblotting with anti-InsP3R1, anti-4.1N, and anti-CASK antibodies. In agreement with recombinant proteinsdata (Fig. 7A), we found that InsP3R1, 4.1N, and CASKspecifically associated with Sdc2, but not with Sdc2-mutbeads (Fig. 7B). The 4.1N-CTD splice variant associatedwith Sdc2 beads in these experiments is unknown, as thepolyclonal 4.1N antibodies used for Western blotting do notdiscriminate between 4.1N� and 4.1N splice variants. Asimilar fraction of InsP3R1 was associated with Scd2 beadsin both cortex (Fig. 7B, third row) and cerebellar (Fig. 7B,forth row) SPM. Despite the indirect nature of the InsP3R1–Sdc2 interaction, it was largely resistant to 1 M NaCl (Fig.7B, third and fourth lanes), suggesting strong associationwithin the native InsP3R1–4.1N–CASK–Syn2 complex.

Discussion

InsP3R1 interactions with neuronal cytoskeleton

Highly developed spectrin/actin lattice is present inpostsynaptic spines of central neurons (Harris and Kater,1994; Kaech et al., 1997). A number of previous reportssuggested interactions between InsP3R1 and cytoskeleton.Biochemical results pointed to the InsP3R1 association withF-actin (Fujimoto et al., 1995) and ankyrin (Bourguignon etal., 1993; Joseph and Samanta, 1993). The InsP3R-4.1Ninteractions described in the present report may be related toInsP3R1–F-actin association (Fujimoto et al., 1995), as 4.1Nprotein binds F-actin (Biederer and Sudhof, 2001). Directassociation of InsP3R1 with ankyrin has been suggested(Bourguignon and Jin, 1995). The ankyrin-binding motif2546GGVGDVLRKPS2556 (Bourguignon and Jin, 1995) islocated within a pore-forming region of the InsP3R1 anddoes not overlap with the 4.1N-binding domain (F2627–R2676) (Fig. 1A). Thus, it is possible that InsP3R1 binds toboth 4.1N and ankyrin proteins via adjacent motifs.

A recent report indicated that the CTD domain of 4.1Nand 4.1G proteins is associated with GluR1 (Shen et al.,2000). In contrast to GluR1, InsP3R1 carboxy-terminalbinds selectively to 4.1N-CTD (Fig. 2). In turn, the 4.1Nisoform is specific for InsP3R1 and binds only weakly toInsP3R2 and InsP3R3 (Figs. 1 and 3). When tested in yeasttwo-hybrid and COS cell localization assays, the InsP3R1carboxy-terminal is specific for 4.1N� alternatively splicedform (Figs. 2 and 4). However, full-length InsP3R1 binds ina similar way to both 4.1N and 4.1N� isoforms in immu-noprecipitation experiments (Figs. 3B and 7A). These re-sults suggest that 4.1N protein may associate with an addi-tional region of InsP3R1 localized outside of the carboxy-termini. This secondary association does not appear to beregulated by alternative splicing of the 4.1N-CTD domain(Figs. 3B and 7A).

InsP3R1 as a part of macromolecular signalingcomplex in PSD

The postsynaptic spines of excitatory synapses containelectron-dense compact material termed postsynaptic den-sity. Recent biochemical and molecular analysis suggestedthat PSD is a macromolecular complex that links plasmamembrane receptors with intracellular signaling compo-nents (Kennedy, 1997; Sheng, 2001; Ziff, 1997). Ultrastruc-tural analysis showed that a significant fraction of hip-pocampal dendritic spines (Spacek and Harris, 1997) andmost cerebellar Purkinje cell spines (Harris and Stevens,1988) contain smooth endoplasmic reticulim (SER). TheInsP3R1 are localized to SER in spines of cerebellar Pur-kinje neurons (Petralia et al., 2001) and present in spines ofsome hippocampal CA1 neurons (Sharp et al., 1993). Ul-trastructural data suggest that some InsP3R1 in cerebellarpostsynaptic spines come into close contact with PSD (Pe-tralia et al., 2001). Recent data suggest that InsP3R-medi-ated Ca2� release in postsynaptic spines of cerebellar Pur-kinje cells is both necessary and sufficient for induction ofLTD at parallel fiber–Purkinje cell synapses (Eilers et al.,1997; Finch and Augustine, 1998; Miyata et al., 2000;Takechi et al., 1998). The role of InsP3R1 in spines ofhippocampal neurons is less clear (Rose and Konnerth,2001; Svoboda and Mainen, 1999).

The amino-terminal of InsP3R1 is associated with theEVH1 domain of mGluR1�/5-binding protein Homer (Tu etal., 1998) (Fig. 8). Homer forms dimers via coiled–coil(CC) region and binds to Shank protein via EVH1 domain(Du et al., 1998; Naisbitt et al., 1999; Tu et al., 1998; Xiaoet al., 2000). The recrutiment of InsP3R1 to spines of hip-pocampal neurons is induced as a result of Homer/Shankoverexpression (Sala et al., 2001). Colocalization of Homerand InsP3R1 in the PSD region of cerebellar neurons hasbeen demonstrated in ultrastructural studies (Petralia et al.,2001). Syndecan-2 proteins are also concentrated in PSD(Ethell and Yamaguchi, 1999; Hsueh and Sheng, 1999;Hsueh et al., 1998). In biochemical experiments we dem-onstrated a possibility of the InsP3R1–4.1N–CASK–Syn2quaternary complex formation in vivo and in vitro (Fig. 7).From these results we hypothesize that fraction of InsP3R1located close to PSD may also form a complex with 4.1N–CASK–Syn2 in postsynaptic spines (Fig. 8).

Previous functional experiments led us to propose that afraction of InsP3R in cells is directly coupled to PIP2 andlocalized within a specialized ER compartment located im-mediately underneath the plasma membrane (Lupu et al.,1998). It is tempting to speculate that the pool of InsP3R1located close to PSD is bound to Homer–mGluR1�/5, PIP2,and 4.1N–CASK–Syn2 (Fig. 8). We point out that theproposed model (Fig. 8) is largely based on biochemicalanalysis of InsP3R–4.1N association in vitro reported in thepresent article. Additional experiments will be needed totest the importance of the InsP3R1–4.1N association forneuronal Ca2� signaling in vivo. For example, examination


of InsP3R1 targeting to PSD and InsP3R1-mediated Ca2�

signaling in synaptic spines of neurons from 4.1N knockoutmice can provide such an insight. These and other experi-ments will be required to test the importance of theInsP3R1–4.1N association for Ca2� signaling in the nervoussystem.

Experimental methods

Yeast two-hybrid methods

The carboxy-terminal region of rat InsP3R1 (Mignery etal., 1990) (amino acids D2590–A2749) was amplified byPCR and cloned into pLexN vector (IC bait). A rat brain(P8-P9) cDNA library in pVP16-3 vector (3 � 105 inde-pendent clones) was a kind gift of Dr. T. Sudhof. The yeasttwo-hybrid screen of rat brain cDNA library with the IC baitwas performed according to published procedures (Fieldsand Song, 1989; Hata et al., 1996). Sixteen prey clonesobtained in the screen were rescued and sequenced. A liquidyeast two-hybrid assay was performed and quantified aspreviously described (Maximov et al., 1999).

Plasmids

Fragments of rat InsP3R1 (Mignery et al., 1990), ratInsP3R2 (Sudhof et al., 1991), rat InsP3R3 (Blondel et al.,

1993), rat 4.1N� protein (Yamakawa et al., 1999) (clone 8),human 4.1N protein (hKIAA0338) (Nagase et al., 1997),and human 4.1G protein (Parra et al., 1998) (ESTAA333370 � P299-D1005) were amplified by PCR, clonedinto corresponding yeast two-hybrid (prey pVP16-3 and baitpLexN), mammalian (HA-pCMV5 or pEGFP-C3), or bac-terial (pGEX-KG) expression vectors and sequenced. Thefollowing InsP3R1 baits in pLexN vector were generated(listed by encoded amino acid and residue numbers): IC,D2590–A2749; IC_G2736X, D2590–G2736; IC3, R2676–A2749; IC4, Q2714–A2749; IC5, D2590–Q2714; IC6,D2590–R2676; IC7, D2590–L2646; IC8, D2590–F2627;IC9, L2646–A2749; IC10, F2627–A2749; IC11, L2606–A2749. The InsP3R2 bait is IC6-2, D2542–R2628, theInsP3R3 bait is IC6-3, D2517–R2603. The 4.1 preys inpVP16-3 vector are (r-rat, h-human): h4.1N-U3, L538–S753; h4.1N-CTD, S753–S881; r4.1N-CTD�, S1423–S1551 (del E1427–K1483); h4.1G-U3-CTD, L675–D1005;h4.1G-CTD, T879–D1005. The 4.1 expression constructs inHA-pCMV5 vector are HA-h4.1N, M1–S881; HA-h4.1N�,M1–S881 (del E757–K813); HA-r4.1N-U3-CTD�, D1254–S1551 (del E1427–K1483); HA-h4.1G-U3-CTD, L675–D1005; HA-h4.1G-CTD, T879–D1005. The InsP3R1 ex-pression constructs in pEGFP-C3 are GFP-IC6, D2590–R2676 and GFP-IC4, Q2714–A2749. The InsP3R1expression construct in pGEX-KG is GST-IC6, D2590–R2676.

Fig. 8. Hypothetical marcomolecular signaling complex at PSD. 4.1N-FERM domain forms a ternary complex with the carboxy-terminal tail of syndecan-2(syn2) and PDZ domain of CASK (present study and Cohen et al. (1998); Ethell and Yamaguchi (1999); Hsueh and Sheng (1999); Hsueh et al., (1998)).Ectodomain of syndecans binds to the extracellular matrix (ECM) (Woods and Couchman, 1998). SABD of the 4.1N protein binds to spectrin–actin latticein the postsynaptic terminal (Biederer and Sudhof, 2001; Harris and Kater, 1994). The 4.1N-CTD domain binds to the carboxy-terminal region of InsP3R1(present study). The amino-terminal domain of InsP3R1 binds to Homer (Tu et al., 1998) and PIP2 (Glouchankova et al., 2000; Lupu et al., 1998). Homerlinks InsP3R1 and mGluR1�/5 (Brakeman et al., 1997; Tu et al., 1998; Xiao et al., 1998). We propose that the described cascade of molecular interactionsmay help to assemble upstream (mGluR1�/5) and downstream (InsP3R1) signaling components of phosphoinositide signaling pathway at PSD. Note thatultrastructural studies (Petralia et al., 2001) show that only a fraction of InsP3R1 in the spines are located sufficiently close to PSD to be involved in directassociation with Homer-mGluR1�/5, PIP2, and 4.1N–syndecan-2.


In vitro binding assay

Rat InsP3R1 (RT1)- and InsP3R3 (RT3)-encoding bacu-loviruses were previously described (Maes et al., 2000; Tuet al., 2002). The rat InsP3R2 (RT2)-encoding baculoviruswas generated from full-length rat InsP3R2 plasmid(Ramos-Franco et al., 2000) using the Bac-to-Bac systemaccording to the manufacturer’s (Invitrogen) instructions.Spodoptera frugiperda (Sf9) cells (100 ml) were infectedwith RT1, RT2, or RT3 viruses and collected by centrifu-gation 48 h postinfection. Collected cells were solubilizedin the extraction buffer A containing 1% CHAPS, 137 mMNaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4

(pH 7.2), 5 mM EDTA, 5 mM EGTA, and protease inhib-itors. The lysate was cleared by centrifugation (100,000g inTL-100) and used in immunoprecipitation experiments.COS7 cells were transfected with HA-4.1N expression con-structs using the DEAE–dextran method. Seventy-twohours after transfection, the cells were collected and solu-bilized in extraction buffer A. Extracts were clarified bycentrifugation, mixed with an equal volume of Sf9 celllysate containing recombinant InsP3R1, InsP3R2, orInsP3R3, and incubated for 2 h at 4°C. The mixture wasprecipitated with anti-HA monoclonal antibody attached toprotein G–Agarose and analyzed by Western blotting withanti-InsP3R1, anti-InsP3R2, and anti-InsP3R3 polyclonal an-tibodies as described. In complementary experiments withInsP3R1 and InsP3R3 the mixture was precipitated withpolyclonal anti-InsP3R1 (or preimmune sera) or anti-InsP3R3 polyclonal antibodies attached to protein A–Sepha-rose beads and analyzed by Western blotting with mono-clonal anti-HA antibodies or anti-InsP3R3 monoclonalantibodies as described.

COS cell localization assay

COS7 cells were grown to �80% confluence on glasscoverslips (coated with polylysine) in DMEM containing10% fetal bovine serum. The cells were cotransfected withHA-4.1N and GFP-IC4/IC6 expression plasmids by theDEAE–dextran method. Seventy-two hours after transfec-tion the cells were fixed in 4% paraformaldehyde (Sigma)containing 4% sucrose, washed three times with PBS, per-meabilized in 0.25% Triton X-100, and stained with mono-clonal anti-HA (1:1000 in 5% BSA blocking solution).Cells were washed three times, incubated with Rhodamine–anti-mouse secondary antibodies, washed with PBS, andmounted in Moweol. Confocal images were taken with aZeiss 600 laser confocal microscope using 63x oil-im-mersed objective and edited with Adobe Photoshop 5.0.

Brain fractionations and immunoprecipitations

The subcellular fractions of total rat brain, of rat braincortex, and of rat brain cerebellum were prepared as previ-ously described (Jones and Matus, 1974). For immunopre-

cipitations the P2 synaptosomal membrane fraction wassolubilized for 1.5 h at 4°C in extraction buffer A. Thelysate was clarified by a 20-min centrifugation at 50,000g(TL-100) and incubated with protein A–Sepharose beadscovered with polyclonal antibodies against rat InsP3R1, rat4.1N protein, or the corresponding preimmune serum.Beads were pelleted by centrifugation, washed three timeswith the extraction buffer, incubated for 30 min with 5 nM[3H]InsP3 (Amersham), pelleted by centrifugation, andrinsed quickly with extraction buffer. The amount of[3H]InsP3 in the final sample was quantified by a beta-counter and presented as cpm. The nonspecific signal, esti-mated from immunoprecipitation with the correspondingpreimmune serum, was substracted from the total counts.GST-IC6 protein used in competitive experiments was ex-pressed in BL21 bacterial strain and purified on GST beadsby standard protocols (Pharmacia).

Affinity chromatography

The peptide corresponding to the sequence of carboxy-terminal tail of rat syndecan-2 (Sdc2, RMRKKDEG-SYDLGERKPSSAAYQKAPTKEFYA) was coupled toNHS-activated Sepharose beads (Amersham) via amino-terminal group. Control peptide (Sdc2-mut, AAA-KAAAAAADLGAAAPSSAAYQKAPTKE) lacked 4.1-FERM and CASK-PDZ binding motifs. For experimentswith recombinant proteins CASK (Hata et al., 1996) andHA-h4.1N proteins were expressed in HEK293 cells byLipofectamine-mediated transfection. InsP3R1 was ex-pressed in Sf9 cells by baculovirus infection. Recombinantproteins were extracted from HEK293 and Sf9 cells inextraction buffer A for 1 h at 4°C. Protein extracts wereclarified by centrifugation for 20 min at 100,000g (TL-100),combined, and incubated with Sdc2 or Sdc2-mut peptidescoupled to Sepharose beads for 16 h at 4°C and 1 h at roomtemperature. Beads were washed with 40 bead volumes ofextraction buffer A and attached proteins were sequentiallyeluted with 1 bead volume of 1 M NaCl and then 1 beadvolume of 1% SDS. Samples were resolved by SDS–PAGEand analyzed by immunoblotting with antibodies againstInsP3R1, CASK, and HA. For experiments with native pro-teins rat cortex or rat cerebellum were homogenized in 320mM sucrose, 25 mM Hepes (pH 7.2). Crude SPM from ratcortex and rat cerebellum were obtained as described (Jonesand Matus, 1974). Obtained SPM were solubilized in ex-traction buffer A and binding to the Sdc2 and Sdc2-mutpeptides was analyzed as for recombinant proteins.

Antibodies

The following monoclonal antibodies were used. Anti-HA(HA.11) was from Covance and anti-synapsin, anti-CASK,and anti-InsP3R3 were from Transduction Labs. SecondaryRhodamine- and FITC-conjugated antibodies were fromJackson Imunoresearch. Polyclonal PSD-95 antibody was a


gift from Dr. T. Sudhof. Rabbit polyclonal anti-InsP3R1antibody T443 was previously described (Kaznacheyeva etal., 1998). Affinity-purified rabbit polyclonal anti-InsP3R2antibody (T2C) was a kind gift from Dr. Gregory Mignery(Ramos-Franco et al., 2000); rabbit polyclonal anti-InsP3R3antibody (IB7124) was generated against RLGFVDVQNC-MSR peptide coupled to KLH and affinity-purified; rabbitpolyclonal anti-4.1N antibody (IB6971) was generatedagainst DPSPEERDKKPQES peptide coupled to KLH; rab-bit polyclonal anti-4.1G antibody (IB7120) was generatedagainst RVVVHKETELAEEGEE peptide coupled to KLH.The specificity of anti-4.1N and anti-4.1G antibodies wastested as described under Results (Fig. 5A and B).

Acknowledgments

We thank Yin Wang, Masaya Okamoto, Shuza Sugita,and Thomas C. Sudhof for advice with yeast two-hybridscreen and the gift of the rat brain cDNA library; Nan Wangand Phyllis Foley for expert technical and administrativeassistance; Dale Elmer, Nan Wang, and Elena Nosyreva forassistance with the experiments; and Nan Wang for RT2baculovirus. We thank Thomas C. Sudhof for the InsP3R1and CASK clones, Gregory Mignery for the InsP3R2 cloneand T2C antibodies, Graem Bell for the InsP3R3 clone,Takahiro Nagase and Kazusa DNA Research Institute forthe KIAA0338 clone, Anne Woods for syndecan 2 cloneand antibodies, and Humbert De Smedt for the RT3 bacu-lovirus. This work was supported by the Robert A. WelchFoundation and NIH R01 NS38082 (I.B.).

Note added in proof. InsP3R1-4.1N association wasrecently reported by Zhang et al. (2003). J. Biol. Chem., inpress.

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Association of the type 1 inositol (1,4,5)-trisphosphate ... of the type 1 inositol (1,4,5)-trisphosphate receptor with 4.1N protein in neurons Anton Maximov,1 Tie-Shan Tang,1 and