Gene structure and genetic localization of the PCLO gene encoding the presynaptic active zone protein Piccolo

Int. J. Devl Neuroscience 20 (2002) 161–171

Gene structure and genetic localization of the PCLO gene encoding the presynaptic active zone protein Piccolo Steven D. Fenster, Craig C. Garner∗ Department of Neurobiology, University of Alabama at Birmingham, 1719 6th Avenue South CIRC 589, Birmingham, AL 35294-0021, USA Received 10 January 2002; received in revised form 3 April 2002; accepted 5 April 2002

Abstract Piccolo belongs to a family of presynaptic cytoskeletal proteins likely to be involved in the assembly and function of presynaptic active zones as sites of neurotransmitter release. Given that abnormalities in the formation of synaptic junctions are thought to contribute to cognitive dysfunction during brain development, we have analyzed and compared the gene structure of the Piccolo gene, PCLO, from humans and mice and determined their chromosomal localization. A comparison of the deduced amino acid sequence of cDNA clones encoding Piccolo from human, mouse, rat and chicken reveals the presence of distinct homology domains. Only subsets of these are also present in the structurally related active zone protein Bassoon indicating that Piccolo and Bassoon perform related but distinct functions at active zones. Characterization of the PCLO gene reveals the presence of 25 coding exons spread over 380 kb of genomic DNA. The human PCLO gene maps to 7q11.23-q21.3, a region of chromosome 7 implicated as a linkage site for autism and Williams Syndrome suggesting that alterations in the expression of Piccolo or the PCLO gene could contribute to developmental disabilities and mental retardation. © 2002 ISDN. Published by Elsevier Science Ltd. All rights reserved. Keywords: Synapse; Cytoskeleton; Active zone; cDNA; PCLO gene

1. Introduction Abnormalities in the formation of central nervous system (CNS) synapses are thought to contribute to cognitive dysfunction during brain development. They are also thought to underlie disorders such as Fragile X, epilepsy, schizophrenia and mental retardation as well as age-related neurodegenerative disorders associated with learning and memory dysfunction such as Alzheimer’s disease, or ataxias occurring in disorders such as Huntington’s disease and amyotrophic lateral sclerosis (for review, see Purpura, 1974, 1975; Becker, 1991; Wolff and Missler, 1993). Abbreviations: BAC, bacterial artificial chromosome; BSN, gene name for the active zone protein Bassoon; C2A/C2B, calcium phospholipid binding domains; cDNA, copy deoxy-ribonucleic acid; CNS, central nervous system; FMRP, Fragile X mental retardation protein; kb, kilobase; NCBI, National Center for Biotechnology Information; PRD, proline rich domain; PDZ, PSD95/SAP90-Disc large-ZO1 homology domain; PCLO, gene name for the active zone protein Piccolo; PCR, polymerase chain reaction; PBH, Piccolo–Bassoon homology domain; RT, reverse transcriptase; SVs, synaptic vesicles ∗ Corresponding author. Tel.: +1-205-975-5573; fax: +1-205-934-6571. E-mail address: [email protected] (C.C. Garner).

CNS synapses of the mammalian brain are highly specialized cellular junctions designed for rapid and regulated signaling between nerve cells and their targets. Morphologically, synapses have been characterized as asymmetric structures composed of a presynaptic bouton filled with synaptic vesicles (SVs), a synaptic cleft and a postsynaptic specialization (see Ziv and Garner, 2001). Our ability to define how different genetic and environmental insults cause cognitive dysfunction and mental retardation requires an understanding of the cellular mechanism that lead to the proper assembly and function of CNS synapses. This requires a molecular description of the constituents of synaptic junctions and the mechanisms used by neurons to correctly sort, traffic, and localize each component. To this end, progress in the last 15 years has led to the identification and characterization of numerous synaptic junctional proteins (see Kennedy, 2000; Garner et al., 2000b, 2002; Sheng, 2001). More recently, cellular studies have begun to provide clues to the molecular and cellular mechanisms underlying CNS synaptogenesis (see Ziv and Garner, 2001; Garner et al., 2002). In particular, optical imaging approaches have shown that CNS synapses can form in less than 2 h (Ahmari et al.,

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2000k; Friedman et al., 2000). Moreover, the presynaptic active zone, the site of SV docking and fusion, has been found to form within 20 min of initial axo-dendritic contact, preceding the formation of the postsynaptic reception apparatus by ∼30 min (Friedman et al., 2000; Ziv and Garner, 2001). Mechanistically, initial active zone formation is thought to be triggered by the fusion of a series of precursor vesicles that carry active zone proteins from the cell soma to the site of nascent synapse formation (Zhai et al., 2001). Two recently characterized synaptic junctional proteins thought to be critical to the formation of CNS synapses and carried on these active zone precursor vesicles are the presynaptic active zone proteins, Piccolo (also called Aczonin) and Bassoon (Zhai et al., 2001; Garner et al., 2000a). Both are structurally related high-molecular weight modular proteins of 550 and 420 kDa, respectively, sharing 10 distinct regions of homology called Piccolo–Bassoon homology domains (PBH) (tom Dieck et al., 1998; Wang et al., 1999; Fenster et al., 2000). Piccolo and Bassoon are specifically localized to the cytoskeletal matrix assembled at the active zone, where they are hypothesized to perform a scaffold function, localizing the machinery essential for SV exo- and endocytosis (See Garner et al., 2000a). With regard to mental retardation and cognitive dysfunction, our analysis of the Bassoon gene (BSN) and its transcripts has revealed the presence of a CAG expansion (tom Dieck et al., 1998; Winter et al., 1999). Similar expansions are found in Huntingtin, ataxins, and Fragile X syndrome (See Reddy and Housman, 1997). Moreover, Bassoon expression is selectively enhanced in a neurodegenerative disorder, multiple system atrophy (Hashida et al., 1998). The analysis of BSN knockout mice shows clear defects in the assembly of CNS synapses and in release of neurotransmitter underscoring the importance of this active zone protein in synaptic function (Altrock et al., 2001). Given the striking sequence similarity between Bassoon and Piccolo (Fenster et al., 2000), it is hypothesized that Piccolo plays a similar important functional role in presynaptic active zones and that defects in the gene encoding Piccolo, PCLO, could contribute to cognitive impairment and mental retardation. In the present study, we have begun to address this issue by characterizing the structure of the PCLO gene in both mouse and human and determining their chromosomal localizations. Our results show that Piccolo is encoded by a gene containing 25 exons spanning over 350 kb of genomic DNA. Furthermore, we have found that the mouse gene is found on the proximal end of chromosome 5, while the human PCLO gene is situated at 7q11.23-q21.3, a loci situated near a large deletion observed in patients with Williams Syndrome, a developmental disorder causing among others mental retardation/cognitive dysfunction (Meng et al., 1998). These studies indicate that abnormal expression or mutations in members of this Piccolo and Bassoon gene family may contribute to disorders of the developing nervous system.

2. Experimental procedures 2.1. Analysis of genomic sequence Genomic nucleotide sequence was obtained from database entries at the National Center for Biotechnology Information (NCBI) and Celera Discovery Systems. Downloaded sequence was analyzed using the Vector NTI Suite 6.0 software package (Informax) for the presence of intron/exon boundaries and exon coding regions. Boundaries were defined by presence of consensus splice donor and acceptor sequence following the GT/AG rule. Genetic loci were extracted from database entries at NCBI and Celera. Alignment of amino acids was also performed using Vector NTI software. 2.2. Isolation and characterization of PCLO-containing genomic clones A 129SVJ mouse genomic library (Stratagene) was screened with a rat cDNA probe (44a2) corresponding to the central region coding of the Piccolo gene using standard hybridization techniques. The probe was labeled with 32 P by random priming (Promega, Madison, WI). Several phage clones were isolated and verified for the presence of PCLO sequence using PCR and sequencing techniques. In addition, a bacterial artificial chromosome (BAC) library was screened by Research Genetics (Huntsville, AL) yielding several large genomic clones spanning the PCLO gene. Also, BAC clones mapped by Celera Discovery Systems corresponding to the mouse PCLO gene were obtained from the BACPAC Resource Center at the Children’s Hospital Oakland Research Institute (Oakland, CA). 2.3. RT-PCR of rat brain RNA For RT-PCR experiments, total RNA was isolated from adult rat brain using Triazol reagent (Invitrogen). Following purification, RNA was resuspended in DEPC-treated water and amplified using a one-step PCR kit (Qiagen) according to manufacturer’s specifications. Briefly, oligonucleotide pairs flanking either the C2A domain (forward primer 5 -TGGAAGGGATGCAAGTATTG-3 ; reverse primer 5 -AGCTGCCATGCTGAGGAATT) or the C2B domain (forward primer 5 -GCAAAACCAGTGTCGCCCAG-3 ; reverse primer 5 -CTCGATCATGTCGACATACCC-3 ) were incubated with 1 ␮g of total RNA for 30 min at 50 ◦ C to synthesize first strand cDNA. Single stranded DNA was then heat denatured for 15 min to inactivate the RT and activate the Taq DNA polymerase. The samples were then subjected to 30–35 rounds of amplification. The PCR reactions were run on 1.5% TBE agarose gels, PCR fragments were excised from the gel and purified via column purification (Qiagen). Isolated fragments were subcloned using the TOPO cloning system (Invitrogen) and sequenced at the UAB sequencing

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facility using an automated ABI 373 DNA sequencer. Sequences were analyzed using Vector NTI Suite 6.0 software.

increase two-fold (Altrock et al., 2001). This increase and the presence of the PBH domains in Piccolo potentially explain the limited synaptic phenotype seen in these mutant mice.

3. Results

3.2. Structure of the PCLO gene

In a previous study, we have isolated cDNA clones encoding the entire coding region of Piccolo from rat brain cDNA libraries (Fenster et al., 2000). This study revealed that Piccolo is high-molecular weight protein of ∼550 kDa that shares a high degree of sequence similarity to a second active zone protein, Bassoon. Interestingly, most of the shared homology between these two active zone proteins is confined to short regions of ∼50–500 aa residues in length and are flanked by sequences that are mostly unrelated (see diagram Fig. 1A). These data indicate that Piccolo and Bassoon are part of a new gene family and that they share a limited level of functional redundancy. This concept is supported by the ability of both to interact via their zinc fingers with the Rab3A acceptor protein PRA1 (Fenster et al., 2000).

To determine the organization of the mouse and human PCLO genes, nucleotide sequences extracted from mouse and human cDNA databases were used to analyze sequence information made available through the human and mouse genome projects at Celera and the NCBI. This analysis allowed us to extract information on the exon and intron structure of the mouse and human PCLO gene. Tables 1 and 2 provide detailed information on the size of each exon and intron as well as the exon/intron boundaries for the mouse and human PCLO genes, respectively. A schematic diagram of the human PCLO gene is shown in Fig. 2 and was found to be nearly identical to the mouse PCLO gene (not shown). These data show that the coding regions of the mouse and human PCLO genes are found on 25 exons spanning over 350 kb of genomic DNA, a size and organization that is very similar to the BSN gene (Winter et al., 1999). A careful analysis of each of the published cDNA sequence encoding the rat, mouse and human sequences reveal that mRNA transcribed from the PCLO gene can be alternatively spliced giving rise to two Piccolo isoforms (Fig. 2). This is accomplished by the use of two different in-frame stop codons present in exons 20 and 25. Such alternative splicing is predicted to generate two isoforms of Piccolo, one of 530 kDa with a single C2A domain and one of 550 kDa with both a C2A and C2B domain. Interestingly, the C2A domain has been shown to be a Ca2+ -phospholipid binding domain and is likely to participate in Ca2+ -dependent signaling in nerve terminals (Gerber et al., 2001). Further analysis by RT-PCR revealed additional splicing within the C2A and C2B region. Using primers flanking either the C2A domain or C2B domain, we were able to amplify multiple DNA fragments. Sequencing of these fragments revealed alternatively spliced transcripts lacking exon 19 (181 nucleotide deletion) and 21 and 22 (210 nucleotide deletion; Fig. 2B). In addition, scanning of rat expressed sequence tags in the NCBI database revealed several cDNA clones lacking exon 16 (27 nucleotide deletion). The removal of exons 16 and/or exon 19 as a result of alternative splicing is expected to generate translational products of Piccolo lacking residues located within the C2A domain thus possibly affecting the ability of these regions to respond to influx of calcium during synaptic activity (see Fig. 2B).

3.1. Regions in Piccolo conserved from chicken to men To gain additional information concerning the structural elements present in Piccolo, we have used the primary nucleotide sequence from rat Piccolo to search both mouse, human and chicken nucleotide sequences at the National Center for Biotechnology (NCBI) and Celera Discovery Systems. In addition, we also isolated several mouse genomic clones from lambda phage and BAC libraries. A sequence alignment of the deduced amino acid sequence for the rat, mouse and human Piccolo proteins is shown in Fig. 1C. To gain further insight into the evolutionary differences between mammalian and avarian Piccolo, we also included the deduced amino acid sequence of chicken Piccolo in our comparison (Fig. 1C). These data show that at the amino acid level, human Piccolo is about 82% identical and 86% similar to both rat and mouse Piccolo sequence. In addition, rat Piccolo is 62% identical and 62% similar to chicken Piccolo. Interestingly, if one scans the length of the Piccolo sequences from these four species, distinct regions with identities approaching 95% can be clearly seen separated by regions with identity below 40%. This feature, seen in other proteins across species (Kindler et al., 1990), strongly points to the presence of discrete functional domains within Piccolo. For simplicity of discussion, we have numbered these 1–6 (see Fig. 1A and B). A comparison of these homology regions to those shared between Piccolo and Bassoon (Fig. 1A) reveals that many, including Piccolo–Bassoon homology (PBH) domains 1, 2, 4, 6, 8 are also detected in the alignment of Piccolo sequences from rat, mouse, chicken, and human. While the PBH domains very likely represent functional redundancy between Piccolo and Bassoon, the regions in Piccolo that are conserved from chicken to rodents to man indicate unique properties in Piccolo. Intriguingly, in BSN knockout mice, Piccolo levels

3.3. Chromosomal localization of the PCLO gene The initial localization of the mouse PCLO gene was accomplished by probing Southern blots of mouse/human somatic cell hybrids with a 32 P labeled rat cDNA clone 44a (Cases-Langhoff et al., 1996). These data show that PCLO is present as a single copy in the very proximal region of mouse chromosome 5 (data not shown). To further verify

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Fig. 1. (Continued).

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Fig. 1. (Continued).

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Table 1 Exon/intron boundaries of mouse PCLO Exon number

Encoded protein domain

Exon size (bp)

5 Splice donor

Intron size

3 Splice acceptor

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Start Q, Zn1 Zn1, Zn2 Zn2 CC1 CC2 CC3 – – PDZ PDZ – – – C2A C2A C2A C2A C2A – – – C2B C2B C2B

239 1393 1407 711 5059 2018 2182 137 91 126 109 68 215 51 125 27 91 75 181 195 142 68 135 146 141

CCATGCACAGgcacgcacag CTTAACTGAGgtgagtgcat CCTGACTGAGgtaagcacta AGACAGGACAgtaagttgtg GTGTCTACAGgtcagtatga GGGCTTCACGgtaagaggga GACAGTGAAGgtaaatggct TCAAGAGCAAgtaagtatga ACAGTTTCAGgtagagcaaa CTTATAGAAGgtaaatatta GTGTAAGACTgtgagttttt CCAAAAGTTGgtaggtgaca CGGCAGCAAGgtgagaagga AGAGATTCAGgtatggaaag CAGGACGAGGgtaagtgcaa AGAATGCAAGgtgagaagga CATGGAGCAGgtatgtcaag CCTTGGAGAGgtaagtctag CCATCCAAGGgtaggagata ATTCAACCAAgtaaaagacg TCCCAAGGATgtaagtggta GACACGGAGGgtaagtgatg CATTTACCAGgtatctactt CTCACTTCAGgtaattgtgt AACGCATTGA 3’ UTR

4.4 kb 17.0 kb 120 kb 5.3 kb 357 bp 29.5 kb 4.3 kb 4.5 kb 27.4 kb 14.6 kb 862 bp 513 bp 5.5 kb 5.0 kb 3.3 kb 9.8 kb 1.9 kb 1.4 kb 1.7 kb 17.4 kb 5.7 kb 30.1 kb 574 bp 1.2 kb

cattttctagGAAACAAGAG ttatttgtagATCAAAGAGT tgtcttgcagATTCAAGAAT actctgctagGAAAAGGAAG tgtatttcagGTGAGGTAAT ttggttatagGCTAAAGGTT gtccccaaagCCTATCACTT tatgtcctagATTCTGCAGA ttcaatgcagGTAATGGGCT gtctttctagGGATGCAAGT gtctttttagGGACCTCAAT tttgttctagTGGATAAGGC actatttcagCCTACCGATG caccttaaagCTTCAAATCA atcaatacagTCAAGTCATG tccaatttagTGTTGAGTAC ccgaatgcagCTCATGAAGA cctttcccagGTATTGATTG ttgtaaccagATATGCAGGT cgtcatgcagCAAAGCCTAC tttgaaacagAGGAAAGATG cttcttctagCTAAAACTCA ttattttcagATTTATATGT tgttttctagATTTTACTTT

Exon sequences are in capital letters; intron sequences are in lowercase letters; letters in bold indicate consensus splice junctions; blank regions represent gaps in the human genome project. Q, glutamine-heptad repeats; Zn1, Zn2, double zinc finger motifs; CC1–3, coiled-coil domains; C2A and C2B, C2 domains; PDZ, PDZ domain. Mouse genomic sequence was obtained from database sequences at Celera Discovery Systems and from isolated mouse genomic clones. Table 2 Exon/intron boundaries of human PCLO Exon number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Encoded protein domain

Exon size (bp)

5 Splice donor

Intron size

3 Splice acceptor

Start Q, Zn1 Zn1,Zn2 Zn2 CC 1 CC 2 CC 3 – – PDZ PDZ – – – C2A C2A C2A C2A C2A – – – C2B C2B C2B

5

CCATGCACAGgcatgcacag TTTAACGGAGgtaagtgcat TTTGACTGAGgtaagttata AGAAAAAACAgtaagttaaa GTTACTACAGgtcagtatga GGGCTATACGgtaagagtga GACAGTGAAGgtaaattggg TCAAGAGCAAgtaagtgcta ACAGTTTCAGgtaaagcaaa CTTATGGAAGgtaagaataa GTGTAAGACTgtgagttttt CCAAAAGCTGgtaggtaaaa CAGCAGCAAGgtgaggattg AGAAATTCAGgtatgaagtt CAGGGAGAGGgtaagtaaaa AGAATGCAAGgtagagttgt CATGGAACAGgtatgtcaag CCTTGGGGAGgtaagcctct CCATCAAAGGgtaggaaata AAAGACGCAAgtaaaagacg TTCCGAGGATgtaagtggtc GACACTGAAGgtaaggtaaa CATCTACCAGgtatctaatt TTCTCTTCAGgtagctctgt ACGCATTTGA 3 UTR

5.9 kb 19.1 kb 167.8 kb 8.8kb 366 bp 34.8 kb 5.8 kb 6.1 kb 23.2 kb >30 kb 505 bp 1081 bp 3.8 kb 3.1 kb 2.5 kb 7.7 kb 1.2 kb 2.2 kb 1.5 kb 16.7 kb 4.1 kb 40 kb 575 bp 1.9 kb

actcttctagGAAACAAGAG ttatttgtagGTAAAAGAGT tttcccacagATTCAAGAAT ccctgcttagGAAAAGGAAG tgtatttcagGTGAGGTAAT ttgattatagACTAAAGGTT gtgtccaaagCCTATCATCT tatgtcttagATTATACAGA tttaatacagGTAATGGATT gtctttctagGGATGCAAGT taatttttagGGACCTCAAT tttgttgtagTGGATAAGGC aatattacagCCTACCGATG tctcctaaagCTTCAAATTA atcaatgcagTCAAGTCATG cccaattcagTGCTGAGTAC aactatatagCTCAAGAAGA tttttttcagGTATTGATTG ttgtgaccagACATGCAGGT caccatgcagCAAAGCCTCC ttctaaacagAGGGAAGATG attctcccagCTAAAACTCA ttattttcagATTTATATGT ttttttctagATTTTACTTT

UTR + 248 1675 1407 717 5083 2015 2188 137 91 126 109 68 215 51 125 27 94 72 181 195 142 74 135 146 141 + 3 UTR

Exon sequences are in capital letters; intron sequences are in lowercase letters; letters in bold indicate consensus splice junctions; blank regions represent gaps in the human genome project. Q, glutamine-heptad repeats; Zn1, Zn2, double zinc finger motifs; CC1–3, coiled-coil domains; C2A and C2B, C2 domains; PDZ, PDZ domain. Human genomic sequence was obtained from three genomic clones of 165, 143, and 98 kb in length (DJO828B12, DJ0784G16, DJ0897G10) at the NCBI and from database sequences at the Celera Human Genome Database.

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Fig. 3. Regional map and chromosomal localization of the human PCLO gene. The left side shows the region of chromosome 7q11.23–7q11.23-q21.3 containing the PCLO gene in relation to the LIMKI (lim kinase 1), WBSCR14 (Williams–Beuren syndrome candidate region 14), and STX1A (syntaxin 1A) genes. All three genes are known to be deleted in Williams Syndrome. The distance is indicated in centimorgans (cM). The diagram on the right shows a magnification of the PCLO gene region and selected genes clustered near the PCLO gene region.

our analysis and more accurately place the chromosomal loci, we examined database sequences at Celera placing the gene at mouse chromosome 5q1. In addition, human BAC clones spanning the PCLO gene analyzed by both the NCBI and Celera have been mapped to chromosome 7q11.23-q21.1 (Fig. 3). Interestingly, several genes situated near this region are deleted in patients with Williams Syndrome. These include LIMKI, STX1A, WBSCR1. Other genes within this cluster include Sema3E and Sema3C, two genes known to have important functions for axon pathfinding in Drosophilia (Liu and Strittmatter, 2001; Fig. 3). Of note, the genes clustered around the human PCLO are nearly identical to those surrounding the mouse PCLO, indicating that mutational and deletion studies of genes within this mouse chromosome 5 region may eventually be important for the study of human diseases associated with the corresponding region in chromosome 7.

4. Discussion In terms of synapse assembly, the active zone has been shown to be one of the first structures to form, acquiring the capacity to dock, fuse and recycle SVs within 10–20 min of initial axo-dendritic contact (Friedman et al., 2000). The rapid formation of the active zone appears to be achieved through the fusion of an 80 nm dense core precursor vesicle that carries numerous components of the active zone (Zhai et al., 2001). Three classes of molecules have thus far been identified on these precursor vesicles. These include cell adhesion molecules such as N-cadherin and N-CAM, components of the SV exocytotic machinery including syntaxin and SNAP-25, as well as structural/cytoskeletal components of the active zone including Piccolo and Bassoon (Zhai et al., 2001). As such, the fusion of these vesicles with the plasma membrane could very quickly bring many of the proteins essential for establishing sites of neurotransmitter

release to these nascent synapses. Intriguingly, these precursor vesicles have an electron dense core suggesting that they may also deposit a collection of extracellular matrix proteins and trophic factors into the nascent synaptic cleft. These molecules could act, similar to agrin and neuregulin at the neuromuscular junction (Sanes and Lichtman, 1999), promoting the differentiation of postsynaptic sites and the recruitment of the correct complement of neurotransmitter receptors. Of the presynaptic molecules thus far characterized, Piccolo and Bassoon are the first to arrive on the scene of nascent synapse formation implicating both in the establishment and function of new synapses. (Friedman et al., 2000; Zhai et al., 2001). Our initial studies on the structure of Piccolo and Bassoon indicate that these two proteins are structurally related multi-domain proteins sharing 10 regions of homology. These observations suggest that Piccolo and Bassoon share some common function at synapses (Dresbach et al., 2001). Studies presented here reveal that Piccolo has additional domains or regions not present in Bassoon. These are readily detected by comparing the amino acid sequences of Piccolo deduced from cDNAs from rat, mouse and human. This analysis shows that six regions of high homology are present in Piccolo, the largest being in the C-terminal half of Piccolo. The presence of homology domains within Piccolo and with Bassoon strongly suggests that Piccolo and Bassoon are modular proteins. Embedded within several of these PBH domains are sequences found in other proteins. For example, PBH1 and PBH2 contain two double zinc-finger motifs. In a number of cytoskeletal proteins, zinc fingers have been shown to be sites of protein–protein interaction (Sanchez-Garcia and Rabbitts, 1994) indicating that in Piccolo and Bassoon these are also protein–protein interaction sites. This concept is supported by biochemical studies showing that these zinc fingers interact with the Rab3A acceptor protein, PRA1 (Fenster et al., 2000). PBH4, 6 and 8 are likely to form coiled-coil structures commonly

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seen as sites of homo- or heterodimerization (Lupas, 1996). The remaining PBH regions share no similarity with known proteins. At the C-terminus of Piccolo, in homology domain 6 (Fig. 1B), a PDZ and two C2 domains can be found, features that are absent in Bassoon. The PDZ domain in Piccolo is classified as a class I domain (Daniels et al., 1998) and is most similar to PDZ stretches in a third active zone protein called RIM (Wang et al., 1997). Binding partners for the PDZ domain are not known. PDZ domains were originally identified in the MAGUK family of scaffold protein as well as in a growing number of cortical cytoskeletal protein that direct the assembly of macromolecular signaling complexes (see Garner et al., 2000b). PDZ domains interact with proteins containing a C-terminal T/SXV motif such as voltage and ligand gated ion channels as well as cell adhesion molecules (see Garner et al., 2000b). This suggests that the PDZ domain in Piccolo may interact with proteins at the active zone plasma membrane. This may serve to localize this active zone protein to the synapse and position its C2 domains near calcium entry sites through voltage-activated N-type calcium channels. The Piccolo C2A domain contains all the consensus aspartate residues required for calcium binding indicating that Piccolo may respond to changing calcium levels in nerve terminals. C2 domains, first identified in protein kinase C are Ca2+ /phospholipid binding elements found in a number of synaptic proteins (Südhof and Rizo, 1996). In synaptotagmin, calcium in a 20–100 ␮M range regulates the interaction of its C2 domains with syntaxin during the SV fusion step (Sugita et al., 1996). Recent studies on the C2A domain of Piccolo have confirmed that it binds Ca2+ (>100 ␮M) and phospholipids (Gerber et al., 2001) indicating that Piccolo may respond to changing calcium levels in nerve terminals. Our analysis of the structure of the PCLO gene and its different transcripts has revealed that mRNA transcribed from the PCLO gene can be alternatively spliced. Of particular interest is the use of two different translational stop sites situated in exons 20 and 25. The net result of this choice in splicing is the generation of two isoforms of Piccolo. The first ends with the C2A domain, while the second has both C2A and C2B domains. The C2B domain is not predicted to bind calcium. Also, our identification of alternative splicing in exons encoding the C2A domain indicates the existence of translational products with C2A domains that may not bind calcium. These data suggest that Piccolo has both calcium dependent and independent functions. Piccolo also contains a number of proline rich domains (PRD). One found near the PBH4 domain binds the actin binding protein profilin (Wang et al., 1999). A second region upstream of the PBH1 region, called the “Q” domain, contains 12 copies of a degenerated decapeptide in tandem array. This region has been found to interact with the actin binding protein mAbp1 (Fenster et al., 2001). These data suggest that Piccolo is functionally important in coordinating the assembly of actin in nerve terminals. In addition, mAbp1 has been implicated in clathrin-mediated

endocytosis indicating that Piccolo may also indirectly be involved in SV endocytosis, a fundamental feature of nerve terminals (Kessels et al., 2001). Taken together the multi-domain structure and restricted localization of Piccolo at sites of neurotransmitter release and vesicle recycling provides compelling evidence that it could serve as a presynaptic scaffold protein on which components of SV endo- and exocytosis can be specifically sequestered in the presynaptic nerve terminal. Our analysis of the gene structure of mouse and human PCLO revealed that both are very large genes containing more than 25 exons spanning over 350 kb of genomic DNA. Gene mapping experiments showed that the mouse gene is found on chromosome 5 at 5q1, while the human gene maps to 7q11.23-q21.1. Interestingly, the human PCLO locus is situated near a region previously shown to be deleted in patients with Williams Syndrome. The heterozygotic deletion found in Williams Syndrome is very large removing up to 2.5 megabases of genomic DNA (Meng et al., 1998) resulting in the loss of many genes (reviewed by Francke, 1999). A number of neuronal expressed genes map to this loci including PCLO and genes for lim kinase 1, syntaxin 1A, sema3A, and sema3E. Phenotypically, Williams Syndrome is characterized by development malformations, impaired cognitive abilities visual–spatial processing in addition to neurological deficits (Morris et al., 1988). Clearly, the loss of PCLO within this locus would not be expected to cause all of these neurological phenotypes, however together with the other synaptic genes, it could potentially contribute to abnormalities in the development of the CNS. In addition, a genome wide screen for autism susceptibility genes identified a region of the long arm (7q) of human chromosome 7 as a potential linkage site (International Molecular Genetic Study of Autism Consortium, 2001). The generation and characterization of PCLO knockout mice will help resolve this issue.

Acknowledgements The work was supported by the National Institutes of Health (P50 HD32901, NIH RO1 NS39471, P01 AG06569 and Civitan International Research Center).

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