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Home , Cadherin, PCDHA4, PCDHAC2, PCDHGC3, Protocadherin

Protocadherin family: diversity, structure, and function Hirofumi Morishita1 and Takeshi Yagi2

Protocadherins are predominantly expressed in the nervous in the superfamily [3]. The sub- system, and constitute the largest subgroup within the cadherin group has only been identified and characterized in superfamily. The recent structural elucidation of the amino- studies from the past decade. These genetic and func- terminal cadherin domain in an archetypal protocadherin tional studies have revealed a divergent cytoplasmic revealed unique and remarkable features: the lack of an domain, as well as six or seven extracellular cadherin interface for homophilic adhesiveness found in classical (EC) domains with low sequence similarities to the EC , and the presence of loop structures specific to the domains of the classical cadherin subgroup. protocadherin family. The unique features of extend to their genomic organization. Recent findings have Knowledge of protocadherin family at the molecular level revealed unexpected allelic and combinatorial regulation has increased profoundly in the past few years from for clustered protocadherins, a major subgroup in the publications of the first detailed structural and functional protocadherin family. The unique structural repertoire and findings, as well as identification of unusual gene regu- unusual gene regulation of the protocadherin family may lation for this family. In the following, we shall contex- provide the molecular basis for the extraordinary diversity of the tualize the emergent insights into the structure and nervous system. function of the protocadherin family that have accumu- Addresses lated in the findings from the past ten years. 1 Division of Neuroscience, Children’s Hospital Boston, Harvard Medical School, 320 Longwood Ave., Boston, MA 02115, USA Diversity and classification 2 KOKORO-Biology Group, Laboratories for Integrated Biology, Graduate School of Frontier Biosciences, Osaka University, 1-3 The term ‘protocadherin’, originally introduced about a Yamadaoka, Suita, Osaka 565-0871, Japan decade ago, is now used in various ways after the recent expansion of the molecular family [4–7]. Our subsequent Corresponding authors: Morishita, Hirofumi use of the term shall be based on phylogenic study of ([email protected]) and Yagi, Takeshi sequences consisting each cadherin domains. ([email protected]) More specifically, our references to protocadherins will exclude the Flamingo/CELSER subfamily, which con- Current Opinion in Cell Biology 2007, 19:584–592 tains seven-pass transmembrane , and FAT sub- This review comes from a themed issue on family, whose elements consist of far more than seven EC Cell to cell contact and extracellular matrix domains [3,8]. Currently, within the protocadherin Edited by Lawrence Shapiro and Barry Honig family, more than 70 different protocadherin (Pcdh) have been identified, The protocadherin family can be largely divided into two groups based on their genomic 0955-0674/$ – see front matter structure: clustered protocadherins, and nonclustered # 2007 Elsevier Ltd. All rights reserved. protocadherins [8](Figure 1). Clustered protocadherins DOI 10.1016/j.ceb.2007.09.006 are consist of the Pcdha, b,andg family, each of which has a specific genomic organization clustered in a small gen- ome locus [9]. With over 50 members, the clustered Introduction protocadherin constitutes the largest subgroup within The recent explosion in genome sequencing has revealed the protocadherin family. The Pcdha was originally dis- the impressive diversity of the cadherin superfamily. To covered in the mouse brain, and has been referred to as date, more than 100 different cadherins have been ident- the cadherin-related neuronal receptor (CNR) [5]. As ified. Originally characterized as calcium-dependent cell other protocadherins do not have a specific clustered adhesion molecules, cadherin molecules are now known genome locus, here we shall collectively refer to them to be involved in many biological processes, including cell as nonclustered protocadherins. Nonclustered protocad- recognition, cell signaling during embryogenesis, and the herins can be divided into two subgroups: Pcdhd, and formation of neural circuits [1,2]. Cadherins are identified solitary protocadherins in the phylogenic tree (Pcdh-12, by the presence of cadherin sequence repeats of about 15, 20, 21)[8]. The Pcdhd family comprises at least nine 110 amino acids. Several subgroups of cadherins can be protocadherins, all of which contain highly conserved defined based on shared properties and sequence sim- motifs (CM1, CM2) in their cytoplasmic domains. Mem- ilarity: the classical (type I) and closely related type II bers of Pcdhd subfamily can be further divided in two cadherins, desmosomal cadherins, and protocadherins subgroups, Pcdhd1 and d2, on the basis of overall (Figure 1). Protocadherins are predominantly expressed homology, number of EC repeats (seven versus six), in the nervous system, and constitute the largest subgroup and conservation of specific amino acid motifs in

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Figure 1

Classification of cadherin superfamily and protocadherin family. (a) Classification and schematic diagram of cadherin superfamily. Classical type I cadherins have a conserved tryptophan (W2) in EC1 domain, and a hydrophobic pocket to accommodate W2 of the other EC1, which are crucial for homophilic adhesiveness. The prodomain will be removed to mediate functional adhesion. Highly related type II cadherins are slightly different from type I cadherins in that they have two conserved tryptophan residues (W2 and W4) and the hydrophobic pocket are correspondingly extensive. The cytoplasmic regions of classical cadherins have binding site which links to the . Desmosomal cadherins are similar to type I cadherins in that they have a conserved W2 and five EC domains, but have distinctive cytoplasmic regions. The protocadherin family is strikingly different from other cadherins in that they neither have W2 nor hydrophobic pocket but they have characteristic disulfide-bonded loop in EC1 domain. Their cytoplasmic regions do not have catenin binding site. The protocadherin family can be divided into two subgroups; clustered, and nonclustered protocadherin based on the genomic organization. Clustered protocadherins have six EC domains. Nonclustered protocadherins have various number of EC domains. Proteins that contain an identifiable cadherin-like domain have been loosely referred to others. (b) Classification of mouse protocadherin family. Protocadherin family can be divided into two groups based on their genomic structures: the clustered, and nonclustered protocadherins. Clustered Protocadherins are consisted of Pcdha, b and g families, which are clustered in a small genome locus. Nonclustered protocadherins can be divided into two subgroups, Pcdhd family, and other solitary protocadherins. All Pcdhd contains highly conserved motifs (CM1, CM2) in their cytoplasmic domains. They can be further divided in two subgroups, d1 and d2. Number of members for each group is also mentioned in the figure. cytoplasmic domains. Nine protocadherins have been interfaces of classical cadherin has been shown to be classified from existing research: Pcdh-1, 7, 9, and 11(X/ localized primarily within the amino-terminal EC1 domain Y) in the d1 subgroup, and Pcdh-8, 10, 17, 18, and 19 in the [11,12]. The new report of the solution protein-structure d2 subgroup. of EC1 domain of protocadherin (CNR/Pcdha4), which was determined by NMR, assisted the elucidation of Protein structure and adhesive property the character of protocadherin proteins compared with In the case of classical cadherins, structural biology has classical cadherins at an atomic level [13,14]. Despite provided fruitful insights to reveal the molecular basis for low sequence similarities between the EC1 domains of [10]. The homophilic adhesive binding CNR/Pcdha4 and of classical cadherins (30% at the www.sciencedirect.com Current Opinion in Cell Biology 2007, 19:584–592 586 Cell to cell contact and extracellular matrix

maximum), the overall topology was similar to classical side chain of W2. The lack of a hydrophobic pocket cadherins: a b-sandwich-like structure composed of two suggests that the homophilic adhesion interface that is packed b-sheets (Figure 2). By contrast, the interconnect- important for classical cadherins does not exist in proto- ing loops of protocadherins were quite divergent from the cadherins [14]. classical cadherins, both in length and in biochemical structure. The structural characterization of the protocadherin EC1 domain also revealed crucial variations mainly in the loop A remarkable finding in the structural elucidation was regions, including the protocadherin-specific disulfide that the CNR/Pcdha4 EC1 domain does not have trypto- bonded Cys-(X)5-Cys motif, and the RGD (Arg-Gly-  phan (W2) or a hydrophobic pocket, which is essential for Asp) motif [14 ](Figure 2). The Cys-(X)5-Cys sequence the adhesiveness of the classical cadherins [11,14] is well conserved among the clustered and non clustered (Figures 2 and 3). Among the classical, type II, and protocadherins, but not in classical cadherins. Structural desmosomal cadherins, the amino acid residues consti- analysis of the amino acid sequence revealed that the tuting the hydrophobic pocket are almost completely Cys-(X)5-Cys sequence is not only exposed to solvent as a conserved [10]. Although the CNR/Pcdh-a4 EC1 domain loop, but also forms a disulfide bond between the cysteine has a hydrophobic cluster in the corresponding region, the amino acids. Generally, the disulfide-bonded loops of pocket is not as deep as that in classical cadherins, in Cys-X5-Cys sequences are involved in the protein-protein which the pocket is large enough to accommodate the interactions of viral . This example suggests

Figure 2

Protein structure of protocadherin EC1 domain. (a) The ribbon diagram of the representative NMR structure, and (b) the schematic Greek key topology of the EC1 domain of mouse Protocadherina4. The b-strands are shown in green (bB, bD, and bE) and blue (bA, bC, bF, and bG), representing the two different b-sheets. In (b) and (c), each loop is colored differently for clarity. Conserved calcium-binding residues and the disulfide-bonded loop between Cys70 and Cys76 are shown in red and yellow, respectively. (c) Structure-based alignment of the CNR/Pcdha4 EC1 domain with the EC1 domains of classical cadherins whose structures have been determined. Secondary structure elements of Pcdha are indicated by green and sky blue arrows, and those of the classical cadherins are indicated by gray arrows. The residues responsible for calcium binding are highlighted in blue; the residues in the adhesion interface of the classical cadherins and the corresponding residues forming a hydrophobic cluster in Pcdha4 are shown in red; the RGD motif conserved in the Pcdha family in shown in green; and the disulfide-bonded sequence between Cys70 and Cys76 is highlighted in orange. Adapted from [14].

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Figure 3

Comparison of adhesive interface of EC1 domain of protocadherin and classical cadherin. (a) Shown are backbone structure models of EC1 domain of protocadherin and classical cadherin viewed from the N terminus. Residues that form the hydrophobic pocket that is important for adhesiveness in classical cadherins (N-cadherin) and the corresponding cluster of hydrophobic residues in Pcdha are highlighted with their side chains shown in red. The short side chains of Ala78 and Ala80 contribute to the formation of the deep hydrophobic space in N-cadherin, whereas the corresponding amino acids in CNR/Pcdha4 (Val82 and Val84) have bulkier side chains that make the cavity smaller. Moreover, some other bulky amino acid residues located in loop BC (Leu26, Leu28, and Leu33) participate in the hydrophobic cluster, filling the hydrophobic cavity and diminishing the open volume of the pocket. (b) Beads aggregation assays can be used to study homophilic adhesive property of protocadherins compared with classical cadherins. The extracellular domains of cadherins were tagged at their C termini with the Fc region of human IgG. Chimeric Fc proteins were captured via their Fc tags on fluorescent beads (0.39 mm) coated with anti-Fc antibody. Visualization of the beads by fluorescence microscopy at the 120-min time point revealed aggregates of the ectodomain of full-length N-cadherin-coated beads (Ncad-Fc), but not of the Pcdha4-coated beads (Pcdha-Fc), and control Fc. Notably, the beads coated with chimeric Fc proteins in which the EC1 domain of N-cadherin is replaced with the EC1 domain of Pcdha4 (PcdhaEC1/Ncad-Fc) exhibited no aggregation. Adapted from [14].

that the Cys-X5-Cys sequence of the protocadherin Pcdhg [18]) are observed, reflecting the protein complex family could function as a novel adhesion interface. formation on the same cell surface. From the current An RGD motif, well known from ligands, is findings, we cannot reliably preclude the possibility that shown in the EC1 of CNR/Pcdha4 to be placed on a protocadherins can express trans-homophilic adhesive loop and exposed to the solvent. activity after forming protein complexes with additional molecules. Consistent with the structural character of protocadherin protein, biochemical assessment of extracellular Pcdha Protocadherins that have been studied exhibit either protein, performed with protein coated bead aggregation exhibit no homophilic adhesion activity, or have been assay that reflect intercellular cell adhesion activity, suggested to mediate weak adhesion based on limited showed little trans-homophilic adhesion activity com- evidence ([14,15,19–21,22] for clustered protocadher- pared with that of classical cadherins [14,15]. Moreover, ins, and [23,24] for nonclustered protocadherins). It is swapping the EC1 domain of N-cadherin with the EC1 unclear whether the weak cell adhesion by some of these domain of CNR/Pcdha4 results in complete loss the protocadherins reflects the true homophilic cell adhesion robust aggregation of full length extracellular N- function at physiological levels of expression or is actually cadherin coated beads, suggesting that the difference involved in other functions such as of adhesion activity between classical cadherin and pro- [25]. tocadherin might be at least in part attributed to the structural difference found in EC1 domains (Figure 3) On the other hand, heterophilic cell adhesion activity has [14]. On the other hand, cis-homodimer (Pcdha-Pcdha been reported between the Pcdha4 and the b1 integrin in [16], Pcdhg-Pcdhg [17]), and cis-heterodimer (Pcdha and an in vitro cell aggregation assay in the HEK293T cell line www.sciencedirect.com Current Opinion in Cell Biology 2007, 19:584–592 588 Cell to cell contact and extracellular matrix

[15]. Remarkably, integrin activation is necessary for cells this does less appear to be the case. Instead, clustered to bind to immobilized EC1 fragments of CNR/Pcdha4 protocadherin transcripts are generated by cis alternative [14]. The RGD motif, which is an essential residue for splicing with multiple promoters (almost including highly integrin-dependent cell adhesion activity, is found among conserved CSE sequence), leading to the production of a mammalian Pcdha family members. Researchers have large number of isoforms with various extracellular yet to conclude whether other protocadherins without domain sequences [39,40]. RGD motif also have heterophilic adhesion activity like Pcdha. Clustered protocadherins are present during neural de- velopment and gradually become enriched at synapses One member of the nonclustered protocadherin can [5,21,41–45], and the expression of Pcdh genes decreases modify cell adhesion by regulating the adhesion activity after neurons mature and become myelinated [46,47]. of a classical cadherin. Xenopus laevis paraxial protocad- Individual Pcdh isoforms do not appear to be expressed herin (PAPC), putative mammalian Pcdh-8 homolog, has in specific brain layers or brain nuclei. In fact, in situ been shown to play an essential role in boundary for- hybridization analyses have shown that single neurons of mation and the cell sorting during the early development the same cell type express different Pcdha [5,48] and of the X. laevis embryos [26]. However, PAPC does not Pcdhg isoforms [40], respectively. participate in early cell sorting through homophilic cell– cell adhesion; instead, PAPC modulates classical C-cad- Recent reports are beginning to elucidate the unique herin adhesion through an unknown mechanism [25,27]. mechanism of Pcdh gene expression. Single-cell analysis of Purkinje cells using multiple RT-PCR reactions [49] Thus, the interpretation of data derived from cell aggre- revealed that the Pcdha and Pcdhg isoforms have unusual gation assays should be a cautious one. Researchers monoallelic and combinatorial expression in individual should be careful to rule out the possibility of heterophilic neurons [48,50](Figure 4). Single-cell PCR analyses of interaction via endogenously expressed adhesion mol- Purkinje cells showed that individual neurons express ecules (e.g. Pcdha), or indirect effects through other apparently random sets of Pcdha1–12 mRNAs, with the adhesion molecules (e.g. PAPC). Indeed, the published majority of cells expressing only two. Remarkably, the results of cell aggregation activities observed by expres- same Pcdha1–12 isoform can be expressed from both sing protocadherins should be re-evaluated with these , but this dual expression is rarely seen, points in mind. suggesting that the choice of promoters is stochastic within each and independent between Protocadherin functions can be regulated by proteolysis. chromosomes [48]. In the follow up study, total allelic Recent research demonstrated the specific cleavage of gene regulation in the Pcdha and g clusters, including the Pcdha, and Pcdhg proteins by ADAM10 and presenilin C-type variable exons (C1–C5) and the PcdhgA and gB [17,22,28,29]. In addition to modulating the cell variable exons in single Purkinje cells were examined adhesion [22] and the formation of Pcdha and Pcdhg [50]. Almost all of the Purkinje cells biallelically cis-heterodimer complex [29], proteolysis of Pcdha and expressed all the C-type isoforms, whereas the Pcdha, Pcdhg generates a cytoplasmic fragment that localizes to PcdhgAandgB isoforms showed differential regulation in the nucleus [17,28,29], and the Pcdhg cytoplasmic each cell with both monoallelic and combinatorial gene domain can transactivate all the Pcdhg promoters (but regulation. These results indicated that different types of not depend on the conserved element (CSE)) [17]. The allelic gene regulation (monoallelic and biallelic) biological role of this process is currently unknown. occurred in the Pcdha and g clusters, and that these genes were spliced into the same constant exons. Gene regulation, expression, and function The genes for the nonclustered protocadherin subfamily In an effort to uncover the mechanisms of monoallelic produce alternative splicing variants, but do not encode expression of the Pcdha genes, long-range cis-regulatory variable extracellular domains, resulting in only small DNA elements in the Pcdha gene cluster have been variations [8]. By contrast, the genes for clustered proto- recently identified [51]. Two of the DNA sequences, cadherins (Pcdha, b and g) are sequentially organized, HS5-1, and HS7, display enhancer activity in reporter generating more than 50 transcripts from the three gene assays. Additionally, HS-5 is necessary for high-level clusters [6,7]. Each of the Pcdha and g gene clusters expression from all Pcdha1–12 promoters, as well as the contain multiple ‘variable’ exons, as well as a set of PcdhaC1 promoter. By contrast, the element is not ‘constant’ exons (Figure 4). The number and sequence required for expression from the PcdhaC2 promoter. of variable exons differ across vertebrate species [9,30– The requirement of the HS5–1 element for the majority 38]. The structural similarity between clustered proto- of the cluster constituents is consistent with the possib- cadherin and immunoglobulin gene raised the possibility ility that monoallelic expression of Pcdha variable exon is that clustered protocadherins undergo DNA rearrange- a consequence of competition between individual vari- ments similar to that of immunoglobulin genes. However, able exon promoters for the regulatory elements. In

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Figure 4

Organization and regulation of protocadherin gene cluster. (a) The genomic organization of mouse Pcdha, Pcdhb, and Pcdhg gene clusters. In the 18c region of mouse chromosome 18, there are three gene clusters of Pcdha, Pcdhb and Pcdhg. The mouse Pcdha gene cluster consists of 14 variable exons and a set of three constant region exons. Mature mRNAs of Pcdha are generated from one of these variable exons and a set of three constant region exons. The PcdhaC1 and C2 variable region exons exhibit greater similarity to PcdhgC3, C4 and C5 rather than to the other Pcdha variable region exons (green boxes). (b) A model of the two distinct types of gene regulation in the Pcdha and Pcdhg clusters in single Purkinje cells. The widths of the arrows indicate the expression levels of each exon. The Pcdha1 to a12 and PcdhgA and gB exons show monoallelic and combinatorial expression. By contrast, the C-type Pcdha and Pcdhg exons are expressed biallelically and constitutively. This model shows one of the example of one Purkinje cell; thus another Purkinje cell expresses a different sets of Pcdha1 to a12 and PcdhgA and gB exons. The r1 and r2 in the C57BL/6 (B6) allele represent relic 1 and relic 2 pseudogenes, respectively. Adapted from [50]. addition to long-range cis-regulatory elements, recent To gain insight into in vivo function of protocadherin wide analysis of the binding sites of family, genetic studies have been conducted [41,54]. insulator protein CCCTC-binding factor (CTCF) Deletion of the entire cluster of Pcdhg genes resulted revealed that CTCF binding sites punctuate the alterna- in partial apoptotic loss of spinal interneurons, and a tive promoters in the Pcdha and Pcdhg cluster locus, reduction in spinal synaptic density [41]. To further raising the possibility of the involvement of insulator investigate the specific roles of Pcdhg in synaptogenesis, elements in the selection of promoters in distinct cells apoptosis can be minimized by removing BAX, or by [52,53]. Further studies aimed at revealing the mechan- using a hypomorphic allele of Pcdhg [54]. In these isms of expression of the Pcdh gene cluster will be mutant mice, the spinal cord still shows decreased synap- particularly valuable for understanding the molecular tic density and the activity of the formed synapses is mechanisms involved in generating the diversity of indi- reduced, providing the first evidence for a role of this vidual neurons. protocadherin in synaptic development. The lack of www.sciencedirect.com Current Opinion in Cell Biology 2007, 19:584–592 590 Cell to cell contact and extracellular matrix

general effects of Pcdhg deficiency on the nervous system 5. Kohmura N, Senzaki K, Hamada S, Kai N, Yasuda R, Watanabe M, Ishii H, Yasuda M, Mishina M, Yagi T: Diversity revealed by a may be because of compensation by Pcdha and b. How- novel family of cadherins expressed in neurons at a synaptic ever, the localized effects of deletion of Pcdhg on inter- complex. Neuron 1998, 20:1137-1151. neurons of the spinal cord also indicate that expression of 6. Wu Q, Maniatis T: A striking organization of a large family of human neural cadherin-like cell adhesion genes. Cell 1999, different members of the protocadherin family might 97:779-790. specify both the survival and synaptic organization of 7. Sugino H, Hamada S, Yasuda R, Tuji A, Matsuda Y, Fujita M, different neuronal populations. 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Hydrophobic Remarkably, recent biochemical and fly genetic findings binding interface for homophilic adhesion was more extensive than the suggested that the diversity of DSCAM provides each case of type1 cadherins to accommodate two conserved tryptophan side chains, which is one in type 1 cadherins. This structure shows good neuron with a unique identity that enables it to dis- contrast to the structure of protocadherin, which lacks tryptophan and tinguish between self and non-self cells [56]. For has little hydrophobic pocket. vertebrates, the DSCAM gene does not encode multiple 13. Umitsu M, Morishita H, Murata Y, Udaka K, Akutsu H, Yagi T, isoforms. From the current findings, extremely interest- Ikegami T: 1H, 13C and 15N resonance assignments of the first cadherin domain of Cadherin-related neuronal receptor ing findings are expected from further research on the (CNR)/protocadherin alpha. J Biomol NMR 2005, 31:365-366. significance of the diversity of protocadherin family in 14. 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