Proteomics Analysis Reveals Overlapping Functions of Clustered Protocadherins
2009; Elsevier BV; Volume: 9; Issue: 1 Linguagem: Inglês
10.1074/mcp.m900343-mcp200
ISSN1535-9484
AutoresMeng-Hsuan Han, Chengyi Lin, Shuxia Meng, Xiaozhong Wang,
Tópico(s)Hedgehog Signaling Pathway Studies
ResumoThe three tandem-arrayed protocadherin (Pcdh) gene clusters, namely Pcdh-α, Pcdh-β, and Pcdh-γ, play important roles in the development of the vertebrate central nervous system. To gain insight into the molecular action of PCDHs, we performed a systematic proteomics analysis of PCDH-γ-associated protein complexes. We identified a list of 154 non-redundant proteins in the PCDH-γ complexes. This list includes nearly 30 members of clustered Pcdh-α, -β, and -γ families as core components of the complexes and additionally over 120 putative PCDH-associated proteins. We validated a selected subset of PCDH-γ-associated proteins using specific antibodies. Analysis of the identities of PCDH-associated proteins showed that the majority of them overlap with the proteomic profile of postsynaptic density preparations. Further analysis of membrane protein complexes revealed that several validated PCDH-γ-associated proteins exhibit reduced levels in Pcdh-γ-deficient brain tissues. Therefore, PCDH-γs are required for the integrity of the complexes. However, the size of the overall complexes and the abundance of many other proteins remained unchanged, raising a possibility that PCDH-αs and PCDH-βs might compensate for PCDH-γ function in complex formation. As a test of this idea, RNA interference knockdown of both PCDH-αs and PCDH-γs showed that PCDHs have redundant functions in regulating neuronal survival in the chicken spinal cord. Taken together, our data provide evidence that clustered PCDHs coexist in large protein complexes and have overlapping functions during vertebrate neural development. The three tandem-arrayed protocadherin (Pcdh) gene clusters, namely Pcdh-α, Pcdh-β, and Pcdh-γ, play important roles in the development of the vertebrate central nervous system. To gain insight into the molecular action of PCDHs, we performed a systematic proteomics analysis of PCDH-γ-associated protein complexes. We identified a list of 154 non-redundant proteins in the PCDH-γ complexes. This list includes nearly 30 members of clustered Pcdh-α, -β, and -γ families as core components of the complexes and additionally over 120 putative PCDH-associated proteins. We validated a selected subset of PCDH-γ-associated proteins using specific antibodies. Analysis of the identities of PCDH-associated proteins showed that the majority of them overlap with the proteomic profile of postsynaptic density preparations. Further analysis of membrane protein complexes revealed that several validated PCDH-γ-associated proteins exhibit reduced levels in Pcdh-γ-deficient brain tissues. Therefore, PCDH-γs are required for the integrity of the complexes. However, the size of the overall complexes and the abundance of many other proteins remained unchanged, raising a possibility that PCDH-αs and PCDH-βs might compensate for PCDH-γ function in complex formation. As a test of this idea, RNA interference knockdown of both PCDH-αs and PCDH-γs showed that PCDHs have redundant functions in regulating neuronal survival in the chicken spinal cord. Taken together, our data provide evidence that clustered PCDHs coexist in large protein complexes and have overlapping functions during vertebrate neural development. The development of neural circuitry involves a complex interplay of cell-cell adhesion, interneuronal signaling, and assembly of intracellular macromolecular protein complexes. A group of protocadherin genes, known as clustered protocadherins (Pcdhs), 1The abbreviations used are:PCDHprotocadherinBNblue nativePBPiggyBacPSDpostsynaptic densityRNAiRNA interferenceSGsucrose gradientSNIPSNAP-25-interacting-protein2Dtwo-dimensionalGFPgreen fluorescent proteinCAMKCa2+/calmodulin-dependent protein kinaseTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineLTQlinear trap quadrupoleshRNAshort hairpin RNAIPimmunoprecipitationFffireflyLucluciferaseRnRenillaPBasePiggyBac transposasePpostnatal dayCMVcytomegalovirus. 1The abbreviations used are:PCDHprotocadherinBNblue nativePBPiggyBacPSDpostsynaptic densityRNAiRNA interferenceSGsucrose gradientSNIPSNAP-25-interacting-protein2Dtwo-dimensionalGFPgreen fluorescent proteinCAMKCa2+/calmodulin-dependent protein kinaseTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineLTQlinear trap quadrupoleshRNAshort hairpin RNAIPimmunoprecipitationFffireflyLucluciferaseRnRenillaPBasePiggyBac transposasePpostnatal dayCMVcytomegalovirus. are among the transmembrane signaling molecules that are implicated in this developmental process (1Benson D.L. Colman D.R. Huntley G.W. Molecules, maps and synapse specificity.Nat. Rev. Neurosci. 2001; 2: 899-909Crossref PubMed Scopus (145) Google Scholar, 2Takeichi M. The cadherin superfamily in neuronal connections and interactions.Nat. Rev. Neurosci. 2007; 8: 11-20Crossref PubMed Scopus (331) Google Scholar). Protocadherins are type I transmembrane proteins, which share significant sequence homology with classic cadherins in their extracellular domain but possess distinct intracellular domains (3Sano K. Tanihara H. Heimark R.L Obata S. Davidson M. St John T. Taketani S. Suzuki S. Protocadherins: a large family of cadherin-related molecules in central nervous system.EMBO J. 1993; 12: 2249-2256Crossref PubMed Scopus (328) Google Scholar, 4Morishita H. Yagi T. Protocadherin family: diversity, structure, and function.Curr. Opin. Cell Biol. 2007; 19: 584-592Crossref PubMed Scopus (164) Google Scholar, 5Halbleib J.M. Nelson W.J. Cadherins in development: cell adhesion, sorting, and tissue morphogenesis.Genes Dev. 2006; 20: 3199-3214Crossref PubMed Scopus (774) Google Scholar).The vertebrate genomes encode nearly 60 protocadherin genes in three gene clusters including Pcdh-α, Pcdh-β, and Pcdh-γ, which are tandem-arrayed on the same chromosome and transcribed in the same direction (6Wu Q. Maniatis T. A striking organization of a large family of human neural cadherin-like cell adhesion genes.Cell. 1999; 97: 779-790Abstract Full Text Full Text PDF PubMed Scopus (541) Google Scholar, 7Noonan J.P. Grimwood J. Schmutz J. Dickson M. Myers R.M. Gene conversion and the evolution of protocadherin gene cluster diversity.Genome Res. 2004; 14: 354-366Crossref PubMed Scopus (92) Google Scholar, 8Wu Q. Comparative genomics and diversifying selection of the clustered vertebrate protocadherin genes.Genetics. 2005; 169: 2179-2188Crossref PubMed Scopus (69) Google Scholar). Individual Pcdh-α and Pcdh-γ transcripts contain a variable exon and a cluster-specific constant exon, whereas Pcdh-βs are encoded by a single variable exon. Clustered Pcdhs are expressed predominantly in the central nervous system, and individual neurons express distinct combinations of Pcdhs through a mechanism involving multiple promoter activation and allelic exclusion (9Kohmura N. Senzaki K. Hamada S. Kai N. Yasuda R. Watanabe M. Ishii H. Yasuda M. Mishina M. Yagi T. Diversity revealed by a novel family of cadherins expressed in neurons at a synaptic complex.Neuron. 1998; 20: 1137-1151Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 10Wang X. Su H. Bradley A. Molecular mechanisms governing Pcdh-gamma gene expression: evidence for a multiple promoter and cis-alternative splicing model.Genes Dev. 2002; 16: 1890-1905Crossref PubMed Scopus (152) Google Scholar, 11Tasic B. Nabholz C.E. Baldwin K.K. Kim Y. Rueckert E.H. Ribich S.A. Cramer P. Wu Q. Axel R. Maniatis T. Promoter choice determines splice site selection in protocadherin alpha and gamma pre-mRNA splicing.Mol. Cell. 2002; 10: 21-33Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 12Frank M. Ebert M. Shan W. Phillips G.R. Arndt K. Colman D.R. Kemler R. Differential expression of individual gamma-protocadherins during mouse brain development.Mol. Cell. Neurosci. 2005; 29: 603-616Crossref PubMed Scopus (79) Google Scholar, 13Zou C. Huang W. Ying G. Wu Q. Sequence analysis and expression mapping of the rat clustered protocadherin gene repertoires.Neuroscience. 2007; 144: 579-603Crossref PubMed Scopus (48) Google Scholar, 14Esumi S. Kakazu N. Taguchi Y. Hirayama T. Sasaki A. Hirabayashi T. Koide T. Kitsukawa T. Hamada S. Yagi T. Monoallelic yet combinatorial expression of variable exons of the protocadherin-alpha gene cluster in single neurons.Nat. Genet. 2005; 37: 171-176Crossref PubMed Scopus (199) Google Scholar, 15Kaneko R. Kato H. Kawamura Y. Esumi S. Hirayama T. Hirabayashi T. Yagi T. Allelic gene regulation of Pcdh-alpha and Pcdh-gamma clusters involving both monoallelic and biallelic expression in single Purkinje cells.J. Biol. Chem. 2006; 281: 30551-30560Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 16Ribich S. Tasic B. Maniatis T. Identification of long-range regulatory elements in the protocadherin-alpha gene cluster.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 19719-19724Crossref PubMed Scopus (93) Google Scholar). PCDH proteins are targeted partly to synapses (9Kohmura N. Senzaki K. Hamada S. Kai N. Yasuda R. Watanabe M. Ishii H. Yasuda M. Mishina M. Yagi T. Diversity revealed by a novel family of cadherins expressed in neurons at a synaptic complex.Neuron. 1998; 20: 1137-1151Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 17Phillips G.R. Tanaka H. Frank M. Elste A. Fidler L. Benson D.L. Colman D.R. Gamma-protocadherins are targeted to subsets of synapses and intracellular organelles in neurons.J. Neurosci. 2003; 23: 5096-5104Crossref PubMed Google Scholar, 18Junghans D. Heidenreich M. Hack I. Taylor V. Frotscher M. Kemler R. Postsynaptic and differential localization to neuronal subtypes of protocadherin beta16 in the mammalian central nervous system.Eur. J. Neurosci. 2008; 27: 559-571Crossref PubMed Scopus (41) Google Scholar, 19Wang X. Weiner J.A. Levi S. Craig A.M. Bradley A. Sanes J.R. Gamma protocadherins are required for survival of spinal interneurons.Neuron. 2002; 36: 843-854Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar), and PCDHs have been shown to mediate both homophilic and heterophilic cell-cell adhesion (12Frank M. Ebert M. Shan W. Phillips G.R. Arndt K. Colman D.R. Kemler R. Differential expression of individual gamma-protocadherins during mouse brain development.Mol. Cell. Neurosci. 2005; 29: 603-616Crossref PubMed Scopus (79) Google Scholar, 20Mutoh T. Hamada S. Senzaki K. Murata Y. Yagi T. Cadherin-related neuronal receptor 1 (CNR1) has cell adhesion activity with beta1 integrin mediated through the RGD site of CNR1.Exp. Cell Res. 2004; 294: 494-508Crossref PubMed Scopus (43) Google Scholar, 21Obata S. Sago H. Mori N. Rochelle J.M. Seldin M.F. Davidson M. St John T. Taketani S. Suzuki S.T. Protocadherin Pcdh2 shows properties similar to, but distinct from, those of classical cadherins.J. Cell Sci. 1995; 108: 3765-3773Crossref PubMed Google Scholar, 22Fernández-Monreal M. Kang S. Phillips G.R. Gamma-protocadherin homophilic interaction and intracellular trafficking is controlled by the cytoplasmic domain in neurons.Mol. Cell. Neurosci. 2009; 40: 344-353Crossref PubMed Scopus (54) Google Scholar). Two functions of clustered PCDHs in neural development have emerged from genetic studies on Pcdh-γ and Pcdh-α clusters. First, PCDHs are essential for the survival of specific neuronal subtypes. A dramatic increase of apoptosis in spinal interneurons and retina ganglion cells has been observed in Pcdh-γ-deleted mice (19Wang X. Weiner J.A. Levi S. Craig A.M. Bradley A. Sanes J.R. Gamma protocadherins are required for survival of spinal interneurons.Neuron. 2002; 36: 843-854Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 23Lefebvre J.L. Zhang Y. Meister M. Wang X. Sanes J.R. {gamma}-Protocadherins regulate neuronal survival but are dispensable for circuit formation in retina.Development. 2008; 135: 4141-4151Crossref PubMed Scopus (110) Google Scholar, 24Prasad T. Wang X. Gray P.A. Weiner J.A. A differential developmental pattern of spinal interneuron apoptosis during synaptogenesis: insights from genetic analyses of the protocadherin-{gamma} gene cluster.Development. 2008; 135: 4153-4164Crossref PubMed Scopus (80) Google Scholar). Similarly, Pcdh-α knockdown by morpholino in zebrafish causes neuronal loss during neurogenesis (25Emond M.R. Jontes J.D. Inhibition of protocadherin-alpha function results in neuronal death in the developing zebrafish.Dev. Biol. 2008; 321: 175-187Crossref PubMed Scopus (32) Google Scholar). Second, PCDHs play a role in the establishment of neuronal connectivity. Abnormal axon convergence of olfactory sensory neurons has been shown in Pcdh-α mutant mice (26Hasegawa S. Hamada S. Kumode Y. Esumi S. Katori S. Fukuda E. Uchiyama Y. Hirabayashi T. Mombaerts P. Yagi T. The protocadherin-alpha family is involved in axonal coalescence of olfactory sensory neurons into glomeruli of the olfactory bulb in mouse.Mol. Cell. Neurosci. 2008; 38: 66-79Crossref PubMed Scopus (99) Google Scholar), and synaptic development is severely impaired in the spinal cord of Pcdh-γ-null and hypomorphic mice (27Weiner J.A. Wang X. Tapia J.C. Sanes J.R. Gamma protocadherins are required for synaptic development in the spinal cord.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 8-14Crossref PubMed Scopus (158) Google Scholar).Despite the importance of PCDHs in vertebrate neural development, it is still unclear how PCDHs mediate their function at the molecular level. Several binding partners of clustered PCDHs have been reported. For example, the Src family tyrosine kinase FYN, neurofilament M, and fascin were found to interact with PCDH-αs (9Kohmura N. Senzaki K. Hamada S. Kai N. Yasuda R. Watanabe M. Ishii H. Yasuda M. Mishina M. Yagi T. Diversity revealed by a novel family of cadherins expressed in neurons at a synaptic complex.Neuron. 1998; 20: 1137-1151Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 28Triana-Baltzer G.B. Blank M. Cytoplasmic domain of protocadherin-alpha enhances homophilic interactions and recognizes cytoskeletal elements.J. Neurobiol. 2006; 66: 393-407Crossref PubMed Scopus (24) Google Scholar). PCDH-αs have a heterophilic, calcium-dependent cell adhesion activity with β1 integrin (20Mutoh T. Hamada S. Senzaki K. Murata Y. Yagi T. Cadherin-related neuronal receptor 1 (CNR1) has cell adhesion activity with beta1 integrin mediated through the RGD site of CNR1.Exp. Cell Res. 2004; 294: 494-508Crossref PubMed Scopus (43) Google Scholar). PCDH-γ isoform B1 can interact with the microtubule-destabilizing protein SCG10 (29Gayet O. Labella V. Henderson C.E. Kallenbach S. The b1 isoform of protocadherin-gamma (Pcdhgamma) interacts with the microtubule-destabilizing protein SCG10.FEBS Lett. 2004; 578: 175-179Crossref PubMed Scopus (14) Google Scholar). PCDH-αs and PCDH-γs are also associated with metalloproteinases and γ-secretases in both extracellular and cytoplasmic domains and undergo proteolytic cleavage (30Haas I.G. Frank M. Véron N. Kemler R. Presenilin-dependent processing and nuclear function of gamma-protocadherins.J. Biol. Chem. 2005; 280: 9313-9319Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 31Hambsch B. Grinevich V. Seeburg P.H. Schwarz M.K. {gamma}-Protocadherins, presenilin-mediated release of C-terminal fragment promotes locus expression.J. Biol. Chem. 2005; 280: 15888-15897Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 32Bonn S. Seeburg P.H. Schwarz M.K. Combinatorial expression of alpha- and gamma-protocadherins alters their presenilin-dependent processing.Mol. Cell. Biol. 2007; 27: 4121-4132Crossref PubMed Scopus (43) Google Scholar). Recently, we demonstrated that PCDH-αs and PCDH-γs interact with two tyrosine kinases, focal adhesion kinase and PYK2, and negatively regulate their activities (33Chen J. Lu Y. Meng S. Han M.H. Lin C. Wang X. alpha- and gamma-Protocadherins negatively regulate PYK2.J. Biol. Chem. 2009; 284: 2880-2890Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). In addition, among clustered PCDHs, PCDH-αs and PCDH-γs appear to be in complex with each other (28Triana-Baltzer G.B. Blank M. Cytoplasmic domain of protocadherin-alpha enhances homophilic interactions and recognizes cytoskeletal elements.J. Neurobiol. 2006; 66: 393-407Crossref PubMed Scopus (24) Google Scholar, 34Murata Y. Hamada S. Morishita H. Mutoh T. Yagi T. Interaction with protocadherin-gamma regulates the cell surface expression of protocadherin-alpha.J. Biol. Chem. 2004; 279: 49508-49516Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Taken together, these data suggest that PCDHs might function in large protein complexes that intersect with multiple intracellular signaling pathways.As an important step to understand molecular action of clustered protocadherins, we performed a systematic proteomics survey of PCDH-γ-associated protein complexes. Here, we show that PCDH-γs are present in large macromolecular complexes of ∼1,000 kDa using both sucrose gradient (SG) ultracentrifugation and two-dimensional blue-native (2D BN)/SDS-PAGE methods. To further define the molecular composition of the complexes, we isolated PCDH-γ-associated complexes by affinity purification and identified proteins through mass spectrometry. From this analysis, we identified 142 putative PCDH-γ-associated proteins. We validated a selected set of the mass spectrometry-identified proteins in the PCDH-γ complexes. A number of PCDH-α and PCDH-β isoforms were found in complex with PCDH-γ in the brain, suggesting that they are present in similar functional complexes. We further confirmed that PCDH-α, -β, and -γ subfamily isoforms can form complexes with each other in HEK293T cells. Moreover, simultaneous knockdown of both PCDH-αs and PCDH-γs induced apoptosis in the developing chicken spinal cord. Thus, our data provide a molecular repertoire of PCDH complexes and demonstrate overlapping functions of clustered PCDHs.DISCUSSIONGenetic studies have demonstrated that clustered PCDHs play important roles in regulating neuronal survival and synaptic connectivity in the central nervous system. To understand the molecular mechanisms of PCDH signaling, in this study, we performed a proteomics profiling of PCDH-γ protein complexes. Our analysis led to identification of nearly 140 PCDH-γ-associated proteins including cytoskeleton components, cell adhesion molecules, vesicle trafficking/transport proteins, and signaling molecules (see supplemental Table S1). Strikingly, the vast majority of PCDH-associated proteins are previously identified by proteomics analysis as components of postsynaptic density complexes. Thus, our list provides molecular evidence that PCDHs interact with synapses. Furthermore, this list will facilitate future studies to dissect the function of clustered PCDHs at the molecular level.Although the verification and functional analysis of each protein on our list are beyond the scope of the current study, it is worth discussing several candidate proteins and their potential links to protocadherin functions. For most of the identified proteins, we do not know whether their associations with PCDHs are direct or indirect. It is interesting to note that 14-3-3, an important signaling adaptor protein (52Jin J. Smith F.D. Stark C. Wells C.D. Fawcett J.P. Kulkarni S. Metalnikov P. O'Donnell P. Taylor P. Taylor L. Zougman A. Woodgett J.R. Langeberg L.K. Scott J.D. Pawson T. Proteomic, functional, and domain-based analysis of in vivo 14–3-3 binding proteins involved in cytoskeletal regulation and cellular organization.Curr. Biol. 2004; 14: 1436-1450Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar), is present in PCDH-γ complexes. This protein has also been identified in our yeast two-hybrid screen using the common cytoplasmic domain of PCDH-γs (data not shown). Furthermore, bacterially expressed 14-3-3 directly interacts with the PCDH-γ cytoplasmic domain in vitro (data not shown). Thus, 14-3-3 is most likely a direct binding partner for PCDH-γs. Other direct target proteins on our list also include previously identified SRC kinases for PCDH-αs (9Kohmura N. Senzaki K. Hamada S. Kai N. Yasuda R. Watanabe M. Ishii H. Yasuda M. Mishina M. Yagi T. Diversity revealed by a novel family of cadherins expressed in neurons at a synaptic complex.Neuron. 1998; 20: 1137-1151Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). On the other hand, some proteins, for example α-catenin and β-catenin, appear to be indirect binding proteins for PCDHs (3Sano K. Tanihara H. Heimark R.L Obata S. Davidson M. St John T. Taketani S. Suzuki S. Protocadherins: a large family of cadherin-related molecules in central nervous system.EMBO J. 1993; 12: 2249-2256Crossref PubMed Scopus (328) Google Scholar). They are present in the complexes likely due to binding to classic cadherins, R-cadherin and N-cadherin. The identification of R-cadherins and N-cadherin associated with PCDH-γs suggests an intriguing possibility of cross-talking between the classic cadherin and clustered protocadherin pathways. Future studies may improve our understanding in this regard. A group of identified proteins including PSD-95 and components of CAMKII kinase complexes has known functions in postsynaptic differentiation (53Kim E. Sheng M. PDZ domain proteins of synapses.Nat. Rev. Neurosci. 2004; 5: 771-781Crossref PubMed Scopus (1209) Google Scholar, 54Colbran R.J. Brown A.M. Calcium/calmodulin-dependent protein kinase II and synaptic plasticity.Curr. Opin. Neurobiol. 2004; 14: 318-327Crossref PubMed Scopus (252) Google Scholar). Noticeably, both PSD-95 and CAMKII-γ exhibited decreased levels in the membrane protein complexes from Pcdh-γ-deficient mice. This observation is consistent with the synaptic deficit observed in Pcdh-γ mutant mice (27Weiner J.A. Wang X. Tapia J.C. Sanes J.R. Gamma protocadherins are required for synaptic development in the spinal cord.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 8-14Crossref PubMed Scopus (158) Google Scholar). Another set of PCDH-γ-associated proteins includes adaptor protein AP2A, AP2B, SV2, clathrin, kinesin 2A, and RAB-interacting proteins, which are clearly important for vesicle transport and trafficking. It has been reported that PCDHs are enriched in intracellular vesicles (17Phillips G.R. Tanaka H. Frank M. Elste A. Fidler L. Benson D.L. Colman D.R. Gamma-protocadherins are targeted to subsets of synapses and intracellular organelles in neurons.J. Neurosci. 2003; 23: 5096-5104Crossref PubMed Google Scholar, 55Phillips G.R. Huang J.K. Wang Y. Tanaka H. Shapiro L. Zhang W. Shan W.S. Arndt K. Frank M. Gordon R.E. Gawinowicz M.A. Zhao Y. Colman D.R. The presynaptic particle web: ultrastructure, composition, dissolution, and reconstitution.Neuron. 2001; 32: 63-77Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar), and our findings might identify a molecular anchor for PCDHs on these vesicles. Nuclear proteins such as histone were also identified in our MS analysis. It is currently unclear whether this is biologically relevant or simply a purification artifact. Interestingly, the proteolytically processed cytoplasmic domains of PCDH-γs and PCDH-αs have been proposed to have a nuclear function like the NOTCH intracellular domain (30Haas I.G. Frank M. Véron N. Kemler R. Presenilin-dependent processing and nuclear function of gamma-protocadherins.J. Biol. Chem. 2005; 280: 9313-9319Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 31Hambsch B. Grinevich V. Seeburg P.H. Schwarz M.K. {gamma}-Protocadherins, presenilin-mediated release of C-terminal fragment promotes locus expression.J. Biol. Chem. 2005; 280: 15888-15897Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 32Bonn S. Seeburg P.H. Schwarz M.K. Combinatorial expression of alpha- and gamma-protocadherins alters their presenilin-dependent processing.Mol. Cell. Biol. 2007; 27: 4121-4132Crossref PubMed Scopus (43) Google Scholar, 56Reiss K. Maretzky T. Haas I.G. Schulte M. Ludwig A. Frank M. Saftig P. Regulated ADAM10-dependent ectodomain shedding of gamma-protocadherin C3 modulates cell-cell adhesion.J. Biol. Chem. 2006; 281: 21735-21744Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar).Perhaps the most revealing finding from our proteomics survey of PCDH-γ complexes is the surprisingly high abundance of PCDH-α and PCDH-β isoforms in the PCDH-γ complexes. Although the interaction between PCDH-α and PCDH-γ was reported previously (28Triana-Baltzer G.B. Blank M. Cytoplasmic domain of protocadherin-alpha enhances homophilic interactions and recognizes cytoskeletal elements.J. Neurobiol. 2006; 66: 393-407Crossref PubMed Scopus (24) Google Scholar, 34Murata Y. Hamada S. Morishita H. Mutoh T. Yagi T. Interaction with protocadherin-gamma regulates the cell surface expression of protocadherin-alpha.J. Biol. Chem. 2004; 279: 49508-49516Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), the significance of this interaction was not well appreciated because the relative abundance of the complexes was not evaluated. Using SG and particularly 2D BN-PAGE techniques, we further demonstrated that clustered PCDHs form stable macromolecular complexes distinct from other synaptic proteins and cell adhesion molecules. To support the notion that different clusters of PCDHs coexist in similar functional complexes and might have overlapping functions, we analyzed complex formation in Pcdh-γ-deficient mice and found that all verified PCDH-γ-associated proteins are present in similar protein complexes with a minority of these proteins showing lower abundance. Therefore, it is possible that other PCDHs compensate for PCDH-γ function in complex formation. RNAi experiments in the chicken spinal cord further strengthen this interpretation by showing that combinations of shRNAs against both PCDH-α and PCDH-γ induced apoptosis, whereas PCDH-α or PCDH-γ shRNAs alone did not. Clustered Pcdh genes are ubiquitously expressed in all neuronal populations. A very attractive hypothesis has been proposed based on the diversity and combinatorial expression patterns of Pcdhs (6Wu Q. Maniatis T. A striking organization of a large family of human neural cadherin-like cell adhesion genes.Cell. 1999; 97: 779-790Abstract Full Text Full Text PDF PubMed Scopus (541) Google Scholar, 57Serafini T. Finding a partner in a crowd: neuronal diversity and synaptogenesis.Cell. 1999; 98: 133-136Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 58Shapiro L. Colman D.R. The diversity of cadherins and implications for a synaptic adhesive code in the CNS.Neuron. 1999; 23: 427-430Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 59Yagi T. Takeichi M. Cadherin superfamily genes: functions, genomic organization, and neurologic diversity.Genes Dev. 2000; 14: 1169-1180PubMed Google Scholar). In this model, cell-specific combinatorial PCDH molecules might provide a surface barcode for establishing synaptic specificity. However, mouse knock-out experiments did not provide conclusive evidence to either affirm or disprove this hypothesis. Although regional and subpopulation-specific phenotypes have been described (19Wang X. Weiner J.A. Levi S. Craig A.M. Bradley A. Sanes J.R. Gamma protocadherins are required for survival of spinal interneurons.Neuron. 2002; 36: 843-854Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 23Lefebvre J.L. Zhang Y. Meister M. Wang X. Sanes J.R. {gamma}-Protocadherins regulate neuronal survival but are dispensable for circuit formation in retina.Development. 2008; 135: 4141-4151Crossref PubMed Scopus (110) Google Scholar, 26Hasegawa S. Hamada S. Kumode Y. Esumi S. Katori S. Fukuda E. Uchiyama Y. Hirabayashi T. Mombaerts P. Yagi T. The protocadherin-alpha family is involved in axonal coalescence of olfactory sensory neurons into glomeruli of the olfactory bulb in mouse.Mol. Cell. Neurosci. 2008; 38: 66-79Crossref PubMed Scopus (99) Google Scholar, 27Weiner J.A. Wang X. Tapia J.C. Sanes J.R. Gamma protocadherins are required for synaptic development in the spinal cord.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 8-14Crossref PubMed Scopus (158) Google Scholar), no pleiotropic effects are observed for either Pcdh-γ- or Pcdh-α-null mice. A plausible explanation for the lack of pleiotropic neuronal phenotype in the Pcdh-α- or Pcdh-γ-deficient mice is the genetic redundancy and dosage compensation among different clustered Pcdhs. Our proteomics data presented in this study provide direct evidence that clustered PCDHs have functional redundancy both biochemically and biologically. Thus, deletion of all clustered Pcdhs in model systems might be necessary to reveal perhaps even more striking neuronal phenotypes during development. The development of neural circuitry involves a complex interplay of cell-cell adhesion, interneuronal signaling, and assembly of intracellular macromolecular protein complexes. A group of protocadherin genes, known as clustered protocadherins (Pcdhs), 1The abbreviations used are:PCDHprotocadherinBNblue nativePBPiggyBacPSDpostsynaptic densityRNAiRNA interferenceSGsucrose gradientSNIPSNAP-25-interacting-protein2Dtwo-dimensionalGFPgreen fluorescent proteinCAMKCa2+/calmodulin-dependent protein kinaseTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineLTQlinear trap quadrupoleshRNAshort hairpin RNAIPimmunoprecipitationFffireflyLucluciferaseRnRenillaPBasePiggyBac transposasePpostnatal dayCMVcytomegalovirus. 1The abbreviations used are:PCDHprotocadherinBNblue nativePBPiggyBacPSDpostsynaptic densityRNAiRNA interferenceSGsucrose gradientSNIPSNAP-25-interacting-protein2Dtwo-dimensionalGFPgreen fluorescent proteinCAMKCa2+/calmodulin-dependent protein kinaseTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineLTQlinear trap quadrupoleshRNAshort hairpin RNAIPimmunoprecipitationFffireflyLucluciferaseRnRenillaPBasePiggyBac transposasePpostnatal dayCMVcytomegalovirus. are among the transmembrane signaling molecules that are implic
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