Sorting out Genes that Regulate Epithelial and Neuronal Polarity
1998; Cell Press; Volume: 94; Issue: 6 Linguagem: Inglês
10.1016/s0092-8674(00)81727-4
ISSN1097-4172
Autores Tópico(s)Cellular transport and secretion
ResumoMuscle contraction is controlled by motor neurons, which are activated by glutamate release in spinal cord. This stimulus triggers action potentials that are propagated down long motor neuron axons to mediate acetylcholine release at distant neuromuscular junctions. This vectorial signaling in a motor neuron requires polarized sorting of proteins to appropriate neuronal domains. Neurotransmitter receptors for glutamate must be targeted to dendrites, while proteins that mediate synthesis and release of acetylcholine are shuttled down axons. Many nonneuronal cells are also polarized, but the two polarized faces are typically much closer than in neurons. For example, intestinal epithelial cells have an apical surface that faces the lumen of the gut and a basolateral surface that contacts the underlying basement membrane. Uptake of nutrients by epithelial cells requires that transporters for glucose and amino acids are expressed selectively at the apical membrane and that Na+/K+ ATPases, which set up ionic gradients essential for resorption, are expressed at the basolateral membrane. Understanding the basis for cellular polarity is important as defects in protein sorting underlie several common human disorders including cystic fibrosis and polycystic kidney disease (8Fish E.M Molitoris B.A N. Engl. J. Med. 1994; 330: 1580-1588Crossref PubMed Scopus (186) Google Scholar). At an anatomical level, the polarities of neurons and epithelial cells appear quite different. In a motor neuron, a meter or more often separates axon terminals from the soma and dendrites, whereas in polarized epithelia, junctional complexes segregate the basolateral and apical membranes, which are only a few micrometers apart. Despite these differences, work pioneered by Dotti and Simons has demonstrated that similar mechanisms mediate polarized sorting of some (but not all) proteins in both epithelial cells and neurons (7Dotti C.G Simons K Cell. 1990; 62: 63-72Abstract Full Text PDF PubMed Scopus (355) Google Scholar). Proteins that occur at the apical membrane of epithelial cells are typically (but not always) targeted to the axon of neurons, whereas proteins localized to the basolateral domain of epithelial cells often have a somatodendritic distribution in neurons (Figure 1). Furthermore, similar molecular mechanisms mediate polarized sorting of some proteins in neurons and epithelial cells. For instance, glycosylphosphatidylinositol-anchored extracellular proteins are selectively targeted to the apical and axonal surfaces through interactions with specialized cellular lipid domains (13Keller P Simons K J. Cell Sci. 1997; 110: 3001-3009Crossref PubMed Google Scholar). Basolateral/somatodendritic targeting does not apparently rely on lipid interactions, but rather is mediated by cytoplasmic sorting signals. Two such targeting sequences, one a tyrosine-based motif and the other a dileucine motif, are both necessary and sufficient for polarized sorting of certain transmembrane proteins in epithelial cells (17Matter K Yamamoto E.M Mellman I J. Cell Biol. 1994; 126: 991-1004Crossref PubMed Scopus (209) Google Scholar, 22Trowbridge I.S Collawn J.F Hopkins C.R Annu. Rev. Cell Biol. 1993; 9: 129-161Crossref PubMed Scopus (696) Google Scholar). And, these same cytosolic targeting sequences determine protein sorting to the somatodendritic domain of neurons (11Jareb M Banker G Neuron. 1998; 20: 855-867Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Despite the identification of these targeting signals, little is known about the protein machinery that recognizes these sequences and mediates basolateral sorting. Insights into fundamental mechanisms for protein targeting in diverse tissues have recently come from studies of protein recruitment to the synapse. Working independently, several groups simultaneously found that PDZ protein motifs mediate targeting of diverse proteins to synaptic sites (2Brenman J.E Chao D.S Xia H Aldape K Bredt D.S Cell. 1995; 82: 743-752Abstract Full Text PDF PubMed Scopus (821) Google Scholar, 14Kim E Niethammer M Rothschild A Jan Y.N Sheng M Nature. 1995; 378: 85-88Crossref PubMed Scopus (877) Google Scholar, 16Kornau H.-C Schenker L.T Kennedy M.B Seeburg P.H Science. 1995; 269: 1737-1740Crossref PubMed Scopus (1574) Google Scholar). PDZ domains are multifunctional protein–protein interaction motifs that often bind to specific sequences at the extreme C termini of target proteins. A prototypical interaction of this type occurs between PDZ domains from PSD-95, a membrane-associated guanylate kinase, and the tail of Shaker-type K+ channels, which terminate in Thr-Asp-Val. Genetic studies in Drosophila have established that this sort of PDZ domain interaction mediates Shaker K+ channel targeting to the neuromuscular junction (21Tejedor F.J Bokhari A Rogero O Gorczyca M Zhang J Kim E Sheng M Budnik V J. Neurosci. 1997; 17: 152-159Crossref PubMed Google Scholar). In addition to targeting ion channels to the synapse, PDZ proteins often function as molecular scaffolds for signal transduction. This role is exemplified by INAD, a penta-PDZ protein that assembles a G protein–coupled signaling cascade in Drosophila photoreceptors (23Tsunoda S Sierralta J Sun Y Bodner R Suzuki E Becker A Socolich M Zuker C.S Nature. 1997; 388: 243-249Crossref PubMed Scopus (540) Google Scholar). Although PDZ domains do not recognize either the tyrosine-based or dileucine motifs classically described for basolateral targeting in epithelial cells, recent studies of vulval cell differentiation in C. elegans demonstrate that PDZ domains nonetheless mediate basolateral targeting (20Simske J.S Kaech S.M Harp S.A Kim S.K Cell. 1996; 85: 195-204Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). Differentiation of vulval epithelial cells from pluripotent precursors is mediated by LIN-3, an EGF-like paracrine signal secreted by the anchor cell on the basal side of the vulval precursor cells. LIN-3 initiates vulval differentiation by activating LET-23, an EGF receptor tyrosine kinase (RTK)-like protein, localized to the basolateral surface of the vulval precursor cells. Activation of LET-23 mediates a classical Ras-linked RTK signal transduction cascade resulting in MAP kinase activation and cellular differentiation. Mutations of lin-3, let-23, or other genes that act directly in the RTK to MAP kinase pathway prevent differentiation and yield a vulvaless phenotype. Genetic analysis of this pathway has served as a powerful tool for identification of proteins that link RTK activation to MAP kinase and cellular differentiation. This pathway is now also proving to be useful for studies of basolateral protein sorting. Because LIN-3 is secreted on the basal side of the vulval precursor cells, this signal can only be received if LET-23 RTK is properly targeted to the basolateral surface (20Simske J.S Kaech S.M Harp S.A Kim S.K Cell. 1996; 85: 195-204Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). This requirement for polarized expression of LET-23 accounts for the phenotypes of lin-2, lin-7, and lin-10 mutant worms. Mutations of these genes do not directly interfere with LET-23 RTK signal transduction, but disrupt basolateral targeting of LET-23 RTK in vulval precursor cells and thereby cause a vulvaless phenotype (as discussed in 12Kaech S.M Whitfield C.W Kim S.K Cell. 1998; 94 (this issue,): 761-771Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). Molecular cloning of LIN-2 and LIN-7 demonstrated that both are PDZ-containing proteins. Whereas LIN-7 is a small protein with little more than a single PDZ domain, LIN-2 contains SH3 and guanylate kinase domains, similar to PSD-95, but also contains an N-terminal domain similar to calcium/calmodulin-dependent protein kinase. Initially, LIN-10 was reported to be a widely expressed protein that lacked a PDZ domain or other features that might explain its role in basolateral targeting of LET-23 (15Kim S.K Horvitz H.R Genes Dev. 1990; 4: 357-371Crossref PubMed Scopus (56) Google Scholar). However, new data indicates that the original identification of lin-10 was incorrect, and that the authentic lin-10 gene occurs nearby on the physical map. Interestingly, genuine LIN-10 is another PDZ protein and contains two such motifs together with a phosphotyrosine-binding domain (as discussed in 12Kaech S.M Whitfield C.W Kim S.K Cell. 1998; 94 (this issue,): 761-771Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). Basolateral localization of LET-23 RTK therefore requires the concerted activity of three PDZ proteins. In an elegant combination of genetic and biochemical studies in this issue of Cell, Kim and colleagues have defined the specific protein interactions between the PDZ proteins LIN-2, LIN-7, and LIN-10 that mediate basolateral sorting of LET-23 (12Kaech S.M Whitfield C.W Kim S.K Cell. 1998; 94 (this issue,): 761-771Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). As the first order of business, the authors show that the PDZ domain of LIN-7 (but not those of LIN-2 or LIN-10) binds to the tail of LET-23. This interaction comes as little surprise because LET-23 terminates in Thr-Cys-Leu, a sequence that might be predicted to bind the LIN-7 PDZ domain. However, the authors take advantage of genetic tools in C. elegans to extend this biochemical observation and clearly show that binding to LIN-7 is essential for basolateral localization and function of LET-23 in vulval precursor cells. What about lin-2 and lin-10? Because mutations in these genes yield a phenotype similar to lin-7 mutations, Kim and colleagues evaluated possible interactions among all three proteins. Interestingly, they find that LIN-2 can bind to both LIN-10 and LIN-7 to form a ternary complex. Independent evidence of a role for the LIN-2/-7/-10 protein complex derives from rigorous biochemical studies by Südhof and colleagues. Südhof’s group previously identified CASK, the mammalian homolog of LIN-2, as a brain-enriched protein whose PDZ domain can bind the C terminus of neurexin (9Hata Y Butz S Südhof T.C J. Neurosci. 1996; 16: 2488-2494Crossref PubMed Google Scholar). In experiments described in this issue of Cell, Südhof’s group performed preparative immunoprecipitations of CASK from brain extracts and found that a single protein, Mint1, occurs stoichiometrically with CASK (4Butz S Okamoto M Südhof T.C Cell. 1998; 94 (this issue,): 773-782Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar). Remarkably, Mint1 is a mammalian homolog of LIN-10. Similar to the data from Kim’s lab, Südhof’s group shows that CASK (LIN-2) binds not only to both Mint1 (LIN-10) but also to Velis, vertebrate homologs of LIN-7 that are found in the EST database. Thus, the LIN-2/-7/-10 complex is conserved from worms to rats and is enriched in mammalian brain. As these three proteins all contain PDZ motifs, one might expect that these domains would mediate the binding interactions that assemble the complex. Indeed, PDZ/PDZ domain interactions mediate binding of neuronal nitric oxide synthase to PSD-95 (3Brenman J.E Chao D.S Gee S.H McGee A.W Craven S.E Santillano D.R Huang F Xia H Peters M.F Froehner S.C Bredt D.S Cell. 1996; 84: 757-767Abstract Full Text Full Text PDF PubMed Scopus (1394) Google Scholar) and assemble the INAD signaling complex (24Xu X.S Choudhury A Li X Montell C J. Cell Biol. 1998; 142: 545-555Crossref PubMed Scopus (193) Google Scholar). However, biochemical studies of the LIN-2/-7/ -10 complex from both C. elegans and mammals demonstrate that PDZ domains are not involved in assembly, which is mediated by novel binding interfaces (4Butz S Okamoto M Südhof T.C Cell. 1998; 94 (this issue,): 773-782Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 12Kaech S.M Whitfield C.W Kim S.K Cell. 1998; 94 (this issue,): 761-771Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). The PDZ domains therefore remain available to recruit other proteins, such as LET-23 RTK, to the complex. Although these studies clearly indicate that LIN-2/ -7/-10 regulate protein targeting, the biochemical mechanisms that mediate sorting by this complex remain uncertain. A fundamental question is whether the LIN-2/ -7/-10 complex directly targets LET-23 to the basolateral membrane, or instead, if the complex selectively retains LET-23 at the basolateral membrane by preventing transcytosis of LET-23 to the apical surface (18Mostov K.E Cardone M.H Bioessays. 1995; 17: 129-138Crossref PubMed Scopus (115) Google Scholar). Determining the cellular localization of the LIN-2/-7/-10 complex could help distinguish between these two mechanisms. Whereas LIN-7 colocalizes with LET-23 at the basolateral membrane of vulval precursor cells (20Simske J.S Kaech S.M Harp S.A Kim S.K Cell. 1996; 85: 195-204Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar), the distribution of LIN-2 and LIN-10 in these cells remains unknown. In neurons, a large pool of LIN-10 occurs in perinuclear structures resembling ER/Golgi (19Rongo C Whitfield C.W Rodal A Kim S.K Kaplan J.M Cell. 1998; 94 (this issue,)Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), suggesting a possible role for LIN-10 in vesicle trafficking. It will also be important to determine how the LIN-2/-7/-10 sorting mechanism interacts with the other basolateral pathways that recognize tyrosine- and dileucine-based cytosolic signals (22Trowbridge I.S Collawn J.F Hopkins C.R Annu. Rev. Cell Biol. 1993; 9: 129-161Crossref PubMed Scopus (696) Google Scholar, 17Matter K Yamamoto E.M Mellman I J. Cell Biol. 1994; 126: 991-1004Crossref PubMed Scopus (209) Google Scholar). As the mammalian homologs of LIN-2/-7/-10 are expressed primarily in brain (4Butz S Okamoto M Südhof T.C Cell. 1998; 94 (this issue,): 773-782Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar), it is surprising that mutations of these genes in C. elegans disrupt vulval development rather than cause primarily a neuronal phenotype. However, experiments by Kaplan and coworkers in this issue of Cell demonstrate that at least one protein in the complex, LIN-10, has a prominent neuronal role in worms (19Rongo C Whitfield C.W Rodal A Kim S.K Kaplan J.M Cell. 1998; 94 (this issue,)Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). These studies investigate the polarized sorting of GLR-1, a C. elegans glutamate receptor. Like mammalian glutamate receptors, GLR-1 in C. elegans is clustered at postsynaptic sites and contains a C-terminal consensus (Thr-Ala-Val) for binding to PDZ proteins. Kaplan and colleagues first ectopically express GLR-1 in vulval precursor epithelial cells and find it localizes to basolateral membranes in a LIN-2/-7/-10-dependent manner. This polarized sorting nicely fits the model (Figure 1) that somatodendritic (postsynaptic) neuronal proteins often occur at the basolateral membrane of epithelial cells (7Dotti C.G Simons K Cell. 1990; 62: 63-72Abstract Full Text PDF PubMed Scopus (355) Google Scholar). More importantly, they find that lin-10 mutations (but not lin-2 or lin-7 mutations) block postsynaptic targeting of GLR-1, implying that LIN-10 is a shared component of the polarized sorting pathway in neurons and epithelial cells. Furthermore, they find that lin-10 mutants have a behavioral defect characteristic of glr-1 mutations; that is, the mutant worms do not retreat normally when stroked on the nose with a human eye lash. Mechanisms for this postsynaptic targeting of GLR-1 by LIN-10 are not yet clear. LIN-10 does not directly bind to GLR-1 and is, therefore, unlikely to work alone in postsynaptic targeting of GLR-1. Unlike basolateral sorting of LET-23 in epithelial cells, postsynaptic targeting of GLR-1 does not require lin-2 or lin-7 (19Rongo C Whitfield C.W Rodal A Kim S.K Kaplan J.M Cell. 1998; 94 (this issue,)Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). This may indicate that related or redundant PDZ proteins substitute for LIN-2 and LIN-7 in C. elegans neurons. It should also be pointed out that the present evidence does not definitively implicate PDZ domain interactions in postsynaptic targeting of GLR-1. First, no synaptic PDZ protein has yet been shown to bind the C terminus of GLR-1. Second, mutations of the PDZ-binding consensus (Thr-Ala-Val) at the C terminus of GLR-1 do not disrupt postsynaptic targeting or function of GLR-1. Third, GLR-1 is postsynaptic in young lin-10 mutant larvae and only becomes mislocalized in older larvae and adults (19Rongo C Whitfield C.W Rodal A Kim S.K Kaplan J.M Cell. 1998; 94 (this issue,)Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Becuase there are multiple interpretations for each of these results, it remains important to identify physiological PDZ proteins that cooperate with LIN-10 in postsynaptic targeting of GLR-1. Unlike the postsynaptic role for LIN-10 in worms, the CASK (LIN-2)/Veli (LIN-7)/Mint1 (LIN-10) complex is proposed to function presynaptically in mammalian neurons (4Butz S Okamoto M Südhof T.C Cell. 1998; 94 (this issue,): 773-782Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar). This suggestion of a presynaptic role is based on the known binding partners for CASK and Mint1. As mentioned above, CASK was originally identified as a binding partner for neurexin, which is thought to have a presynaptic role in regulating both synaptic adhesion and transmitter release. A presynaptic function for Mint1 is suggested by its interaction with Munc18-1, a synaptic vesicle trafficking protein. In addition to its presynaptic roles in adhesion and vesicle trafficking, the CASK/Veli/Mint1 complex may also serve related functions at postsynaptic and nonsynaptic sites. CASK, for instance, is concentrated at both pre- and postsynaptic sites in brain and binds to syndecan-2, a heparin sulfate proteoglycan (5Cohen A.R Wood D.F Marfatia S.M Walther Z Chishti A.H Anderson J.M J. Cell Biol. 1998; 142: 129-138Crossref PubMed Scopus (310) Google Scholar, 10Hsueh Y Yang F Kharazia V Naisbitt S Cohen A.R Weinberg J Sheng M J. Cell Biol. 1998; 142: 139-151Crossref PubMed Scopus (277) Google Scholar). While the anatomical localization of Mint1 has not yet been analyzed in brain, a role in the ER/Golgi is suggested by studies showing that Mint1 binds to and regulates processing of β-amyloid precursor protein (1Borg J.P Yang Y De Taddéo-Borg M Margolis B Turner R.S J. Biol. Chem. 1998; 273: 14761-14766Crossref PubMed Scopus (179) Google Scholar). As Mint1 is homologous to LIN-10 and can restore postsynaptic localization of GLR-1 in lin-10 mutants (19Rongo C Whitfield C.W Rodal A Kim S.K Kaplan J.M Cell. 1998; 94 (this issue,)Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), it is tempting to speculate that Mint1 might also regulate clustering of vertebrate glutamate receptors, which are known to bind to PDZ proteins at the synapse (6Dong H O’Brien R.J Fung E.T Lanahan A.A Worley P.F Huganir R.L Nature. 1997; 386: 279-284Crossref PubMed Scopus (726) Google Scholar). While many such interesting questions remain, these three papers establish that LIN-2/-7/-10 form an evolutionarily conserved protein complex and mediate fundamental aspects of protein targeting in neurons and epithelial cells. Future genetic and biochemical studies of these proteins in both invertebrates and mammals promise to help sort out protein sorting.
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