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Formation of the gap junction nexus: binding partners for connexins
Journal of Physiology - Paris 96 (2002) 243–249 www.elsevier.com/locate/jphysparis
Formation of the gap junction nexus: binding partners for connexins Heather S. Duffya, Mario Delmarb, David C. Spraya,* a
Department of Neuroscience, Albert Einstein College of Medicine, 1410 Pelham Pkwy S., Bronx, NY 10461, USA b SUNY Upstate Medical University, Syracuse, NY 13210, USA
Abstract Gap junctions are the morphological correlates of direct cell–cell communication and are formed of hexameric assemblies of gap junction proteins (connexins) into hemichannels (or connexons) provided by each coupled cell. Gap junction channels formed by each of the connexin subtypes (of which there are as many as 20) display different properties, which have been attributed to differences in amino acid sequences of gating domains of the connexins. Recent studies additionally indicate that connexin proteins interact with other cellular components to form a protein complex termed the Nexus. This review summarizes current knowledge regarding the protein–protein interactions involving of connexin proteins and proposes hypothesized functions for these interactions. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Astrocyte; CNS; Synapses
Regions of cell–cell and cell–substrate interactions and of intercellular communication are specialized membrane microdomains containing complexes of proteins fulfilling these roles and linking these domains to the cytoskeleton. Such complexes include occluding junctions (zonula occludens), anchoring junctions (zonula adherens and hemidesmosomes/desmosomes) and communicating junctions (chemical and electrotonic synapses, the latter of which are gap junctions, or zonula communicans). With the exception of gap junctions, each of these membrane domains has been known for some time to involve complex interactions between multiple protein binding partners [33,34,19]. Traditionally, gap junction proteins (connexins) have been considered as simple pore-forming proteins that exhibit little or no interaction with other cellular components. Thus, much of the work on gap junctions has focused on the structure and function of individual connexins [29]. Evidence is now accumulating that this view of the gap junction has been too narrow and that connexins have rich interactions with a myriad of other proteins that may turn out to be important in multiple aspects of gap junction biology including function, regulation, and even structure. Connexins are four transmembrane domain (tetraspan) proteins with cytoplasmically localized amino * Corresponding author. Tel.: +1-718-430-2537; fax: +1-718-4308594. E-mail address: [email protected] (D.C. Spray).
terminus, cytoplasmic loop and carboxyl terminal domains (Fig. 1). It is these intracellular domains that are the most variable in amino acid sequence among the different connexins, with the cytoplasmic loop and carboxyl terminal domains conferring most of the diversity among connexin subtypes. The presence of these variable sequences, some of which contain known signaling domain motifs (for one example see Fig. 2) implies that these regions may be important in differential connexin function. Studies now show that there are variations in the protein-protein interactions of these domains and it is these differing protein–protein interactions that may confer distinctive functional or regulatory specificity to gap junctions formed of the individual connexins. The study of interactions of proteins with connexins have been somewhat hampered by the difficulty in extracting connexins from their location at the appositional membranes between cells. Interactions have been studied in a variety of ways including immunolocalization, immunoprecipitation, and binding assays of one type or another. Each of these has its advantages and limitations. Immunolocalization of other proteins with connexins is readily possible using immunofluorescence confocal microscopy because the connexins are localized in specific membrane regions, namely sites of cell–cell contact. However, overlapping staining must then be followed with rigorous biochemistry to ensure that the co-localization is not simply fortuitous. There are multiple biochemical techniques that have been routinely used in the study of connexin–protein interactions.
0928-4257/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0928-4257(02)00012-8
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Fig. 1. Schematic of a standard connexin molecule. There are two extracellular loop, designated E1 and E2, and three distinct intracellular domains, the amino terminus (NT), the cytoplasmic loop (CL) and the carboxyl terminus (CT). The extracellular loops and transmembrane domains are highly conserved while the cytoplasmic domains vary between connexins, particularly in the regions of the CL and the CT.
Fig. 2. Schematic of the amino acid sequence of the Cx43 carboxyl terminal domain. Regions of known binding motifs are highlighted in gray. Two regions for potential interactions with signaling molecules dominate the Cx43 carboxyl terminal domain, a proline rich region from amino acid 274–283 and a serine rich region from amino acid 359–369.
These include co-immunoprecipitation studies with connexin antibodies and binding assays to connexinGST fusion proteins. When all works well, these techniques yield important information as to what proteins are associated, either directly or within a complex, with the connexin of interest used as a ‘‘capture’’ ligand. The major difficulty in applying these techniques to the study of connexin–protein interactions is the low protein levels of connexins found in many cell types. Improving the affinity of the connexin–substrate linkage, enrichment for connexin in cell extracts, and pooling of purified complexes may provide useful improvements in this technique. Recently we have begun using the technique of mirror resonance (MR) to quantify the kinetics of binding between Cx43 and known binding partners and as a tool to identify novel connexin binding partners [5]. Mirror
resonance records the interaction of binding partners in real time, thus allowing direct measurements of K(association) and K(dissociation) and calculation of the KD (from Kdiss/Kass or from steady state response amplitudes) for interactions observed. One example of such an experiment has involved covalent linkage of the carboxyl terminal domain of Cx43 (Cx43CT) to MR cuvettes as a ‘‘capture’’ ligand, and measurement of responses to addition of lysates of wild type murine astrocytes to the Cx43CT. Such studies have indicated the existence of proteins in astrocytes that bind directly to the Cx43CT (Fig. 3). The next step in such studies is to elute the binding partners from the MR cuvette following binding for identification using MALDI-TOF mass spectroscopy. Although the above demonstration of the existence of cytoplasmic binding partners for Cx43 in astrocytes was performed using a technique that is novel in the gap junction field, it is consistent with recent reports in other cell types, indicating that Cx43 has a number of binding partners, many of which have been localized within astrocytes. In addition, other connexins have also been shown to interact with other proteins, and it seems clear that the application of improved ‘‘fishing’’ strategies will greatly expand this list. The sections that follow summarize the state of knowledge regarding connexin–protein interactions and provide hypotheses regarding possible functions of these interactions.
1. Tight junction associated proteins Among the first types of proteins that were shown to interact with connexins are those traditionally thought to interact as part of the tight junction complex and in the case of ZO-1, adherens junctions as well [14]. Occludin was first described as an integral membrane protein involved in the formation of the tight junctional
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Fig. 3. Binding of astrocyte lysates to the carboxy terminal domain of Cx43. Panel A shows the trace of a single experiment showing the binding of astrocyte lysate in real time. Arrows designate the addition of astrocyte lysate to the cuvette (arrow 1), rinse with phosphate buffered saline with 1% Tween20 (Sigma Chemicals, St. Louis MO) (arrow 2), and regeneration of the cuvette using 10 mM HCl (arrow 3). Panel B shows the concentration dependence of the binding of astrocyte lysate components to the Cx43 carboxyl terminal domain. The Kd estimated for the total of the unknown binding partners is 17 ng/ml showing high affinity binding of some component of astrocytes to the Cx43 carboxyl terminal domain.
strands [8]. Similarly, claudins have been described as a primary component of tight junctions [10]. Zonula occludens-1, a cytoplasmic scaffolding protein, was hypothesized to play a key role in the localization of tight junction proteins within the tight junction strand [9]. Each of these has now been described to associate with connexins as well. 1.1. Zonula occludens-1 (ZO-1) ZO-1 was the first tight junction associated protein to be shown to interact with connexins. Reports showed
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that connexin43 (Cx43), the primary connexin subtype found in many tissues including fibroblasts and myocytes, directly interacted with ZO-1 [31,13]. The site of interaction between Cx43 and ZO-1 was shown to occur through the second PDZ domain of ZO-1 and the carboxyl terminal end of Cx43. The carboxyl terminal of Cx43 contains a potential consensus sequence for a PDZ binding domain (DLEI, Fig. 2) that has been hypothesized to be important in the Cx43-ZO-1 interaction. This association of Cx43 with ZO-1 has been confirmed in Sertoli cells in the testes [2], and in astrocytes [4]. Cx43 is apparently not the only connexin that links with ZO-1. Two groups have recently reported that Cx45 also interacts with ZO-1 [17,20], potentially stabilizing heteromeric complexes of Cx43. Interestingly, both Cx43 and Cx45 are alpha subtype gap junction proteins. Examination of the sequences of the other alpha connexins suggests that there may be multiple alpha connexins with the potential to interact with ZO-1. In contrast, the beta and gamma connexins appear not to have the PDZ binding domain consensus sequences at their most terminal carboxyl regions, implying that these connexins are less likely to interact with ZO-1 in the typical PDZ-carboxyl terminus manner. The role of the interaction between connexins and ZO-1 is unclear but several hypotheses have been proposed. Toyofuku et al. have suggested that one potential role is in the trafficking of Cx43 to the intercalated discs of the heart, thereby aiding in the formation of gap junctions in these regions. Although Cx43-ZO-1 linkage may contribute to this process, studies by our group and others [7,27] have shown that carboxyl terminal truncations of connexin43 are still trafficked to the membrane and form functional channels. This suggests that the most terminal end of the protein is not required for formation of gap junctions at cell membranes. However, it should be mentioned that the question of whether efficiency of trafficking is altered in the absence of the Cx43–ZO-1 interaction has not yet been addressed. Another potential role for the interaction of ZO-1 with Cx43 is to act as a scaffold for other proteins, thereby bringing them into close contact with either the gap junction proteins themselves or with molecules that pass through the gap junction plaques. However, it also remains to be determined if ZO-1 helps form such a multimeric complex at the site of gap junctions. 1.2. Occludin Occludin is an integral membrane tight junction associated protein that was thought to be restricted in function to the tight junctional complex. It, like the connexins, is also a four transmembrane domain protein. Using freeze fracture immunogold labeling occludin has been shown to make up some of the particles found in tight junctional strands and both total levels
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and phosphorylation state of occludin have been proposed to confer the barrier function of tight junctions [6,25]. Recent studies using cultured hepatocytes show that occludin is also associated with gap junctional proteins, in particular connexin32 [18]. Examination of freeze-fracture replicas of these cells shows many small gap junctional plaques localized near tight junctional strands, and co-immunoprecipitation studies on cultured hepatocytes Western blotted with Cx32 provided evidence for an association between occludin and Cx32. Immunofluorescence studies have co-localized these proteins at cell membranes, suggesting that they may interact at the membranes but determination of whether there is Cx32 in tight junction strands, occludin in gap junctional plaques, or both awaits freeze-fracture immunogold studies to try to localize these proteins to the junctions that they form within membranes. Cx26 has also been reported to interact with occludin in polarized sheets of a human intestinal cell line T84 through an interaction with the coiled-coil domain of occludin [28]. The occludin-Cx26 interaction was not confirmed by Kojima et al. in hepatocytes [18], indicating either that there may be cell specific interactions between this protein and the connexins or that the occludin domain used for the connexin binding experiments may be masked in ‘‘native’’ occludin. 1.3. Claudins An additional tetraspan family of proteins, the claudins, has also been found to associate with connexins. Tsukita’s group first described the claudin family of integral membrane proteins that are components of tight junctional complexes [26]. There are a number of members of the claudin family, each of which is cell specifically localized. In their examination of cultured hepatocytes, Kojima et al. found that along with an association with occludin, Cx32 also associated with claudin-1 [18], a primary hepatocyte claudin subtype; as with the occludin interaction, the locus of this interaction is unclear. It has been hypothesized that the interaction allows for the formation of a scaffold of proteins localized near sites of cell contact, thereby localizing signaling proteins that may be regulated by signals passed through the gap junctions. Another type of interaction between connexins and claudins has also been reported, a reciprocal regulation of the two proteins. In a cell type that is normally devoid of tight junctions, astrocytes, treatment with the pro-inflammatory cytokine Interleukin-1b causes a loss of Cx43 [15] with a concurrent upregulation of the previously absent protein claudin-1 [4]. Whether this is due to a single transcriptional regulator or to two independent events is unknown, but the outcome at the membrane is a morphological switch from gap junctions to
rudimentary protein strands containing elements of the correct size and structure to be tight junctional proteins (Fig. 4). It was hypothesized that by limiting both intercellular communication via gap junctions and bulk flow through the extracellular space, the switch from gap junctions to tight junction-like strands may help limit the size of lesions due to astrocyte damage.
2. Adherens junction associated proteins The function of adherens junctions is both in holding appositional cells together, an ‘‘adhesive’’ function, and in intracellular signaling cascades important in cell motility and division. They are composed of a complex of proteins localized to appositional membranes. Very recently there have been reports of interactions of connexins with members of one protein family found in the adherens junctional complex, the catenins. Examination of the role of Wnt-1 signaling in cardiac myocytes revealed a direct interaction between Cx43 and the adherens junction associated protein, b-catenin [1]. This interaction was hypothesized to be important in Wnt-1 regulation of Cx43 transcription and to provide feedback for regulating the extent of junctional communication between cardiac myocytes. Another catenin family member found to be associated with Cx43 is p120 catenin. A recent study by Xu et al. showed co-localization of Cx43 with p120 catenin, an Armadillo protein involved in modulation of cell motility [35]. In this study neural crest cells were shown to co-express Cx43 and p120 catenin, proteins that were regulated in a similar fashion by the Wnt-1 signaling pathway, and these proteins co-localized at sites of cell– cell contact. The importance of this interaction in development is not yet known but a signaling role for Cx43 in development has been hypothesized. 2.1. Cytoskeletal proteins To date, there has been only one report of direct interaction between a connexin and a cytoskeletal protein. This study used GST binding assays and immunolocalization to show binding between Cx43 and the cytoskeletal protein tubulin [12]. They determined that a juxtamembrane region of the Cx43 carboxyl terminal domain was required for this interaction but that disruption of microtubules with nocodazole had no apparent effect on either Cx43 localization at the membrane or on gross channel function. While this in no way rules out a function for the Cx43-tubulin interaction, it does indicate that this interaction may be less important than interactions that directly regulate the channel, such as the Cx43 interaction with src (below).
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Fig. 4. Ultrastructural examination of control and IL-1b treated human astrocytes. In control cells (panel A) large gap junctions were found between cultured astrocytes. In IL-1b treated cells these gap junctions were absent, instead rudimentary strands of intramembranous particles were seen (Reprinted with permission, Duffy et al., 2000, J. of Neuroscience 20 RC114).
3. Src proteins Of substantial interest for the past several years has been the interaction of Cx43 with the proto-oncogenic signaling protein Src. Src is a non-receptor tyrosine kinase that, upon activation, is an effector molecule of multiple signaling pathways such as those involving MAPK and PKC. In normal cells src resides in the inactive form due to phosphorylation of tyrosine 527. Release of this phosphorylation and subsequent phosphorylation within src’s SH2/SH3 domains results in activation of src, thereby allowing for tyrosine phosphorylation of its targets [3]. The exact mechanism of gating of Cx43 by src has been the topic of some debate. In a report that appeared over a decade ago, a kinase-active form of src (pp60vsrc) was reported to require the tyrosine phosphorylation of Cx43 in order to inhibit cell–cell communication in the oocyte expression system [30]. In 1995 Alan Lau’s group reported that ppv60-src directly interacts with Cx43 in mammalian cells, concurrent with phosphorylation of Y247 and Y265 in the carboxyl terminal domain of Cx43 [23,16]. They subsequently showed that interactions of Cx43 proline-rich regions are also important in the interactions of Cx43 with the SH2 and SH3 domains of v-src [22]. Subsequent work showed partial co-localization of Cx43 and ppv-src in fibroblasts and showed that these two proteins could be reciprocally co-immunoprecipitated from pp60v-src transformed cells [24]. In 1999 Bruce Nicholson’s group reported that in Xenopus oocytes the interaction of the SH3 domain was important for the regulation of the Cx43 channel by pp60v-src but that serine, rather than tyrosine phosphorylation was required [36]. Subsequent studies have corroborated the early work by Swenson and clearly show that the phosphorylation of the tyrosines is an important phosphorylation step in the loss of gap junctional communication induced by interaction of pp60v-src with Cx43 [21,22].
Regulation of Cx43 channel opening and closure may be only part of the function of the interaction of src with connexin43. Recent evidence has indicated that the normal cellular (c-src) form of this oncogene also interacts with Cx43, but only when activated by phosphorylation [11], supporting findings by Alan Lau’s group on v-src and the early work by Swenson et al. [16,22,24,30]. This activated c-src appears to interact with Cx43 at the SH3 domain, and causes phosphorylation of Cx43. Interestingly this may have a role other than that of directly regulation of the Cx43 channel. Toyofuku et al. showed that interaction of Cx43 with c-src is also capable of changing the Cx43-protein interactions such that that binding of c-src to Cx43 disrupts the interaction of Cx43 with the scaffolding protein ZO-1 [32]. This suggests that one role of c-src interaction with Cx43 may involve regulation of the composition of the protein complex at the Cx43 Nexus.
4. Conclusions The study of protein–protein interactions of connexins is a newly emerging field, thus while a number of binding partners are known (Fig. 5), the extent of diversity and binding affinities of proteins within the Nexus of different connexins is awaiting clarification. As indicated above, the finding that composition of the Nexus may change in response to intracellular stimuli indicates that the Nexus is a dynamic structure that changes as the needs of the cell, and any interconnected cells, change in response to environmental cues. The ultimate goal of these studies is not just to identify the connexin binding partners, but also to determine under which physiological conditions and in which intracellular compartment along their trafficking pathway these partners alter their association with connexins. Thus, the gap junction Nexus may be visualized as a scaffold for a whole host of signaling, structural, and cytoskeletal
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Fig. 5. Schematic showing the known binding partners for connexins. Individual connexins are shown with the specific binding partners that each has been shown to have. The exact location of where each protein binds is not known yet, although many appear to interact with the carboxyl terminal domains of the conne xins.
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Formation of the gap junction nexus: binding partners for connexins Heather S. Duffya, Mario Delmarb, David C. Spraya,* a
Department of Neuroscience, Albert Einstein College of Medicine, 1410 Pelham Pkwy S., Bronx, NY 10461, USA b SUNY Upstate Medical University, Syracuse, NY 13210, USA
Abstract Gap junctions are the morphological correlates of direct cell–cell communication and are formed of hexameric assemblies of gap junction proteins (connexins) into hemichannels (or connexons) provided by each coupled cell. Gap junction channels formed by each of the connexin subtypes (of which there are as many as 20) display different properties, which have been attributed to differences in amino acid sequences of gating domains of the connexins. Recent studies additionally indicate that connexin proteins interact with other cellular components to form a protein complex termed the Nexus. This review summarizes current knowledge regarding the protein–protein interactions involving of connexin proteins and proposes hypothesized functions for these interactions. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Astrocyte; CNS; Synapses
Regions of cell–cell and cell–substrate interactions and of intercellular communication are specialized membrane microdomains containing complexes of proteins fulfilling these roles and linking these domains to the cytoskeleton. Such complexes include occluding junctions (zonula occludens), anchoring junctions (zonula adherens and hemidesmosomes/desmosomes) and communicating junctions (chemical and electrotonic synapses, the latter of which are gap junctions, or zonula communicans). With the exception of gap junctions, each of these membrane domains has been known for some time to involve complex interactions between multiple protein binding partners [33,34,19]. Traditionally, gap junction proteins (connexins) have been considered as simple pore-forming proteins that exhibit little or no interaction with other cellular components. Thus, much of the work on gap junctions has focused on the structure and function of individual connexins [29]. Evidence is now accumulating that this view of the gap junction has been too narrow and that connexins have rich interactions with a myriad of other proteins that may turn out to be important in multiple aspects of gap junction biology including function, regulation, and even structure. Connexins are four transmembrane domain (tetraspan) proteins with cytoplasmically localized amino * Corresponding author. Tel.: +1-718-430-2537; fax: +1-718-4308594. E-mail address: [email protected] (D.C. Spray).
terminus, cytoplasmic loop and carboxyl terminal domains (Fig. 1). It is these intracellular domains that are the most variable in amino acid sequence among the different connexins, with the cytoplasmic loop and carboxyl terminal domains conferring most of the diversity among connexin subtypes. The presence of these variable sequences, some of which contain known signaling domain motifs (for one example see Fig. 2) implies that these regions may be important in differential connexin function. Studies now show that there are variations in the protein-protein interactions of these domains and it is these differing protein–protein interactions that may confer distinctive functional or regulatory specificity to gap junctions formed of the individual connexins. The study of interactions of proteins with connexins have been somewhat hampered by the difficulty in extracting connexins from their location at the appositional membranes between cells. Interactions have been studied in a variety of ways including immunolocalization, immunoprecipitation, and binding assays of one type or another. Each of these has its advantages and limitations. Immunolocalization of other proteins with connexins is readily possible using immunofluorescence confocal microscopy because the connexins are localized in specific membrane regions, namely sites of cell–cell contact. However, overlapping staining must then be followed with rigorous biochemistry to ensure that the co-localization is not simply fortuitous. There are multiple biochemical techniques that have been routinely used in the study of connexin–protein interactions.
0928-4257/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0928-4257(02)00012-8
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Fig. 1. Schematic of a standard connexin molecule. There are two extracellular loop, designated E1 and E2, and three distinct intracellular domains, the amino terminus (NT), the cytoplasmic loop (CL) and the carboxyl terminus (CT). The extracellular loops and transmembrane domains are highly conserved while the cytoplasmic domains vary between connexins, particularly in the regions of the CL and the CT.
Fig. 2. Schematic of the amino acid sequence of the Cx43 carboxyl terminal domain. Regions of known binding motifs are highlighted in gray. Two regions for potential interactions with signaling molecules dominate the Cx43 carboxyl terminal domain, a proline rich region from amino acid 274–283 and a serine rich region from amino acid 359–369.
These include co-immunoprecipitation studies with connexin antibodies and binding assays to connexinGST fusion proteins. When all works well, these techniques yield important information as to what proteins are associated, either directly or within a complex, with the connexin of interest used as a ‘‘capture’’ ligand. The major difficulty in applying these techniques to the study of connexin–protein interactions is the low protein levels of connexins found in many cell types. Improving the affinity of the connexin–substrate linkage, enrichment for connexin in cell extracts, and pooling of purified complexes may provide useful improvements in this technique. Recently we have begun using the technique of mirror resonance (MR) to quantify the kinetics of binding between Cx43 and known binding partners and as a tool to identify novel connexin binding partners [5]. Mirror
resonance records the interaction of binding partners in real time, thus allowing direct measurements of K(association) and K(dissociation) and calculation of the KD (from Kdiss/Kass or from steady state response amplitudes) for interactions observed. One example of such an experiment has involved covalent linkage of the carboxyl terminal domain of Cx43 (Cx43CT) to MR cuvettes as a ‘‘capture’’ ligand, and measurement of responses to addition of lysates of wild type murine astrocytes to the Cx43CT. Such studies have indicated the existence of proteins in astrocytes that bind directly to the Cx43CT (Fig. 3). The next step in such studies is to elute the binding partners from the MR cuvette following binding for identification using MALDI-TOF mass spectroscopy. Although the above demonstration of the existence of cytoplasmic binding partners for Cx43 in astrocytes was performed using a technique that is novel in the gap junction field, it is consistent with recent reports in other cell types, indicating that Cx43 has a number of binding partners, many of which have been localized within astrocytes. In addition, other connexins have also been shown to interact with other proteins, and it seems clear that the application of improved ‘‘fishing’’ strategies will greatly expand this list. The sections that follow summarize the state of knowledge regarding connexin–protein interactions and provide hypotheses regarding possible functions of these interactions.
1. Tight junction associated proteins Among the first types of proteins that were shown to interact with connexins are those traditionally thought to interact as part of the tight junction complex and in the case of ZO-1, adherens junctions as well [14]. Occludin was first described as an integral membrane protein involved in the formation of the tight junctional
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Fig. 3. Binding of astrocyte lysates to the carboxy terminal domain of Cx43. Panel A shows the trace of a single experiment showing the binding of astrocyte lysate in real time. Arrows designate the addition of astrocyte lysate to the cuvette (arrow 1), rinse with phosphate buffered saline with 1% Tween20 (Sigma Chemicals, St. Louis MO) (arrow 2), and regeneration of the cuvette using 10 mM HCl (arrow 3). Panel B shows the concentration dependence of the binding of astrocyte lysate components to the Cx43 carboxyl terminal domain. The Kd estimated for the total of the unknown binding partners is 17 ng/ml showing high affinity binding of some component of astrocytes to the Cx43 carboxyl terminal domain.
strands [8]. Similarly, claudins have been described as a primary component of tight junctions [10]. Zonula occludens-1, a cytoplasmic scaffolding protein, was hypothesized to play a key role in the localization of tight junction proteins within the tight junction strand [9]. Each of these has now been described to associate with connexins as well. 1.1. Zonula occludens-1 (ZO-1) ZO-1 was the first tight junction associated protein to be shown to interact with connexins. Reports showed
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that connexin43 (Cx43), the primary connexin subtype found in many tissues including fibroblasts and myocytes, directly interacted with ZO-1 [31,13]. The site of interaction between Cx43 and ZO-1 was shown to occur through the second PDZ domain of ZO-1 and the carboxyl terminal end of Cx43. The carboxyl terminal of Cx43 contains a potential consensus sequence for a PDZ binding domain (DLEI, Fig. 2) that has been hypothesized to be important in the Cx43-ZO-1 interaction. This association of Cx43 with ZO-1 has been confirmed in Sertoli cells in the testes [2], and in astrocytes [4]. Cx43 is apparently not the only connexin that links with ZO-1. Two groups have recently reported that Cx45 also interacts with ZO-1 [17,20], potentially stabilizing heteromeric complexes of Cx43. Interestingly, both Cx43 and Cx45 are alpha subtype gap junction proteins. Examination of the sequences of the other alpha connexins suggests that there may be multiple alpha connexins with the potential to interact with ZO-1. In contrast, the beta and gamma connexins appear not to have the PDZ binding domain consensus sequences at their most terminal carboxyl regions, implying that these connexins are less likely to interact with ZO-1 in the typical PDZ-carboxyl terminus manner. The role of the interaction between connexins and ZO-1 is unclear but several hypotheses have been proposed. Toyofuku et al. have suggested that one potential role is in the trafficking of Cx43 to the intercalated discs of the heart, thereby aiding in the formation of gap junctions in these regions. Although Cx43-ZO-1 linkage may contribute to this process, studies by our group and others [7,27] have shown that carboxyl terminal truncations of connexin43 are still trafficked to the membrane and form functional channels. This suggests that the most terminal end of the protein is not required for formation of gap junctions at cell membranes. However, it should be mentioned that the question of whether efficiency of trafficking is altered in the absence of the Cx43–ZO-1 interaction has not yet been addressed. Another potential role for the interaction of ZO-1 with Cx43 is to act as a scaffold for other proteins, thereby bringing them into close contact with either the gap junction proteins themselves or with molecules that pass through the gap junction plaques. However, it also remains to be determined if ZO-1 helps form such a multimeric complex at the site of gap junctions. 1.2. Occludin Occludin is an integral membrane tight junction associated protein that was thought to be restricted in function to the tight junctional complex. It, like the connexins, is also a four transmembrane domain protein. Using freeze fracture immunogold labeling occludin has been shown to make up some of the particles found in tight junctional strands and both total levels
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and phosphorylation state of occludin have been proposed to confer the barrier function of tight junctions [6,25]. Recent studies using cultured hepatocytes show that occludin is also associated with gap junctional proteins, in particular connexin32 [18]. Examination of freeze-fracture replicas of these cells shows many small gap junctional plaques localized near tight junctional strands, and co-immunoprecipitation studies on cultured hepatocytes Western blotted with Cx32 provided evidence for an association between occludin and Cx32. Immunofluorescence studies have co-localized these proteins at cell membranes, suggesting that they may interact at the membranes but determination of whether there is Cx32 in tight junction strands, occludin in gap junctional plaques, or both awaits freeze-fracture immunogold studies to try to localize these proteins to the junctions that they form within membranes. Cx26 has also been reported to interact with occludin in polarized sheets of a human intestinal cell line T84 through an interaction with the coiled-coil domain of occludin [28]. The occludin-Cx26 interaction was not confirmed by Kojima et al. in hepatocytes [18], indicating either that there may be cell specific interactions between this protein and the connexins or that the occludin domain used for the connexin binding experiments may be masked in ‘‘native’’ occludin. 1.3. Claudins An additional tetraspan family of proteins, the claudins, has also been found to associate with connexins. Tsukita’s group first described the claudin family of integral membrane proteins that are components of tight junctional complexes [26]. There are a number of members of the claudin family, each of which is cell specifically localized. In their examination of cultured hepatocytes, Kojima et al. found that along with an association with occludin, Cx32 also associated with claudin-1 [18], a primary hepatocyte claudin subtype; as with the occludin interaction, the locus of this interaction is unclear. It has been hypothesized that the interaction allows for the formation of a scaffold of proteins localized near sites of cell contact, thereby localizing signaling proteins that may be regulated by signals passed through the gap junctions. Another type of interaction between connexins and claudins has also been reported, a reciprocal regulation of the two proteins. In a cell type that is normally devoid of tight junctions, astrocytes, treatment with the pro-inflammatory cytokine Interleukin-1b causes a loss of Cx43 [15] with a concurrent upregulation of the previously absent protein claudin-1 [4]. Whether this is due to a single transcriptional regulator or to two independent events is unknown, but the outcome at the membrane is a morphological switch from gap junctions to
rudimentary protein strands containing elements of the correct size and structure to be tight junctional proteins (Fig. 4). It was hypothesized that by limiting both intercellular communication via gap junctions and bulk flow through the extracellular space, the switch from gap junctions to tight junction-like strands may help limit the size of lesions due to astrocyte damage.
2. Adherens junction associated proteins The function of adherens junctions is both in holding appositional cells together, an ‘‘adhesive’’ function, and in intracellular signaling cascades important in cell motility and division. They are composed of a complex of proteins localized to appositional membranes. Very recently there have been reports of interactions of connexins with members of one protein family found in the adherens junctional complex, the catenins. Examination of the role of Wnt-1 signaling in cardiac myocytes revealed a direct interaction between Cx43 and the adherens junction associated protein, b-catenin [1]. This interaction was hypothesized to be important in Wnt-1 regulation of Cx43 transcription and to provide feedback for regulating the extent of junctional communication between cardiac myocytes. Another catenin family member found to be associated with Cx43 is p120 catenin. A recent study by Xu et al. showed co-localization of Cx43 with p120 catenin, an Armadillo protein involved in modulation of cell motility [35]. In this study neural crest cells were shown to co-express Cx43 and p120 catenin, proteins that were regulated in a similar fashion by the Wnt-1 signaling pathway, and these proteins co-localized at sites of cell– cell contact. The importance of this interaction in development is not yet known but a signaling role for Cx43 in development has been hypothesized. 2.1. Cytoskeletal proteins To date, there has been only one report of direct interaction between a connexin and a cytoskeletal protein. This study used GST binding assays and immunolocalization to show binding between Cx43 and the cytoskeletal protein tubulin [12]. They determined that a juxtamembrane region of the Cx43 carboxyl terminal domain was required for this interaction but that disruption of microtubules with nocodazole had no apparent effect on either Cx43 localization at the membrane or on gross channel function. While this in no way rules out a function for the Cx43-tubulin interaction, it does indicate that this interaction may be less important than interactions that directly regulate the channel, such as the Cx43 interaction with src (below).
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Fig. 4. Ultrastructural examination of control and IL-1b treated human astrocytes. In control cells (panel A) large gap junctions were found between cultured astrocytes. In IL-1b treated cells these gap junctions were absent, instead rudimentary strands of intramembranous particles were seen (Reprinted with permission, Duffy et al., 2000, J. of Neuroscience 20 RC114).
3. Src proteins Of substantial interest for the past several years has been the interaction of Cx43 with the proto-oncogenic signaling protein Src. Src is a non-receptor tyrosine kinase that, upon activation, is an effector molecule of multiple signaling pathways such as those involving MAPK and PKC. In normal cells src resides in the inactive form due to phosphorylation of tyrosine 527. Release of this phosphorylation and subsequent phosphorylation within src’s SH2/SH3 domains results in activation of src, thereby allowing for tyrosine phosphorylation of its targets [3]. The exact mechanism of gating of Cx43 by src has been the topic of some debate. In a report that appeared over a decade ago, a kinase-active form of src (pp60vsrc) was reported to require the tyrosine phosphorylation of Cx43 in order to inhibit cell–cell communication in the oocyte expression system [30]. In 1995 Alan Lau’s group reported that ppv60-src directly interacts with Cx43 in mammalian cells, concurrent with phosphorylation of Y247 and Y265 in the carboxyl terminal domain of Cx43 [23,16]. They subsequently showed that interactions of Cx43 proline-rich regions are also important in the interactions of Cx43 with the SH2 and SH3 domains of v-src [22]. Subsequent work showed partial co-localization of Cx43 and ppv-src in fibroblasts and showed that these two proteins could be reciprocally co-immunoprecipitated from pp60v-src transformed cells [24]. In 1999 Bruce Nicholson’s group reported that in Xenopus oocytes the interaction of the SH3 domain was important for the regulation of the Cx43 channel by pp60v-src but that serine, rather than tyrosine phosphorylation was required [36]. Subsequent studies have corroborated the early work by Swenson and clearly show that the phosphorylation of the tyrosines is an important phosphorylation step in the loss of gap junctional communication induced by interaction of pp60v-src with Cx43 [21,22].
Regulation of Cx43 channel opening and closure may be only part of the function of the interaction of src with connexin43. Recent evidence has indicated that the normal cellular (c-src) form of this oncogene also interacts with Cx43, but only when activated by phosphorylation [11], supporting findings by Alan Lau’s group on v-src and the early work by Swenson et al. [16,22,24,30]. This activated c-src appears to interact with Cx43 at the SH3 domain, and causes phosphorylation of Cx43. Interestingly this may have a role other than that of directly regulation of the Cx43 channel. Toyofuku et al. showed that interaction of Cx43 with c-src is also capable of changing the Cx43-protein interactions such that that binding of c-src to Cx43 disrupts the interaction of Cx43 with the scaffolding protein ZO-1 [32]. This suggests that one role of c-src interaction with Cx43 may involve regulation of the composition of the protein complex at the Cx43 Nexus.
4. Conclusions The study of protein–protein interactions of connexins is a newly emerging field, thus while a number of binding partners are known (Fig. 5), the extent of diversity and binding affinities of proteins within the Nexus of different connexins is awaiting clarification. As indicated above, the finding that composition of the Nexus may change in response to intracellular stimuli indicates that the Nexus is a dynamic structure that changes as the needs of the cell, and any interconnected cells, change in response to environmental cues. The ultimate goal of these studies is not just to identify the connexin binding partners, but also to determine under which physiological conditions and in which intracellular compartment along their trafficking pathway these partners alter their association with connexins. Thus, the gap junction Nexus may be visualized as a scaffold for a whole host of signaling, structural, and cytoskeletal
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Fig. 5. Schematic showing the known binding partners for connexins. Individual connexins are shown with the specific binding partners that each has been shown to have. The exact location of where each protein binds is not known yet, although many appear to interact with the carboxyl terminal domains of the conne xins.
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