Formation of the gap junction nexus: binding partners for connexins

Heather S. Duffya, Mario Delmarb, David C. Spraya,*aDepartment of Neuroscience, Albert Einstein College of Medicine, 1410 Pelham Pkwy S., Bronx, NY 10461, USA

bSUNY 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 gapjunction proteins (connexins) into hemichannels (or connexons) provided by each coupled cell. Gap junction channels formed byeach 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 withother cellular components to form a protein complex termed the Nexus. This review summarizes current knowledge regarding theprotein–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 interactionsand of intercellular communication are specializedmembrane microdomains containing complexes of pro-teins fulfilling these roles and linking these domains tothe cytoskeleton. Such complexes include occludingjunctions (zonula occludens), anchoring junctions(zonula adherens and hemidesmosomes/desmosomes)and communicating junctions (chemical and electro-tonic synapses, the latter of which are gap junctions, orzonula communicans). With the exception of gap junc-tions, each of these membrane domains has been knownfor some time to involve complex interactions betweenmultiple protein binding partners [33,34,19].Traditionally, gap junction proteins (connexins) have

been considered as simple pore-forming proteins thatexhibit little or no interaction with other cellular com-ponents. Thus, much of the work on gap junctions hasfocused on the structure and function of individualconnexins [29]. Evidence is now accumulating that thisview of the gap junction has been too narrow and thatconnexins have rich interactions with a myriad of otherproteins that may turn out to be important in multipleaspects of gap junction biology including function, reg-ulation, and even structure.Connexins are four transmembrane domain (tetra-

span) proteins with cytoplasmically localized amino

terminus, cytoplasmic loop and carboxyl terminaldomains (Fig. 1). It is these intracellular domains thatare the most variable in amino acid sequence among thedifferent connexins, with the cytoplasmic loop and car-boxyl terminal domains conferring most of the diversityamong connexin subtypes. The presence of these vari-able sequences, some of which contain known signalingdomain motifs (for one example see Fig. 2) implies thatthese regions may be important in differential connexinfunction. Studies now show that there are variations inthe protein-protein interactions of these domains and itis these differing protein–protein interactions that mayconfer distinctive functional or regulatory specificity togap junctions formed of the individual connexins.The study of interactions of proteins with connexins

have been somewhat hampered by the difficulty inextracting connexins from their location at the apposi-tional membranes between cells. Interactions have beenstudied in a variety of ways including immunolocaliza-tion, immunoprecipitation, and binding assays of onetype or another. Each of these has its advantages andlimitations. Immunolocalization of other proteins withconnexins is readily possible using immunofluorescenceconfocal microscopy because the connexins are localizedin specific membrane regions, namely sites of cell–cellcontact. However, overlapping staining must then befollowed with rigorous biochemistry to ensure that theco-localization is not simply fortuitous. There are mul-tiple biochemical techniques that have been routinelyused in the study of connexin–protein interactions.

0928-4257/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.

PI I : S0928-4257(02 )00012-8

Journal of Physiology - Paris 96 (2002) 243–249

www.elsevier.com/locate/jphysparis

* Corresponding author. Tel.: +1-718-430-2537; fax: +1-718-430-

8594.

E-mail address: spray@aecom.yu.edu (D.C. Spray).

These include co-immunoprecipitation studies withconnexin antibodies and binding assays to connexin-GST fusion proteins. When all works well, these tech-niques yield important information as to what proteinsare associated, either directly or within a complex, withthe connexin of interest used as a ‘‘capture’’ ligand. Themajor difficulty in applying these techniques to thestudy of connexin–protein interactions is the low pro-tein levels of connexins found in many cell types.Improving the affinity of the connexin–substrate link-age, enrichment for connexin in cell extracts, and pool-ing of purified complexes may provide usefulimprovements in this technique.Recently we have begun using the technique of mirror

resonance (MR) to quantify the kinetics of bindingbetween Cx43 and known binding partners and as a toolto identify novel connexin binding partners [5]. Mirror

resonance records the interaction of binding partnersin real time, thus allowing direct measurements ofK(association) and K(dissociation) and calculation of the KD(from Kdiss/Kass or from steady state response ampli-tudes) for interactions observed. One example of suchan experiment has involved covalent linkage of the car-boxyl terminal domain of Cx43 (Cx43CT) to MR cuv-ettes as a ‘‘capture’’ ligand, and measurement ofresponses to addition of lysates of wild type murineastrocytes to the Cx43CT. Such studies have indicatedthe existence of proteins in astrocytes that bind directlyto the Cx43CT (Fig. 3). The next step in such studies isto elute the binding partners from the MR cuvette fol-lowing binding for identification using MALDI-TOFmass spectroscopy.Although the above demonstration of the existence of

cytoplasmic binding partners for Cx43 in astrocytes wasperformed using a technique that is novel in the gapjunction field, it is consistent with recent reports in othercell types, indicating that Cx43 has a number of bindingpartners, many of which have been localized withinastrocytes. In addition, other connexins have also beenshown to interact with other proteins, and it seems clearthat the application of improved ‘‘fishing’’ strategies willgreatly expand this list. The sections that follow sum-marize the state of knowledge regarding connexin–pro-tein interactions and provide hypotheses regardingpossible functions of these interactions.

1. Tight junction associated proteins

Among the first types of proteins that were shown tointeract with connexins are those traditionally thoughtto interact as part of the tight junction complex and inthe case of ZO-1, adherens junctions as well [14].Occludin was first described as an integral membraneprotein involved in the formation of the tight junctional

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.

244 H.S. Duffy et al. / Journal of Physiology - Paris 96 (2002) 243–249

strands [8]. Similarly, claudins have been described as aprimary component of tight junctions [10]. Zonulaoccludens-1, a cytoplasmic scaffolding protein, washypothesized to play a key role in the localization oftight junction proteins within the tight junction strand[9]. Each of these has now been described to associatewith connexins as well.

1.1. Zonula occludens-1 (ZO-1)

ZO-1 was the first tight junction associated protein tobe shown to interact with connexins. Reports showed

that connexin43 (Cx43), the primary connexin subtypefound in many tissues including fibroblasts and myo-cytes, directly interacted with ZO-1 [31,13]. The site ofinteraction between Cx43 and ZO-1 was shown to occurthrough the second PDZ domain of ZO-1 and the car-boxyl terminal end of Cx43. The carboxyl terminal ofCx43 contains a potential consensus sequence for aPDZ binding domain (DLEI, Fig. 2) that has beenhypothesized to be important in the Cx43-ZO-1 inter-action. This association of Cx43 with ZO-1 has beenconfirmed in Sertoli cells in the testes [2], and in astro-cytes [4]. Cx43 is apparently not the only connexin thatlinks with ZO-1. Two groups have recently reportedthat Cx45 also interacts with ZO-1 [17,20], potentiallystabilizing heteromeric complexes of Cx43. Interest-ingly, both Cx43 and Cx45 are alpha subtype gap junc-tion proteins. Examination of the sequences of the otheralpha connexins suggests that there may be multiplealpha connexins with the potential to interact withZO-1. In contrast, the beta and gamma connexinsappear not to have the PDZ binding domain consensussequences at their most terminal carboxyl regions,implying that these connexins are less likely to interactwith 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 pro-posed. Toyofuku et al. have suggested that one poten-tial role is in the trafficking of Cx43 to the intercalateddiscs of the heart, thereby aiding in the formation of gapjunctions in these regions. Although Cx43-ZO-1 linkagemay contribute to this process, studies by our group andothers [7,27] have shown that carboxyl terminal trunca-tions of connexin43 are still trafficked to the membraneand form functional channels. This suggests that themost terminal end of the protein is not required forformation of gap junctions at cell membranes. However,it should be mentioned that the question of whetherefficiency of trafficking is altered in the absence of theCx43–ZO-1 interaction has not yet been addressed.Another potential role for the interaction of ZO-1 withCx43 is to act as a scaffold for other proteins, therebybringing them into close contact with either the gapjunction proteins themselves or with molecules that passthrough the gap junction plaques. However, it alsoremains to be determined if ZO-1 helps form such amultimeric complex at the site of gap junctions.

1.2. Occludin

Occludin is an integral membrane tight junctionassociated protein that was thought to be restricted infunction to the tight junctional complex. It, like theconnexins, is also a four transmembrane domain pro-tein. Using freeze fracture immunogold labeling occlu-din has been shown to make up some of the particlesfound in tight junctional strands and both total levels

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 buf-

fered 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 astro-

cyte 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.

H.S. Duffy et al. / Journal of Physiology - Paris 96 (2002) 243–249 245

and phosphorylation state of occludin have been pro-posed to confer the barrier function of tight junctions[6,25]. Recent studies using cultured hepatocytes showthat occludin is also associated with gap junctionalproteins, in particular connexin32 [18]. Examination offreeze-fracture replicas of these cells shows manysmall gap junctional plaques localized near tightjunctional strands, and co-immunoprecipitation stud-ies on cultured hepatocytes Western blotted withCx32 provided evidence for an association betweenoccludin and Cx32. Immunofluorescence studies haveco-localized these proteins at cell membranes, sug-gesting that they may interact at the membranes butdetermination of whether there is Cx32 in tight junc-tion strands, occludin in gap junctional plaques, orboth awaits freeze-fracture immunogold studies to tryto localize these proteins to the junctions that theyform within membranes.Cx26 has also been reported to interact with occludin

in polarized sheets of a human intestinal cell line T84through an interaction with the coiled-coil domain ofoccludin [28]. The occludin-Cx26 interaction was notconfirmed by Kojima et al. in hepatocytes [18], indicat-ing either that there may be cell specific interactionsbetween this protein and the connexins or that theoccludin domain used for the connexin binding experi-ments may be masked in ‘‘native’’ occludin.

1.3. Claudins

An additional tetraspan family of proteins, the clau-dins, has also been found to associate with connexins.Tsukita’s group first described the claudin family ofintegral membrane proteins that are components oftight junctional complexes [26]. There are a number ofmembers of the claudin family, each of which is cellspecifically localized. In their examination of culturedhepatocytes, Kojima et al. found that along with anassociation with occludin, Cx32 also associated withclaudin-1 [18], a primary hepatocyte claudin subtype; aswith the occludin interaction, the locus of this interac-tion is unclear. It has been hypothesized that the inter-action allows for the formation of a scaffold of proteinslocalized near sites of cell contact, thereby localizingsignaling proteins that may be regulated by signals pas-sed through the gap junctions.Another type of interaction between connexins and

claudins has also been reported, a reciprocal regulationof the two proteins. In a cell type that is normallydevoid of tight junctions, astrocytes, treatment with thepro-inflammatory cytokine Interleukin-1b causes a lossof Cx43 [15] with a concurrent upregulation of the pre-viously absent protein claudin-1 [4]. Whether this is dueto a single transcriptional regulator or to two indepen-dent events is unknown, but the outcome at the mem-brane is a morphological switch from gap junctions to

rudimentary protein strands containing elements ofthe correct size and structure to be tight junctionalproteins (Fig. 4). It was hypothesized that by limitingboth intercellular communication via gap junctionsand bulk flow through the extracellular space, theswitch from gap junctions to tight junction-likestrands may help limit the size of lesions due toastrocyte damage.

2. Adherens junction associated proteins

The function of adherens junctions is both in holdingappositional cells together, an ‘‘adhesive’’ function, andin intracellular signaling cascades important in cellmotility and division. They are composed of a complexof proteins localized to appositional membranes. Veryrecently there have been reports of interactions of con-nexins with members of one protein family found in theadherens junctional complex, the catenins. Examinationof the role of Wnt-1 signaling in cardiac myocytesrevealed a direct interaction between Cx43 and theadherens junction associated protein, b-catenin [1]. Thisinteraction was hypothesized to be important in Wnt-1regulation of Cx43 transcription and to provide feed-back for regulating the extent of junctional communi-cation between cardiac myocytes.Another catenin family member found to be asso-

ciated with Cx43 is p120 catenin. A recent study by Xuet al. showed co-localization of Cx43 with p120 catenin,an Armadillo protein involved in modulation of cellmotility [35]. In this study neural crest cells were shownto co-express Cx43 and p120 catenin, proteins that wereregulated in a similar fashion by the Wnt-1 signalingpathway, and these proteins co-localized at sites of cell–cell contact. The importance of this interaction indevelopment is not yet known but a signaling role forCx43 in development has been hypothesized.

2.1. Cytoskeletal proteins

To date, there has been only one report of directinteraction between a connexin and a cytoskeletal pro-tein. This study used GST binding assays and immuno-localization to show binding between Cx43 and thecytoskeletal protein tubulin [12]. They determined thata juxtamembrane region of the Cx43 carboxyl terminaldomain was required for this interaction but that dis-ruption of microtubules with nocodazole had noapparent effect on either Cx43 localization at themembrane or on gross channel function. While this inno way rules out a function for the Cx43-tubulininteraction, it does indicate that this interaction may beless important than interactions that directly regulatethe channel, such as the Cx43 interaction with src(below).

246 H.S. Duffy et al. / Journal of Physiology - Paris 96 (2002) 243–249

3. Src proteins

Of substantial interest for the past several years hasbeen the interaction of Cx43 with the proto-oncogenicsignaling protein Src. Src is a non-receptor tyrosinekinase that, upon activation, is an effector molecule ofmultiple signaling pathways such as those involvingMAPK and PKC. In normal cells src resides in theinactive form due to phosphorylation of tyrosine 527.Release of this phosphorylation and subsequent phos-phorylation within src’s SH2/SH3 domains results inactivation of src, thereby allowing for tyrosine phos-phorylation of its targets [3].The exact mechanism of gating of Cx43 by src has

been the topic of some debate. In a report that appearedover a decade ago, a kinase-active form of src (pp60v-src) was reported to require the tyrosine phosphoryla-tion of Cx43 in order to inhibit cell–cell communicationin the oocyte expression system [30]. In 1995 Alan Lau’sgroup reported that ppv60-src directly interacts withCx43 in mammalian cells, concurrent with phosphor-ylation of Y247 and Y265 in the carboxyl terminaldomain of Cx43 [23,16]. They subsequently showed thatinteractions of Cx43 proline-rich regions are alsoimportant in the interactions of Cx43 with the SH2 andSH3 domains of v-src [22]. Subsequent work showedpartial co-localization of Cx43 and ppv-src in fibro-blasts and showed that these two proteins could bereciprocally co-immunoprecipitated from pp60v-srctransformed cells [24]. In 1999 Bruce Nicholson’s groupreported that in Xenopus oocytes the interaction of theSH3 domain was important for the regulation of theCx43 channel by pp60v-src but that serine, rather thantyrosine phosphorylation was required [36]. Subsequentstudies have corroborated the early work by Swensonand clearly show that the phosphorylation of the tyr-osines is an important phosphorylation step in the lossof gap junctional communication induced by interactionof pp60v-src with Cx43 [21,22].

Regulation of Cx43 channel opening and closure maybe only part of the function of the interaction of srcwith connexin43. Recent evidence has indicated that thenormal cellular (c-src) form of this oncogene also inter-acts with Cx43, but only when activated by phosphor-ylation [11], supporting findings by Alan Lau’s groupon v-src and the early work by Swenson et al.[16,22,24,30]. This activated c-src appears to interactwith Cx43 at the SH3 domain, and causes phosphor-ylation of Cx43. Interestingly this may have a role otherthan that of directly regulation of the Cx43 channel.Toyofuku et al. showed that interaction of Cx43 withc-src is also capable of changing the Cx43-proteininteractions such that that binding of c-src to Cx43 dis-rupts the interaction of Cx43 with the scaffolding pro-tein ZO-1 [32]. This suggests that one role of c-srcinteraction with Cx43 may involve regulation of thecomposition of the protein complex at the Cx43 Nexus.

4. Conclusions

The study of protein–protein interactions of con-nexins is a newly emerging field, thus while a number ofbinding partners are known (Fig. 5), the extent ofdiversity and binding affinities of proteins within theNexus of different connexins is awaiting clarification. Asindicated above, the finding that composition of theNexus may change in response to intracellular stimuliindicates that the Nexus is a dynamic structure thatchanges as the needs of the cell, and any interconnectedcells, change in response to environmental cues. Theultimate goal of these studies is not just to identify theconnexin binding partners, but also to determine underwhich physiological conditions and in which intracellularcompartment along their trafficking pathway these part-ners alter their association with connexins. Thus, the gapjunction Nexus may be visualized as a scaffold for awhole host of signaling, structural, and cytoskeletal

Fig. 4. Ultrastructural examination of control and IL-1b treated human astrocytes. In control cells (panel A) large gap junctions were foundbetween cultured astrocytes. In IL-1b treated cells these gap junctions were absent, instead rudimentary strands of intramembranous particles wereseen (Reprinted with permission, Duffy et al., 2000, J. of Neuroscience 20 RC114).

H.S. Duffy et al. / Journal of Physiology - Paris 96 (2002) 243–249 247

binding partners that dance a well coordinated ballet ofinteractions as cells change their minute to minute needfor cell–cell communication.

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