Structural Changes in the Carboxyl Terminus of the Gap Junction Protein Connexin43 Indicates Signaling between Binding Domains for c-Src and Zonula Occludens-1

Abstract

Regulation of cell-cell communication by the gap junction protein connexin43 can be modulated by a variety of connexin-associating proteins. In particular, c-Src can disrupt the connexin43 (Cx43)-zonula occludens-1 (ZO-1) interaction, leading to down-regulation of gap junction intercellular communication. The binding sites for ZO-1 and c-Src correspond to widely separated Cx43 domains (ϳ100 residues apart); however, little is known about the structural modifications that may allow information to be transferred over this distance. Here, we have characterized the structure of the connexin43 carboxyl-terminal domain (Cx43CT) to assess its ability to interact with domains from ZO-1 and c-Src. NMR data indicate that the Cx43CT exists primarily as an elongated random coil, with two regions of ␣-helical structure. NMR titration experiments determined that the ZO-1 PDZ-2 domain affected the last 19 Cx43CT residues, a region larger than that reported to be required for Cx43CT-ZO-1 binding. The c-Src SH3 domain affected Cx43CT residues Lys-264 -Lys-287, Ser-306 -Glu-316, His-331-Phe-337, Leu-356 -Val-359, and Ala-367-Ser-372. Only region Lys-264 -Lys-287 contains the residues previously reported to act as an SH3 binding domain. The specificity of these interactions was verified by peptide competition experiments. Finally, we demonstrated that the SH3 domain could partially displace the Cx43CT-PDZ-2 complex. These studies represent the first structural characterization of a connexin domain when integrated in a multimolecular complex. Furthermore, we demonstrate that the structural characteristics of a disordered Cx43CT are advantageous for signaling between different binding partners that may be important in describing the mechanism of channel closure or internalization in response to pathophysiological stimuli.Gap junction channels serve to directly interconnect the cytoplasm of neighboring cells, allowing the passage of moderately small ions, metabolites, and signaling molecules. Mammalian gap junction channels are formed by as many as 21 different connexin proteins (1). Of these, connexin43 (Cx43) 1 is the most completely characterized in terms of channel gating properties (2-4), phosphorylation sites (5-7), mechanisms of pH sensitivity (8 -11), and overall molecular structure (12). Cx43 is the most abundant gap junction protein in various tissues, including heart and brain. Cx43 null mice have been extensively investigated, with important differences being found as compared with wild types with regard to numerous processes, including cardiac developmental abnormalities, electrical synchrony in the heart, spreading depression in brain, as well as global gene expression changes in heart and astrocytes (13)(14)(15)(16)(17)(18)(19)(20).Connexin molecules are tetraspan membrane proteins, with both amino and carboxyl termini within the cytoplasm. Although the structure of the membrane-spanning portions of Cx43 has been solved to a resolution of about 7.5 Å (in the membrane plane) using electron crystallography (12), a constr...

Interaction between connexin35 and zonula occludens-1 and its potential role in the regulation of electrical synapses

“…This discrepancy is ascribable to differences in the experimental conditions: (i) the full length of the cytoplasmic C terminus was tested in that study (changes in conformation upstream to the PDZ binding domain could alter binding) (27), and (ii) in contrast with our studies, the C terminus was used as ligand. The shorter C-terminal Cx43 peptide (Cx43CT 20 ) used in our study competed against the interaction between (full length) Cx43CT and PDZ2 of ZO-1 in NMR studies (27), which is consistent with our observations. The ZO-1 PDZ domains we used for SPR analysis were human sequences, and the domain structure of human and teleost ZO-1 are very similar (37).…”

“…4E was neither concentration-dependent nor reproducible. The lack of interaction of Cx35CT 15 with the PDZ2 domain did not result from a lack of instrumental sensitivity, as a peptide corresponding to the last 20 aa of the Cx43 C terminus (Cx43CT 20 ; PSSRASSRASSRPRPDDLEI), which includes the reported PDZ2 binding motif (27,33), bound with higher affinity to the PDZ2 domain of ZO-1 in a concentrationdependent manner ( Fig. 4F; one run on a chip with two levels of ligand).…”

Modulation of connexin expression and gap junction communication in astrocytes by the gram‐positive bacterium S. aureus

“…Similarly, our results with the p38 MAPK inhibitor SB202190 indicate that the MAPK pathway is responsible, in part, for the inhibitory effects of S. aureus on functional GJC in astrocytes. However, the participation of other kinases that have been shown to interact with Cx43, such as PKC (Martinez et al, 2002;Reynhout et al, 1992), casein kinase 1 (Cooper and Lampe, 2002), PKA (Burghardt et al, 1995;TenBroek et al, 2001), and c-Src Sorgen et al, 2004) cannot be excluded in modulating the S. aureus-induced decrease in Cx43 expression, and warrant further investigation.…”

The Second PDZ Domain of Zonula Occludens-1 Is Dispensable for Targeting to Connexin43 Gap Junctions

“…Yet recent studies have demonstrated that the interaction of Cx43 with ZO-1 appears to involve conserved residues in Cx43 that are upstream of the canonical C-terminal PDZ binding motif (Fanning et al 2007;Sorgen et al 2004), and further indicate that Cx43 C-termini might interface with ZO-1 homodimerized by PDZ2 interactions (Fanning et al 2007). Thus it remains possible that abrogation of PDZ-mediated Cx43-ZO-1 interaction by Cterminal tagging of Cx43 is due not only to masking of the terminal isoleucine residue (Giepmans and Moolenaar 1998), but also results from disruption of critical residues further upstream (and/or loss of ZO-1 dimerization).…”

“…ZO-1 has been shown to interact with several connexins, but the best characterized is Cx43 (Giepmans 2004). The second PDS95/dlg/ZO-1 (PDZ2) domain of ZO-1 is known to interact with the cytoplasmic C-terminal residues of Cx43 (Giepmans and Moolenaar 1998;Sorgen et al 2004). However, the physiological significance of direct PDZ2-mediated interaction between ZO-1 and Cx43 has remained elusive.…”