A tyrosine-based sorting signal is involved in connexin43 stability and gap junction turnover

Abstract

The gap junction protein connexin43 is known to have a rapid turnover, involving degradation by both the proteasomal and lysosomal systems, but the structural features of connexin43 that govern these actions are not known. The connexin43 C-terminal sequence contains a proline-rich region corresponding to the consensus of a protein-protein interaction PY-motif (xPPxY), and an overlapping putative tyrosine-based sorting signal (Yxxphi; =hydrophobic), known to play a role in the intracellular trafficking of many membrane proteins. As both motifs may control turnover of connexin43, we used a combination of metabolic radiolabelling, immuno-precipitation and functional assays to determine the possible role of these motifs in controlling degradation of human connexin43 expressed in SKHep1 cells. Mutation V289D in the tyrosine-based sorting motif increased the steady-state pool of connexin43 by approximately 3.5-fold, while mutation P283L in the PY-motif produced a comparatively modest augmentation (1.7-fold). No additive effect was observed when the overlapping tyrosine was mutated. In pulse-chase experiments, the Y286A substitution increased the half-life of connexin43 from 2 to 6 hours, indicating that the increased steady-state levels reflected reduced protein degradation. Moreover, expression at the junctional membrane, as well as gap junction-mediated intercellular communication (GJC), were nearly abolished by lysosomal inhibitors and Brefeldin A in cells expressing wild-type connexin43, but were unaffected in the tyrosine mutant. These results provide strong evidence that the tyrosine-based motif of human connexin43 is a prime determinant controlling connexin43 stability, and consequently GJC, by targeting connexin43 for degradation in the endocytic/lysosomal compartment

Defective Epidermal Barrier in Neonatal Mice Lacking the C-Terminal Region of Connexin43

“…Cx43K258stop protein displayed a prolonged half-life time. This is in accordance with data on human Cx43 by Thomas et al (2003), showing increased Cx43 half-life of 2-6 h when residue tyrosine 286 was mutated. The persistence of mutant Cx43 gap junction channels in the upper layers of the epidermis seems to interfere with terminal kerationcyte differentiation and complete closure of the epidermal permeability barrier before birth.…”

Ubiquitylation of the gap junction protein connexin-43 signals its trafficking from early endosomes to lysosomes in a process mediated by Hrs and Tsg101

“…Modulation of gap junction degradation is considered to be an important mechanism by which the level of intercellular communication via gap junctions is regulated (Berthoud et al, 2004;Laird, 2005;Musil et al, 2000;Thomas et al, 2003). However, the molecular machinery involved in the degradation of gap junctions has remained poorly understood.…”

Internalization of Large Double-Membrane Intercellular Vesicles by a Clathrin-dependent Endocytic Process

“…Alternatively, conserved putative Dab2 binding motifs of the type XPXY are present in the C terminus of Cx43 (P 283 PGY 286 ) and at least eight additional mouse and human Cxs (including hCx31.9 and its mouse orthologue mCx30.2, h/mCx32, h/mCx37, h/mCx45, h/mCx46, h/mCx47, h/mCx50, and hCx59), suggesting a potential direct interaction of Dab2 with a number of Cxs. Notably, mutation of critical amino acid residues within and around the putative Cx43-Dab2 binding site (P 283 , Y 286 , and V 289 ) significantly increased the half-life and the PM localization of Cx43 (Thomas et al, 2003), suggesting a pivotal role for Dab2 in GJ internalization.…”

“…Alternatively, conserved putative Dab2 binding motifs of the type XPXY are present in the C terminus of Cx43 (P 283 PGY 286 ) and at least eight additional mouse and human Cxs (including hCx31.9 and its mouse orthologue mCx30.2, h/mCx32, h/mCx37, h/mCx45, h/mCx46, h/mCx47, h/mCx50, and hCx59), suggesting a potential direct interaction of Dab2 with a number of Cxs. Notably, mutation of critical amino acid residues within and around the putative Cx43-Dab2 binding site (P 283 , Y 286 , and V 289 ) significantly increased the half-life and the PM localization of Cx43 (Thomas et al, 2003), suggesting a pivotal role for Dab2 in GJ internalization.In addition to its clathrin-adaptor function, Dab2 can associate via its C-terminal serine-and proline-rich region with the C-terminal globular tail of the minus-end-directed actin motor myo6. This association facilitates transport of The GTPase dynamin is also recruited to GJs, resulting in double-membrane protrusion/invagination, neck restriction, and double-membrane scission.…”