Abstract
We previously demonstrated that the gap junction protein connexin43 is translated as a 42-kD protein (connexin43-NP) that is efficiently phosphorylated to a 46,000-Mr species (connexin43-P2) in gap junctional communication-competent, but not in communication-deficient, cells. In this study, we used a combination of metabolic radiolabeling and immunoprecipitation to investigate the assembly of connexin43 into gap junctions and the relationship of this event to phosphorylation of connexin43. Examination of the detergent solubility of connexin43 in communication-competent NRK cells revealed that processing of connexin43 to the P2 form was accompanied by acquisition of resistance to solubilization in 1% Triton X-100. Immunohistochemical localization of connexin43 in Triton-extracted NRK cells demonstrated that connexin43-P2 (Triton-insoluble) was concentrated in gap junctional plaques, whereas connexin43-NP (Triton-soluble) was predominantly intracellular. Using either a 20 degrees C intracellular transport block or cell-surface protein biotinylation, we determined that connexin43 was transported to the plasma membrane in the Triton-soluble connexin43-NP form. Cell-surface biotinylated connexin43-NP was processed to Triton-insoluble connexin43-P2 at 37 degrees C. Connexin43-NP was also transported to the plasma membrane in communication defective, gap junction-deficient S180 and L929 cells but was not processed to Triton-insoluble connexin43-P2. Taken together, these results demonstrate that gap junction assembly is regulated after arrival of connexin43 at the plasma membrane and is temporally associated with acquisition of insolubility in Triton X-100 and phosphorylation to the connexin43-P2 form.
Highlights
We previously demonstrated that the gap junction protein connexin43 is translated as a 42-kD protein that is efficiently phosphorylated to a 46,000-M* species in gap junctional communication-competent, but not in communication-deficient, cells
Certain cell lines that are severely deficient in junctional communication constitutively synthesize Connexin43 but neither process it to the P2 form nor accumulate Connexin43 in visible gap junctional plaques
Processing of Connexin43 to the P2 form is not required for transport of Connexin43 to the cell surface but is tightly correlated with incorporation of Connexin43 intojunctional plaques, a fact suggesting that terminal phosphorylation of Connexin43 may be involved in a later step in gap junctional plaque assembly or in functional processes
Summary
We previously demonstrated that the gap junction protein connexin is translated as a 42-kD protein (connexin43-NP) that is efficiently phosphorylated to a 46,000-M* species (connexin43-P2) in gap junctional communication-competent, but not in communication-deficient, cells. Connexin43-NP was transported to the plasma membrane in communication defective, gap junction-deficient S180 and L929 cells but was not processed to Triton-insoluble connexin43-P2. In NRK and other communication-competent cells, Connexin undergoes a dramatic posttranslational change in Triton X-100 solubility that is temporally associated both with phosphorylation of connexin to tile P2 form and with assembly of Connexin into gap junctional plaques. Both acquisition of Triton insolubility and phosphorylation of Connexin occur (at least in part) after transport of Connexin to the plasma membrane. Processing of Connexin to the P2 form is not required for transport of Connexin to the cell surface but is tightly correlated with incorporation of Connexin intojunctional plaques, a fact suggesting that terminal phosphorylation of Connexin may be involved in a later step in gap junctional plaque assembly or in functional processes
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
