The role of connexin43-interacting protein, CIP85 in the trafficking of connexin43

Abstract

Connexin43 (Cx43) forms hemichannels and gap junction channels that are essential for the homeostatic maintenance of most cells. Dynamic regulation of Cx43-containing channels at the plasma membrane is achieved through the rapid turnover of Cx43. Thus, the balance between Cx43 turnover and synthesis controls the long-term availability of Cx43 protein. The Cx43 lifecycle is directed by interactions between Cx43 and a large number of proteins that control various aspects of Cx43 trafficking, channel formation, and degradation. One such protein, Cx43-interacting protein of 85 kDa (CIP85) was identified by yeast-two hybrid screening, and its interaction with Cx43 was characterized. CIP85 contains a Src homology 3 (SH3) domain that binds to prolinerich regions of target proteins including Cx43, a RUN domain that may play a role in Ras-like GTPase signaling, a short coiled-coil protein-binding motif and a TBC domain that may exhibit GTPase activator activity toward Rab5 and regulate endocytosis. Previously, pulse-chase assays showed that overexpression of CIP85 appeared to induce the turnover of Cx43 through the lysosomal pathway in a manner dependent upon the interaction between CIP85 and Cx43. I have helped generate mouse monoclonal antibodies against the SH3 domain and N-terminal region of CIP85, which I used to demonstrate the conservation of CIP85 and its association with Cx43 in cells from several mammalian species. My data confirm that CIP85 interacts with Cx43 at the plasma membrane, and demonstrate the interaction of CIP85 with Cx43 and clathrin at the plasma membrane. I also observed that interaction with CIP85 increases the internalization of Cx43 plaques from the plasma membrane and the rate of Cx43 turnover. Overall, these data demonstrate a role for CIP85 in Cx43 endocytic trafficking and further substantiate its role in Cx43 turnover. This knowledge of CIP85-mediated regulation of Cx43 may help to identify a novel therapeutic target to counteract the loss of Cx43 or impairment of Cx43-GJIC that disrupt normal cell functions, and are associated with many human diseases including cancer.