Dileucine-like motifs in the C-terminal tail of connexin32 control its endocytosis and assembly into gap junctions

Abstract

Defects in assembly of gap junction-forming proteins, called connexins (Cxs), are observed in a variety of cancers. Connexin32 (Cx32; also known as GJB1) is expressed by the polarized cells in epithelia. We discovered two dileucine-based motifs, which govern the intracellular sorting and endocytosis of transmembrane proteins, in the C-terminal tail of Cx32 and explored their role in regulating its endocytosis and gap junction-forming abilities in pancreatic and prostate cancer cells. One motif, designated as LI, was located near the juxtamembrane domain, whereas the other, designated as LL, was located distally. We also discovered a non-canonical motif, designated as LR, in the C-terminal tail. Our results showed that rendering these motifs non-functional had no effect on the intracellular sorting of Cx32. However, rendering the LL or LR motif nonfunctional enhanced the formation of gap junctions by inhibiting Cx32 endocytosis by the clathrin-mediated pathway. Rendering the LI motif nonfunctional inhibited gap junction formation by augmenting the endocytosis of Cx32 via the LL and LR motifs. Our studies have defined distinct roles of these motifs in regulating the endocytosis of Cx32 and its gap junction-forming ability.