Double‐membrane gap junction internalization requires the clathrin‐mediated endocytic machinery

Abstract

Direct cell-cell communication mediated by plasma membrane-spanning gap junction (GJ) channels is vital to all aspects of cellular life. Obviously, GJ intercellular communication (GJIC) requires precise regulation, and it is known that controlled biosynthesis and degradation, and channel opening and closing (gating) are exploited. We discovered that cells internalize GJs in response to various stimuli. Here, we report that GJ internalization is a clathrin-mediated endocytic process that utilizes the vesicle-coat protein clathrin, the adaptor proteins adaptor protein complex 2 and disabled 2, and the GTPase dynamin. To our knowledge, we are first to report that the endocytic clathrin machinery can internalize double-membrane vesicles into cells.

Protein Kinase Cδ-mediated Phosphorylation of Connexin43 Gap Junction Channels Causes Movement within Gap Junctions followed by Vesicle Internalization and Protein Degradation

“…The latter is consistent with findings by Leithe et al (50) that proteasomal degradation occurs after stimulation of PKC with TPA. Cx43 has been shown to co-localize with clathrin, AP-2, Dab2, and dynamin2 at gap junction plaques (51)(52)(53). Additionally, the loss of dynamin GTPase activity or the reduction of dynamin2, clathrin, AP-2, or Dab2 proteins inhibited the internalization of Cx43, as observed by a significant decrease in the number of annular junctions.…”

Structural Studies of the Nedd4 WW Domains and Their Selectivity for the Connexin43 (Cx43) Carboxyl Terminus

“…The NMR solution structure of WW3 has already been solved albeit in complex with a small peptide (46). All 1 H, 15 N, and 13 C backbone resonances and most side chain and aromatic groups of the WW1 and WW2 domains (97 and 95% of the expected resonances, respectively) were assigned in 1ϫ PBS (pH 7.4) at 25°C (Fig. 1C).…”

“…The rat Nedd4 WW domains WW1 (Nedd4 245-281 ), WW2 (Nedd4 401-437 ), and WW3 (Nedd4 458 -494 ) were cloned into the bacterial expression vector pGEX-KT (GST-tagged; Amersham Biosciences) using standard PCR methods and transformed for overexpression in BL21(DE3) cells (unlabeled, 15 N-, or 13 C, 15 N-labeled) (Agilent Technologies). Purification was conducted as described previously for a recombinant GSTtagged protein with the tag being removed by thrombin digestion (32)(33)(34).…”

“…The initial step leading to degradation, internalization from the plasma membrane, is a complex process because docked connexons are unable to be separated under physiological conditions (9). Consequently, removal from the plasma membrane is directionally regulated as internalization of the channels within one of the coupled cells forms a double membrane macrostructure called an annular gap junction or connexosome (10); clathrin and other endocytic adaptors have been shown to be involved in this process (11)(12)(13)(14). Degradation of connexins involves both the lysosomal (endosome or autophagy) and proteasomal pathways (15)(16)(17).…”

The connexin43-interacting protein, CIP85, mediates the internalization of connexin43 from the plasma membrane

“…Immunofl uorescence (IF) and electron microscopy studies of connexins in endosomal structures (Murray et al, 1981;Naus et al, 1993;Sasaki & Garant, 1986) and IF microscopy evidence of connexins colocalized with endosomal markers such as 54 K. Cochrane et al EEA1, Rab5, and Rab7 (Boassa et al, 2010;Gilleron et al, 2008;Govindarajan et al, 2010;Leithe et al, 2006;Leithe et al, 2009;Segretain et al, 2003) suggest that connexins are internalized from the plasma membrane by endocytic mechanisms. This is also supported by an evidence that the knockdown of proteins required for clathrin-mediated endocytosis such as clathrin, dynamin2, adapter protein complex-2 (AP-2), and Disabled-2 (Dab2) inhibited the internalization of connexin43 (Cx43) (Gilleron et al, 2011;Gumpert et al, 2008;Piehl et al, 2007). Furthermore, it has been shown that gap junctions, which are aggregated in plaques at the plasma membrane, can be internalized into one of the two adjoining cells as unique double-membrane endocytic structures called annular gap junctions (AGJ) or connexosomes (Gumpert et al, 2008;Piehl et al, 2007;Gilleron et al, 2011;Falk et al, 2009).…”