Pathways regulating the trafficking and turnover of pannexin1 protein and the role of the C-terminal domain

J Biol Chem. 2011 Aug 5;286(31):27639-53. doi: 10.1074/jbc.M111.260711. Epub 2011 Jun 9.

Abstract

Pannexin1 (Panx1) is an integral membrane protein comprised of three species as follows: an unglycosylated core-Gly0, a high mannose-Gly1, and a complex glycosylated Gly2 species. Although Panx1 channels mediate several cellular responses, the domain regulating its oligomerization and cell surface trafficking and the mechanisms governing its internalization and degradation have not been identified. This study characterizes the role of the Panx1 C-tail domain by truncating the polypeptide at residue 307 and expressing the mutant in BICR-M1R(k) and HEK-293T cells. Enzymatic digestion and immunolabeling assays revealed that the Panx1(T307)-RFP was glycosylated primarily to the high mannose species consistent with its retention in the endoplasmic reticulum. Co-expression of Panx1(T307)-RFP with Panx1 followed by co-immunoprecipitation assays revealed that the mutant and Panx1 could interact, whereas biotinylation assays showed that this interaction inhibited Panx1 from maturing into the Gly2 species and reaching the cell surface. Additional inhibitor studies indicated that the degradation of the mutant was via proteasomes, whereas Panx1 was degraded by lysosomes. Analysis of the pathways important in Panx1 internalization revealed partial co-distribution of Panx1 with many molecular constituents of the endocytic machinery that include clathrin, AP2, dynamin II, caveolin-1, and caveolin-2. However, co-immunoprecipitation assays together with the disruption of lipid rafts by methyl-β-cyclodextrin suggest that Panx1 does not engage this endocytic machinery. Furthermore, dominant-negative and pharmacological studies revealed that Panx1 internalization was dynamin II-independent. Collectively, these results indicate that the oligomerization and trafficking of Panx1 are regulated by the C-terminal domain, whereas internalization of long lived Panx1 channels occurs in a manner that is distinct from classical endocytic pathways.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Biopolymers / metabolism
  • Biotin / metabolism
  • Cell Line
  • Cell Line, Tumor
  • Connexins / chemistry
  • Connexins / metabolism*
  • DNA Primers
  • Endocytosis
  • Glycosylation
  • Humans
  • Immunohistochemistry
  • Immunoprecipitation
  • Nerve Tissue Proteins / chemistry
  • Nerve Tissue Proteins / metabolism*
  • Protein Transport
  • Rats
  • Transferrin / metabolism

Substances

  • Biopolymers
  • Connexins
  • DNA Primers
  • Nerve Tissue Proteins
  • PANX1 protein, human
  • Transferrin
  • pannexin 1, rat
  • Biotin