4.6 Article

Effects of C-terminal Modifications of GEC1 Protein and γ-Aminobutyric Acid Type A (GABAA) Receptor-associated Protein (GABARAP), Two Microtubule-associated Proteins, on κ Opioid Receptor Expression

Journal

JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 286, Issue 17, Pages 15106-15115

Publisher

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M111.230896

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Funding

  1. National Institutes of Health [R01 DA17302, P30 DA13429]

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We demonstrated previously that GEC1, a member of the microtubule-associated protein (MAP) family, bound to the human kappa opioid receptor (hKOPR) and promoted hKOPR cell surface expression by facilitating its trafficking along the secretory pathway. GABA(A) receptor-associated protein (GABARAP), a GEC1 analog, also enhanced KOPR expression, but to a lesser extent. The MAP family proteins undergo cleavage of their C-terminal residue(s), and the exposed conserved glycine forms conjugates with phosphatidylethanolamine, which associate with membranes. Here, we examined whether such modifications were required for GEC1 and GABARAP to enhance hKOPR expression. When transiently transfected into CHO or Neuro2A cells, GEC1 and GABARAP were cleaved at the C termini. G116A mutation alone or combined with deletion of Lys(117) in GEC1 (GEC1-A) or Leu(117) in GABARAP (GABARAP-A) blocked their C-terminal cleavage, indicating that the conserved Gly(116) is necessary for C-terminal modification. The two GEC1 mutants enhanced hKOPR expression to similar extents as the wildtype GEC1; however, the two GABARAP mutants did not. Immunofluorescence studies showed that HA-GEC1, HA-GEC1-A, and HA-GABARAP were distributed in a punctate manner and co-localized with KOPR-EGFP in the Golgi apparatus, whereas HA-GABARAP-A did not. Pulldown assay of GST-KOPR-C-tail with HA-GEC1 or HA-GABARAP revealed that GEC1 had stronger association with KOPR-C-tail than GABARAP. These results suggest that because of its stronger binding for hKOPR, GEC1 is able to be recruited by hKOPR sufficiently without membrane association via its C-terminal modification; however, due to its weaker affinity for the hKOPR, GABARAP appears to require C-terminal modifications to enhance KOPR expression.

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