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

Abstract

We demonstrated previously that GEC1, a member of the microtubule-associated protein (MAP) family, bound to the human 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-Ctail 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. opioid receptor (KOPR) 2 is one of the three major types of opioid receptors mediating the effects of opioid drugs and endogenous peptides. The effects of KOPR activation in vivo include anti-nociception (especially for visceral chemical pain), anti-pruritic, water diuresis, and psychotomimetic effects (1). The KOPR agonist nalfurafine (TRK-820) is used clinically in Japan for the treatment of uremic pruritus in kidney dialysis patients (2). KOPR antagonists may be useful for curbing cocaine craving and as anti-anxiety drugs (3, 4). In addition, it has been proposed that KOPR agonists may be useful in treating mania, as antagonists as anti-depressants, and as partial agonists for the management of bipolar disorder (5).We have demonstrated that the protein glandular epithelial cell 1 (GEC1) interacts directly with the C-terminal domain of the KOPR by hydrophobic interactions (6, 7). The interaction increases cell surface expression of the KOPR by enhancing the conversion of the glycosylated intermediates to fully glycosylated forms of the receptor, indicating facilitation of trafficking from the endoplasmic reticulum to Golgi to plasma membranes (7).GEC1 was first cloned as an early estrogen-induced mRNA from guinea pig endometrial glandular epithelial cells (8). Its deduced amino a...

GABARAPL1 (GEC1): Original or copycat?

“…Moreover, Gabarapl1 displays a higher affinity for KOR and, unlike Gabarap, can associate with and enhance the expression of this receptor without being processed at its G116 residue and associated to membranes. 58 These data suggest a different role of these proteins in the transport of the kappa opioid receptor. The residues within the opioid receptor that are necessary for Gabarapl1 interaction are located within a FPXXM motif at its C terminus.…”

GABARAPL1 is required for increased EGFR membrane expression during hypoxia

“…Several proteins, including EGFR can be upregulated during hypoxia [17] and require transportation to the membrane. Since the GABARAP family is implicated in several processes like anterograde trafficking of several receptors like the Kappa Opioid Receptor [19], angiotensin [28,29]and GABA-A receptor [30] we questioned the role of GABARAPL1 in EGFR trafficking. EGFR and other growth factor receptors are often amplified or mutated in cancer and may promote hypoxia tolerance [16,31].…”

“…For example, GABARAPL1 promotes cell surface expression of the Kappa Opioid Receptor (KOR) by facilitating its trafficking along the secretory pathway. Accordingly, knockdown of GABARAPL1 decreases the surface expression of KOR [19]. Binding of GABARAPL1 to the KOR is based on hydrophobic interactions, and the N-terminal region of GABARAPL1 is important for binding to tubulin and surface expression of the KOR [20].…”

GABARAPL1 antibodies: Target one protein, get one free!

“…2B, lane 3), producing only one signal corresponding to the with a higher percentage of acrylamide in the gel and a longer migration time. 3,13 Immunohistological analysis of the endogenous proteins, however, does not provide an easy alternative to the necessity of specific antibodies. Indeed, many researchers have encountered this problem of cross-reactivity with both their 'homemade' and commercially bought antisera and purified antibodies raised against either GABARAPL1 or GABARAP.…”