Abstract
We identified a truncated form (38-117) of GEC1 that interacts with the C-tail of the human kappa opioid receptor (hKOR) by yeast two-hybrid screening. GEC1-(38-117) did not interact with the C-tail of the mu or delta opioid receptors. GEC1, a 117-amino acid protein (Pellerin, I., Vuillermoz, C., Jouvenot, M., Ordener, C., Royez, M., and Adessi, G. L. (1993) Mol. Cell Endocrinol. 90, R17-R21), is highly homologous to GABARAP, GATE-16, and Apg8/aut7, all members of the microtubule associated protein (MAP) family. In pull-down assays, GST-GEC1 interacted directly with the hKOR C-tail, full-length hKOR, and tubulin. When expressed in Chinese hamster ovary (CHO) cells, GEC1 co-immunoprecipitated with FLAG-hKOR. Expression of GEC1 greatly increased total and cell-surface KOR but not mu or delta opioid receptors. GEC1 expression slightly reduced U50,488H-promoted down-regulation, without affecting ligand binding affinity, receptor-G protein coupling, or U50,488H-induced desensitization and internalization. HA-GEC1 expressed in CHO cells was localized in the Golgi apparatus and endoplasmic reticulum (ER). When cells were pulsed with [35S]Met/Cys, GEC1 expression enhanced the level of the mature form (55-kDa band) of FLAG-hKOR at 4, 8, and 22 h after pulse without affecting the precursors (39- and 45-kDa bands), indicating that GEC1 facilitates trafficking of FLAG-hKOR from the ER/Golgi to plasma membranes. GEC1 interacted with N-ethylmaleimide-sensitive factor (NSF) in pull-down assays and co-immunoprecipitated with NSF in rat brain extracts. The interaction with NSF may contribute to GEC1 effects. This is the first report on biological functions of GEC1 and the first demonstration that a GPCR interacts with a protein of the MAP family. The interaction is important for trafficking of the receptor in the biosynthesis pathway.
Highlights
In recent years, GPCRs have been found to interact with many proteins besides G proteins, and such non-G protein interactions are unique to individual receptors and have been shown to play significant roles in signal transduction, receptor regulation, or receptor biogenesis
We identified proteins interacting with the human opioid receptor by yeast two-hybrid screening of a human brain cDNA library using the cytoplasmic tail (C-tail) as the bait
The full-length GEC1 did not interact with the hKOR C-tail in the yeast two-hybrid assay, presumably due to its binding to tubulin, and microtubules, which may prevent AD-GEC1 fusion protein from entering the nucleus
Summary
GPCRs have been found to interact with many proteins besides G proteins, and such non-G protein interactions are unique to individual receptors and have been shown to play significant roles in signal transduction, receptor regulation, or receptor biogenesis (for reviews, see Refs. 7–9). GEC1 cDNA was originally cloned in 1993 from guinea-pig endometrial glandular epithelial cells as an early estrogen-induced mRNA [12]. This protein was named GABAA receptor-associated protein-like 1 [13] and Apg8L [14]. GEC1 belongs to a family of microtubules-associated proteins (MAPs) that includes, in addition, GABAA receptor-associated protein (GABARAP) [15], Golgi-associated ATPase enhancer of 16 kDa (GATE-16) ( named GABARAPL2) [16], and the yeast protein Apg8p/Aut7 [17]. Interaction of the Human Opioid Receptor with GEC1 across the species cloned to date, including frog (AAH72921), mouse (NP_065615), guinea pig (AAL32264), and human (Q9H0R8), indicating that GEC1 is highly conserved in evolution. The wide distribution of this family of proteins suggests that they have important biological functions in cells
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