GEC1 interacts with the kappa opioid receptor and enhances expression of the receptor.
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.
Chen
Chongguang
C
Department of Pharmacology and Center for Substance Abuse Research, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Li
Jian-Guo
JG
Chen
Yong
Y
Huang
Peng
P
Wang
Yulin
Y
Liu-Chen
Lee-Yuan
LY
D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer / Q000494:pharmacology; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D018712:Analgesics, Non-Narcotic / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004967:Estrogens / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D005982:Glutathione Transferase / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D007700:Kinetics; D008715:Methionine / Q000737:chemistry; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000378:metabolism* / Q000502:physiology*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017473:Receptors, Opioid, kappa / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014404:Tubulin / Q000737:chemistry; D020798:Two-Hybrid System Techniques
D048868:Adaptor Proteins, Signal Transducing; D018712:Analgesics, Non-Narcotic; D018076:DNA, Complementary; D004967:Estrogens; C430503:GABARAPL1 protein, human; D008869:Microtubule-Associated Proteins; D012333:RNA, Messenger; D017473:Receptors, Opioid, kappa; D011993:Recombinant Fusion Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D014404:Tubulin; D006639:Histidine; D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; D008715:Methionine; D005982:Glutathione Transferase; D006026:Glycoside Hydrolases; D003545:Cysteine; D005033:Ethylmaleimide