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The Chaperone Protein Calnexin Regulates Cell Surface Expression of both D 1 and D 2 Dopamine Receptors
Author(s) -
Free R Benjamin,
Hazelwood Lisa A,
Spalding Heather N,
Cabrera David M,
Sibley David R
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a247
Subject(s) - calnexin , microbiology and biotechnology , receptor , chemistry , er retention , chaperone (clinical) , hek 293 cells , unfolded protein response , immunoprecipitation , confocal microscopy , cell , endoplasmic reticulum , biology , biochemistry , gene , calreticulin , medicine , pathology , mutant
Interacting proteins play a vital role in the regulation of dopamine receptor (DAR) trafficking and regulation. Coupling co‐immunoprecipitation (co‐IP) assays for DARs with mass spectrometry (MS)‐based peptide sequencing, we identified the chaperone protein calnexin (CNX) as an interacting protein for both D 1 and D 2 DARs. The CNX‐DAR interactions were confirmed with Western analysis and reverse IP experiments. CNX has been characterized as an ER retention protein for glycosylated membrane proteins that facilitates their correct folding and delivery to the cell surface. To determine the influence of CNX on DAR expression, we conducted assays in HEK293T cells using a variety of CNX modifying conditions. Treatment of cells with the tunicamycin or castanospermine, both of which inhibit CNX‐DAR interactions, results in a decrease in cell surface DAR expression as determined by both functional and receptor binding assays. Confocal fluorescence microscopy reveals the accumulation of D 1 ‐GFP and D 2 ‐YFP receptors to internal stores following treatment with these drugs. Over‐expression of CNX also results in a marked decrease in both D 1 and D 2 DAR expression as determined by both second messenger and receptor binding assays. This is likely due to an increase in ER retention since confocal microscopy revealed clustering of DARs in the ER which were co‐localized with an ER marker protein. Taken together, these data suggest that optimal DAR‐CNX interactions (neither too little nor too much) critically regulate D 1 and D 2 receptor trafficking and expression at the cell surface. Supported by: NIH