The Chaperone Protein Calnexin Regulates Cell Surface Expression of both D1 and D2 Dopamine Receptors

• Published in: The FASEB journal 2006-03, Vol.20 (4), p.A247-A247
• Main Authors: Free, R Benjamin, Hazelwood, Lisa A, Spalding, Heather N, Cabrera, David M, Sibley, David R
• Format: Article
• Language: English
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.20.4.A247

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 D1 and D2 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 D1‐GFP and D2‐YFP receptors to internal stores following treatment with these drugs. Over‐expression of CNX also results in a marked decrease in both D1 and D2 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 D1 and D2 receptor trafficking and expression at the cell surface. Supported by: NIH