Abstract 5253: Temperature-Dependent Interactions of α2C-Adrenergic Receptor with Molecular Chaperones
α2C adrenergic receptor (α2C-AR) is poorly transported to the cell surface in fibroblasts and in vascular smooth muscle cells. Cold exposure greatly enhances receptor plasma membrane targeting and contributes to the aberrant vasoconstriction observed in Raynaud Phenomenon. To test if differential interactions with molecular chaperones may explain the temperature-dependent α2C-AR plasma membrane expression, we used HEK293T cells transfected with GFP-tagged α2C-AR. Fluorescence microscopy analysis demonstrated that at 37°C the receptor was extensively co-localized with endoplasmic reticulum (ER) and Golgi markers. In contrast, after exposure to 30°C for 18 h, α2C-AR was predominantly present at the plasma membrane. Radioligand binding experiments showed that low temperature increased the receptor plasma membrane levels by 69±7% (n=16). Blocking receptor internalization using hyperosmolar medium or co-transfection with a dynamin negative mutant did not change the cell surface receptor levels, suggesting that reduced α2C-AR plasma membrane expression at 37°C is due to impaired receptor export. This conclusion was confirmed by the effects of pharmacological chaperones, DMSO (1%) and glycerol (2%), which significantly enhanced α2C-AR cell surface levels at 37°C, but not at 30°C (n=6). Further, co-immunoprecipitation experiments demonstrated that interaction of α2C-AR with BiP/GRP78 was decreased with 57±11% (n=3) at 30°C. In contrast, the α2C-AR interactions with two other molecular chaperones, calnexin and calreticulin, were similar at 37°C and 30°C. In addition, the interactions of α2C-AR with β-cop, a subunit of coatomer coat protein I, were reduced by 68±9% (n=4) at 30°C, indicating that the retrograde traffic from Golgi to ER is also preventing the receptor transport to the cell surface at 37°C. Overall, these data demonstrate that differential interactions with specific chaperones along the biosynthetic pathway are involved in the temperature-dependent α2C-AR expression to the plasma membrane. Aberrant cellular levels of BiP/GRP78 and β-cop may represent useful biomarkers to detect individuals susceptible to Raynaud Phenomenon attacks.