Modulation of α‐ 2C Adrenergic Receptor intracellular trafficking by arginine‐based motifs

The upregulation of α 2C ‐adrenergic receptor (α 2C ‐AR) plasma membrane levels by exposure to cold contributes to the enhanced vasoconstriction observed in Raynaud Phenomenon. To understand this particular intracellular trafficking, we used α 2C ‐AR transfected HEK293T. Exposure to 30°C significantly enhanced α 2C ‐AR plasma membrane levels (69 ± 10 %), as determined by [ 3 H]‐RX821002 binding. Sequence analysis demonstrated that eleven putative arginine based motifs (RXR) are embedded in in the C‐terminus and in the third intracellular loop of α 2C ‐AR, which were shown in other proteins to determine endoplasmic reticulum retention. Deletion of these RXR motifs had no effect on the α 2C ‐AR plasma membrane levels at 37°C, but it accentuates the upregulation at 30°C. Using alanine‐mutagenesis we identified two specific arginine clusters, which are augmenting the receptor trafficking at 30°C. This conclusion is supported by the absence of these motifs in the opossum α 2C ‐AR which lacks the temperature‐sensitive traffic. Also, one specific arginine cluster is shifted to the left in rat and mice α 2C ‐AR, which displayed a reduced augmentation of the receptor levels at the plasma membrane after exposure to 30°C (37 ± 6 % and 32 ± 5 %). Our data indicate that strict spatial positioning of arginine‐based motifs embedded in α 2C ‐AR is essential in prevention of exaggerated vasoconstriction to cold. NIH GrantP20RR018766‐08