Down-regulation of the alpha-2C adrenergic receptor: Involvement of a serine/threonine motif in the third cytoplasmic loop

Abstract

Background: The mechanisms by which alpha-2 adrenergic receptors are downregulated following chronic exposure to agonist are not well understood. Interestingly, the human alpha-2C receptor does not downregulate, whereas the opossum alpha-2C receptor does downregulate. A comparison of the amino acid sequence of the third intracellular loop of these two receptors shows that the opossum alpha-2C receptor contains a potential G protein-coupled receptor kinase (GRK)phosphorylation motif (EESSTSE) with four hydroxyl residues, whereas the human alpha-2C receptor motif only contains two hydroxyl residues (DESSAAAAE). Because a similar acidic serinerich motif (EESSSSD) in the human alpha-2 adrenergic receptor has been demonstrated to be phosphorylated by GRK and all four serines are required for desensitization of the receptor, we sought to determine whether the EESSTSE sequence was involved in the downregulation of the alpha-2C adrenergic receptor. Results: Site-directed mutagenesis was used to mutate the opossum alpha-2C receptor to SSVA and AAVA in place of the SSTS wild-type sequence. Downregulation experiments on CHO cells transfected with the receptors demonstrated that neither of the mutated receptors downregulated following 24 h exposure to norepinephrine, whereas the wild-type receptor downregulated to 65 ± 10% of the control. Conclusions: These results indicate that a motif with four hydroxyl amino acid residues in an acidic environment is important for downregulation of the opossum alpha-2C adrenergic receptor. Because these are potential GRK phosphorylation sites, we suggest that GRK phosphorylation may be involved in alpha-2C adrenergic receptor downregulation. © 2002 Deupree et al; licensee BioMed Central Ltd.