Submitted on April 27, 2006
Revised on September 15, 2006
Salmeterol Stimulation Dissociates 
2-Adrenergic Receptor Phosphorylation and Internalization
Robert H Moore1*, Ellen E Millman1, Veronica Godines1, Nicola A Hanania2, Tuan M Tran3, Hui Peng4, Burton F Dickey5, Brian J Knoll6, and Richard B Clark3
1 Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA,
2 Department of Pulmonary and Critical Care Medicine, Baylor College of Medicine, Houston, Texas, USA,
3 Department of Integrative Biology and Pharmacology, University of Texas Health Sciences Center, Houston, Texas, USA,
4 Department of Biology and Biochemistry, University of Houston, Houston, Texas, USA,
5 Division of Pulmonary Medicine, M.D. Anderson Cancer Center, Houston, Texas, USA,
6 Department of Pharmacology and Pharmaceutical Sciences, University of Houston, Houston, Texas, USA
* To whom correspondence should be addressed. E-mail: rmoore{at}bcm.tmc.edu.
Salmeterol is a long-acting 
2-adrenergic receptor (2AR) agonist commonly used in the treatment of asthma and chronic obstructive pulmonary disease. It differs from other -agonists in that it has a very low intrinisic efficacy, especially when compared to the other available long-acting -agonist, formoterol. Receptor desensitization and downregulation has been described with the chronic use of -agonists. This effect may not be the same with all -agonists and may be related to their stabilization of altered receptor states. The extreme hydrophobicity and high affinity quasi-irreversible binding of salmeterol have rendered studies examining the mechanisms by which it mediates receptor desensitization, downregulation, and internalization difficult. We determined the capacity of salmeterol to induce 2AR endocytosis, G protein-coupled receptor kinase (GRK)-site phosphorylation, degradation, and -arrestin2 translocation in HEK293 cells as compared to other agonists of varying intrinsic efficacies. Despite stimulating GRK-mediated phosphorylation of Ser355,356 after 30 min and 18 h to an extent similar to that observed with agonists of high intrinsic efficacy such as epinephrine and formoterol, salmeterol did not induce any significant 2AR internalization or degradation and was incapable of stimulating the translocation of EGFP--arrestin2 to the cell surface. Salmeterol-induced receptor endocytosis was rescued, at least in part, by the overexpression of EGFP--arrestin2. Our data indicate that salmeterol binding induces an active receptor state that is unable to recruit -arrestin or undergo significant endocytosis or degradation despite stimulating considerable GRK-site phosphorylation. Defects in these components of salmeterol-induced receptor desensitization may be important determinants of its sustained bronchodilation with chronic use.
