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Salmeterol Stimulation Dissociates ₂-Adrenergic Receptor Phosphorylation and Internalization

Department of Pediatrics and Department of Pulmonary and Critical Care Medicine, Baylor College of Medicine; Department of Integrative Biology and Pharmacology, University of Texas Health Sciences Center-Houston; Department of Biology and Biochemistry and Department of Pharmacology and Pharmaceutical Sciences, University of Houston; and Division of Pulmonary Medicine, M.D. Anderson Cancer Center, Houston, Texas

Correspondence and requests for reprints should be addressed to Robert H. Moore, M.D., Department of Pediatrics, Baylor College of Medicine, 6621 Fannin, CCC1040, Houston, TX 77030. 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 with the other available long-acting -agonist, formoterol. Receptor desensitization and down-regulation 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, down-regulation, 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 with 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 significant ₂AR internalization or degradation and was incapable of stimulating the translocation of enhanced green fluorescent protein-–arrestin2 chimera (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.

Key Words: ₂-adrenergic receptor • salmeterol • phosphorylation • internalization • desensitization

CLINICAL RELEVANCE Salmeterol is commonly used in the treatment of asthma and COPD, yet its molecular pharmacologic properties are poorly understood. This work provides information regarding the pharmacologic properties of salmeterol at the cellular and molecular levels.