Salmeterol stimulation dissociates β₂-adrenergic receptor phosphorylation and internalization

Salmeterol is a long-acting β₂-adrenergic receptor (β₂AR) agonist commonly used in the treatment of asthma and chronic obstructive pulmonary disease. It differs from other β-agonists in that it has a very low intrinisic efficacy, especially when compared with the other available long-acting β-agonist, formoterol. Receptor desensitization and down-regulation has been described with the chronic use of β-agonists. This effect may not be the same with all β-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 β₂AR endocytosis, g protein-coupled receptor kinase (GRK)-site phosphorylation, degradation, and β-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 β₂AR internalization or degradation and was incapable of stimulating the translocation of enhanced green fluorescent protein-β-arrestin2 chimera (EGFP-β-arrestin2) to the cell surface. Salmeterol-induced receptor endocytosis was rescued, at least in part, by the overexpression of EGFP-β-arrestin2. Our data indicate that salmeterol binding induces an active receptor state that is unable to recruit β-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.