Brian J. Knoll

Partial agonists and G protein-coupled receptor desensitization

Weak or partial agonists induce less desensitization of G protein-coupled receptors (GPCRs) than do strong agonists. However, there have been few attempts to relate partial agonism quantitatively with the various parameters of agonist-induced desensitization, and to elucidate the mechanisms involved. Our understanding of how the treatment of cells and tissues with partial agonists affects their capacity to activate receptors is based on continued progress in defining partial agonism and the mechanisms of desensitization in which protein kinases, phosphatases, endocytosis and recycling play various roles. In this review, current research concerning partial-agonist-induced desensitization of GPCRs and the nature of partial agonism is summarized, and an attempt is made to put the existing knowledge into a working hypothesis concerning the mechanisms that account for the reduced desensitization in response to partial agonists.

Salmeterol-induced desensitization, internalization and phosphorylation of the human β2-adrenoceptor

Partial agonists of the β2‐adrenoceptor which activate adenylyl cyclase are widely used as bronchodilators for the relief of bronchoconstriction accompanying many disease conditions, including bronchial asthma. The bronchodilator salmeterol has both a prolonged duration of action in bronchial tissue and the ability to reassert this activity following the temporary blockade of human β2‐adrenoceptors with antagonist. We have compared the activation and desensitization of human β2‐adrenoceptor stimulation of adenylyl cyclase induced by salmeterol, adrenaline and salbutamol in a human lung epithelial line, BEAS‐2B, expressing β2‐adrenoceptor levels of 40–70 fmol mg−1, and in human embryonic kidney (HEK) 293 cell lines expressing 2–10 pmol mg−1. The efficacy observed for the stimulation of adenylyl cyclase by salmeterol was only ≈percnt;10% of that observed for adrenaline in BEAS‐2B cells expressing low levels of β2‐adrenoceptor, but similar to adrenaline in HEK 293 cells expressing very high levels of receptors. Salmeterol pretreatment of these cells induced a rapid and stable activation of adenylyl cyclase activity which resisted extensive washing and β2‐adrenoceptor antagonist blockade, consistent with binding to a receptor exosite and/or to partitioning into membrane lipid. The desensitization and internalization of β2‐adrenoceptors induced by the partial agonists salmeterol and salbutamol were considerably reduced relative to the action of adrenaline. Consistent with these observations, the initial rate of phosphorylation of the receptor induced by salmeterol and salbutamol was much reduced in comparison to adrenaline. Our data suggest that the reduction in the rapid phase of desensitization of β2‐adrenoceptors after treatment with salmeterol or salbutamol is caused by a decrease in the rate of β2‐adrenoceptor kinase (βARK) phosphorylation and internalization. In contrast, the rate of cyclic AMP‐dependent protein kinase (PKA)‐mediated phosphorylation by these partial agonists appears to be similar to adrenaline.

Specific changes in β2-adrenoceptor trafficking kinetics and intracellular sorting during downregulation

Agonist-activated beta2-adrenoceptors rapidly internalize and then recycle to the cell surface, however chronic agonist eventually causes receptor downregulation. To characterize beta2-adrenoceptor trafficking kinetics and intracellular sorting during downregulation, human embryonic kidney cells expressing epitope-tagged receptors were examined by radioligand binding with (+/-)-[3H]4-(3-tertiarybutylamino-2-hydroxypropoxy)-benzimidazole- 2-on hydrochloride ([3H]CGP12177) and immunofluorescence microscopy. The first-order receptor recycling rate constant declined after 18 h of agonist compared with 15 min (0.05 min(-1) vs. 0.12 min(-1)), thus increasing the intracellular transit time (20.0 min vs. 8.3 min). There was also a reduction in the rate of receptor endocytosis and a decline in the total number of receptors. Although the intracellular receptor fraction did not increase between 15 min and 18 h of agonist, some receptors moved irreversibly into a protease-containing compartment while retaining radioligand binding activity. Our results indicate that beta2-adrenoceptor downregulation is associated principally with an increased intracellular transit time during recycling. This could promote the diversion of receptors into protease-containing compartments, where there is an irreversible commitment to downregulation prior to loss of radioligand binding activity.