β2-Adrenergic Receptor Down-regulation

Abstract

Sustained activation of most G protein-coupled receptors causes a time-dependent reduction of receptor density in intact cells. This phenomenon, known as downregulation, is believed to depend on a ligand-promoted change of receptor sorting from the default endosomeplasma membrane recycling pathway to the endosomelysosome degradation pathway. This model is based on previous studies of epidermal growth factor (EGF) receptor degradation and implies that receptors need to be endocytosed to be down-regulated.In stable clones of L cells expressing ␤ 2 -adrenergic receptors (␤ 2 ARs), sustained agonist treatment caused a time-dependant decrease in both ␤ 2 AR binding sites and immuno-detectable receptor. Blocking ␤ 2 AR endocytosis with chemical treatments or by expressing a dominant negative mutant of dynamin could not prevent this phenomenon. Specific blockers of the two main intracellular degradation pathways, lysosomal and proteasomeassociated, were ineffective in preventing ␤ 2 AR downregulation. Further evidence for an endocytosis-independent pathway of ␤ 2 AR down-regulation was provided by studies in A431 cells, a cell line expressing both endogenous ␤ 2 AR and EGF receptors. In these cells, inhibition of endocytosis and inactivation of the lysosomal degradation pathway did not block ␤ 2 AR down-regulation, whereas EGF degradation was inhibited. These data indicate that, contrary to what is currently postulated, receptor endocytosis is not a necessary prerequisite for ␤ 2 AR down-regulation and that the inactivation of ␤ 2 ARs, leading to a reduction in binding sites, may occur at the plasma membrane.A recurrent theme in G protein-coupled receptor physiology is that the intensity of the functional response to hormones wanes over time despite the continuous presence of the stimulus. This phenomenon of hormonal tolerance, also known as desensitization, reflects multiple molecular mechanisms of receptor regulation. For most receptors, the predominant mechanism of desensitization is the phosphorylation-dependent uncoupling from G proteins (1). For some receptors, such as the m3-muscarinic acetylcholine receptor, desensitization is the consequence of receptor endocytosis, which decreases the number of surface receptors that may be activated by the hormone (2). Desensitization may also be caused by down-regulation, the ligand-dependent reduction of total receptor number, as in the case of thrombin receptors. Thrombin proteolytic activity cleaves the amino terminus of the receptor, unmasking a new amino-terminal peptide (3); this peptide activates the receptor irreversibly, promoting its endocytosis and its subsequent degradation in lysosomes (4).Desensitization of ␤ 2 -adrenergic receptors (␤ 2 ARs) 1 is mostly dependent on rapid phosphorylation by the cAMP-dependent protein kinase and G protein-coupled receptor kinases (5-7). However, although the ␤ 2 AR is less rapidly affected by downregulation than the thrombin receptor, long term agonist-promoted ␤ 2 AR down-regulation significantly contributes to the desensitiz...

The Extracellular N Terminus of the Endothelin B (ETB) Receptor Is Cleaved by a Metalloprotease in an Agonist-dependent Process

“…42). The cleavage site within the ␤ 2 -adrenergic receptor has not been determined (43). In case of the TSH receptor, spontanous release of the highly glycosylated extracellular N terminus was observed (44).…”

“…The ligand-dependence of this process favor the involvement in receptor activation or down-regulation. However, the proteolysis does not destroy receptor function and the overall structure, as is the case for the vasopressin V 2 receptor or the ␤ 2 -adrenergic receptor (42,43). Rather it generates a new N terminus, similar to the PAR family, although the new N terminus (SLAPAE) of the ET B receptor does not share sequence similarity with that of the of PAR1, PAR2, and PAR4 receptors (SFLLRN/TFLLR, SLIGRL, and GYPGKF, respectively).…”

Resistance of the Human β1-Adrenergic Receptor to Agonist-induced Ubiquitination

“…In one study proteasomal but not lysosomal inhibitors reduced the down-regulation of and ␦ opioid receptors (14), whereas in another, lactacystin, a very specific proteasomal inhibitor, did not impair agonist-induced internalization, lysosomal targeting, and degradation of the ␦ opioid receptor (15). Some of these studies found that steadystate receptor levels increase in unstimulated cells treated with proteasomal inhibitors (14,17). The presence of proteasomal inhibitors also enhances the ubiquitination of some GPCRs (14,16).…”

“…E-64 and leupeptin inhibit lysosomal cysteine proteases, and leupeptin also inhibits serine proteases (32). ALLN, MG132, and lactacystin block proteasomal activity, with lactacystin having the greatest specificity (17). Degradation of ␤ 1 AR expressed in BHK and HEK 293 cells was determined using the surface biotinylation procedure described above.…”

“…Agonist-promoted degradation of CXCR4 receptors is blocked by inhibitors of lysosomal but not proteasomal function, and the receptors are sorted to lysosomes (13). Proteasomal inhibitors are reported to either reduce (12) or have no effect (17) on agonist-mediated internalization and degradation of ␤ 2 AR. In one study proteasomal but not lysosomal inhibitors reduced the down-regulation of and ␦ opioid receptors (14), whereas in another, lactacystin, a very specific proteasomal inhibitor, did not impair agonist-induced internalization, lysosomal targeting, and degradation of the ␦ opioid receptor (15).…”