Internalization and Down-regulation of Human Muscarinic Acetylcholine Receptor m2 Subtypes

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...

Section: Discussionmentioning

confidence: 99%

β2-Adrenergic Receptor Down-regulation

Jockers¹,

Angers²,

Silva³

et al. 1999

“…Phosphorylated receptors then interact with cytoplasmic proteins termed ␤-arrestins, which interfere with receptor-G protein interaction, favoring receptor endocytosis, thus inhibiting the signal (49,50). Generally muscarinic receptor-linked PLD activity is inhibited rapidly within 2 min, but the completion of muscarinic receptor phosphorylation and internalization events are required for a longer time (51,52). Therefore, muscarinic receptor-linked PLD activity might be inhibited by another mechanism.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of Muscarinic Receptor-linked Phospholipase D Activation by Association with Tubulin

Chae

Lee

et al. 2005

Mammalian phospholipase D (PLD) is considered a key enzyme in the transmission signals from various receptors including muscarinic receptors. PLD activation is a rapid and transient process, but a negative regulator has not been found that inhibits signal-dependent PLD activation. Here, for the first time, we report that tubulin binding to PLD 2 is an inhibition mechanism for muscarinic receptor-linked PLD 2 activation. Tubulin was identified in an immunoprecipitated PLD 2 complex from COS-7 cells by peptide mass fingerprinting. The direct interaction between PLD 2 and tubulin was found to be mediated by a specific region of PLD 2 (amino acids 476 -612). PLD 2 was potently inhibited (IC 50 <10 nM) by tubulin binding in vitro. In cells, the interaction between PLD 2 and tubulin was increased by the microtubule disrupting agent nocodazole and reduced by the microtubule stabilizing agent Taxol. Moreover, PLD 2 activity was found to be inversely correlated with the level of monomeric tubulin. In addition, we found that interaction with and the inhibition of PLD 2 by monomeric tubulin is important for the muscarinic receptor-linked PLD signaling pathway. Interaction between PLD 2 and tubulin was increased only after 1-2 min of carbachol stimulation when carbachol-stimulated PLD 2 activity was decreased. The expression of the tubulin binding region of PLD 2 blocked the later decrease in carbachol-induced PLD activity by masking tubulin binding. Taken together, these results indicate that an increase in local membrane monomeric tubulin concentration inhibits PLD 2 activity, and provides a novel mechanism for the inhibition of muscarinic receptor-induced PLD 2 activation by interaction with tubulin. Mammalian phospholipase D (PLD)1 hydrolyzes membrane phosphatidylcholine to generate phosphatidic acid and choline. PLD activity is dramatically activated in response to a variety of signals, including hormones, neurotransmitters, and growth factors (1). PLD products, phosphatidic acid itself or the hydrolyzed product diacylglycerol, have been known to act as intracellular lipid second messengers and to be involved in multiple physiological events, such as, the promotion of mitogenesis, stimulation of respiratory bursts, secretory processes, and actin cytoskeletal reorganization (2-7). Therefore, signal-dependent PLD activity must be tightly controlled in cells.Many reports have been issued about the mechanisms of receptor-mediated PLD activation. Although the mammalian PLD isozymes, PLD 1 and PLD 2 , have some different regulatory properties, in general, agonist-induced PLD is activated by various protein kinases, including protein kinase C, proteintyrosine kinase, and the MAP kinase family, in addition to small G proteins of the ARF, Rho, and Ras families (8 -13). The signal-dependent activation of PLD is rapid and transient. Although, the activation kinetics depend on the stimulus and cell type, the PLD signal is usually diminished within 10 min (14). However, the mechanisms involved in PLD signal inhibition remain unknow...

“…The adenylyl cyclase-coupled m2-muscarinic receptor was one of the first receptors to be shown to be an in vitro substrate for the GRKs (31). Phosphorylation at sites on the third intracellular loop is thought to mediate m2-muscarinic receptor internalization (6,32). m3-muscarinic receptors contained in urea-treated membranes have previously been shown to be phosphorylated by purified GRK-2 and -3 but not GRK-5 and -6 (11).…”

Section: Discussionmentioning

confidence: 99%

“…Originally associated with receptor desensitization (1,2), GPCR phosphorylation has now been implicated in a number of processes including receptor internalization (3)(4)(5)(6) and as a molecular switch that determines coupling to specific signaling pathways (7,8). The receptor-specific kinases involved are generally considered to belong to the G-protein-coupled receptor kinase (GRK) family which are characterized by their sequence homology to rhodopsin kinase (GRK-1) and that include the extensively studied ␤-adrenergic receptor kinases 1 and 2 (GRK-2 and -3, respectively) (2,9).…”

mentioning

confidence: 99%

Phosphorylation and Regulation of a Gq/11-coupled Receptor by Casein Kinase 1α

Budd

McDonald

Tobin

2000

Agonist-mediated receptor phosphorylation by one or more of the members of the G-protein receptor kinase (GRK) family is an established model for G-protein-coupled receptor (GPCR) phosphorylation resulting in receptor desensitization. Our recent studies have, however, suggested that an alternative route to GPCR phosphorylation may be an operation involving casein kinase 1␣ (CK1␣). In the current study we investigate the involvement of CK1␣ in the phosphorylation of the human m3-muscarinic receptor in intact cells. We show that expression of a catalytically inactive mutant of CK1␣, designed to act in a dominant negative manner, inhibits agonist-mediated receptor phosphorylation by ϳ40% in COS-7 and HEK-293 cells. Furthermore, we present evidence that a peptide corresponding to the third intracellular loop of the m3-muscarinic receptor (Ser 345 -Leu 463 ) is an inhibitor of CK1␣ due to its ability to both act as a pseudo-substrate for CK1␣ and form a high affinity complex with CK1␣. Expression of this peptide was able to reduce both basal and agonist-mediated m3-muscarinic receptor phosphorylation in intact cells. These results support the notion that CK1␣ is able to mediate GPCR phosphorylation in an agonist-dependent manner and that this may provide a novel mechanism for GPCR phosphorylation. The functional role of phosphorylation was investigated using a mutant of the m3-muscarinic receptor that showed an ϳ80% reduction in agonist-mediated phosphorylation. Surprisingly, this mutant underwent agonist-mediated desensitization suggesting that, unlike many GPCRs, desensitization of the m3-muscarinic receptor is not mediated by receptor phosphorylation. The inositol (1,4,5)-trisphosphate response did, however, appear to be dramatically potentiated in the phosphorylation-deficient mutant indicating that phosphorylation may instead control the magnitude of the initial inositol phosphate response. It is now well established that G-protein-coupled receptor (GPCR)1 phosphorylation is a general phenomenon that controls specific key signaling properties of receptors. Originally associated with receptor desensitization (1, 2), GPCR phosphorylation has now been implicated in a number of processes including receptor internalization (3-6) and as a molecular switch that determines coupling to specific signaling pathways (7,8). The receptor-specific kinases involved are generally considered to belong to the G-protein-coupled receptor kinase (GRK) family which are characterized by their sequence homology to rhodopsin kinase (GRK-1) and that include the extensively studied ␤-adrenergic receptor kinases 1 and 2 (GRK-2 and -3, respectively) (2, 9). Reconstitution experiments using purified, or partially purified, receptors have demonstrated that in addition to the ␤ 2 -adrenergic receptor a number of GPCRs including muscarinic ((10 -12), substance P (13), bradykinin B 2 (14), and adenosine A 3 receptors (15) can act as GRK substrates. Furthermore, a GRK-2 dominant negative mutant (16) has been widely employed to probe the role of en...