Chris J. van Koppen

Regulation of Muscarinic Acetylcholine Receptor Sequestration and Function by β-Arrestin

After activation, agonist-occupied G protein-coupled receptors are phosphorylated by G protein-coupled receptor kinases and bind cytosolic β-arrestins, which uncouple the receptors from their cognate G proteins. Recent studies on the β2-adrenergic receptor have demonstrated that β-arrestin also targets the receptors to clathrin-coated pits for subsequent internalization and activation of mitogen-activated protein kinases. We and others have previously shown that muscarinic acetylcholine receptors (mAChRs) of the m1, m3, and m4 subtype require functional dynamin to sequester into HEK-293 tsA201 cells, whereas m2 mAChRs sequester in a dynamin-independent manner. To investigate the role of β-arrestin in mAChR sequestration, we determined the effect of overexpressing β-arrestin-1 and the dominant-negative inhibitor of β-arrestin-mediated receptor sequestration, β-arrestin-1 V53D, on mAChR sequestration and function. Sequestration of m1, m3, and m4 mAChRs was suppressed by 60–75% in cells overexpressing β-arrestin-1 V53D, whereas m2 mAChR sequestration was affected by less than 10%. In addition, overexpression of β-arrestin-1 V53D as well as dynamin K44A significantly suppressed m1 mAChR-mediated activation of mitogen-activated protein kinases. Finally, we investigated whether mAChRs sequester into clathrin-coated vesicles by overexpressing Hub, a dominant-negative clathrin mutant. Although sequestration of m1, m3, and m4 mAChRs was inhibited by 50–70%, m2 mAChR sequestration was suppressed by less than 10%. We conclude that m1, m3, and m4 mAChRs expressed in HEK-293 tsA201 cells sequester into clathrin-coated vesicles in a β-arrestin- and dynamin-dependent manner, whereas sequestration of m2 mAChRs in these cells is largely independent of these proteins.

Molecular diversity of sphingolipid signalling

Sphingolipid breakdown products are now being recognized to play a dual role in cellular signalling, acting as intracellular as well as extracellular signalling molecules. Both types of action may even be found with one sphingolipid species. The recent demonstration of G protein‐coupled receptors with high affinity for sphingosine 1‐phosphate and sphingosylphosphorylcholine has been followed by the discovery of several novel sphingolipid actions, such as regulation of heart rate, oxidative burst, neurite retraction or platelet activation. Ligand profiles and concentration‐response relationships suggest the existence of putative sphingolipid receptor subtypes. Against this background, several observations on supposed sphingolipid second messenger actions deserve a new evaluation.

Essential role of dynamin in internalization of M2 muscarinic acetylcholine and angiotensin AT1A receptors.

Most G protein-coupled receptors (GPCRs), including the M1 muscarinic acetylcholine receptor (mAChR), internalize in clathrin-coated vesicles, a process that requires dynamin GTPase. The observation that some GPCRs like the M2 mAChR and the angiotensin AT1A receptor (AT1AR) internalize irrespective of expression of dominant-negative K44A dynamin has led to the proposal that internalization of these GPCRs is dynamin-independent. Here, we report that, contrary to what is postulated, internalization of M2mAChR and AT1AR in HEK-293 cells is dynamin-dependent. Expression of N272 dynamin, which lacks the GTP-binding domain, or K535M dynamin, which is not stimulatable by phosphatidylinositol 4,5-bisphosphate, strongly inhibits internalization of M1 and M2 mAChRs and AT1ARs. Expression of kinase-defective K298M c-Src or Y231F,Y597F dynamin (which cannot be phosphorylated by c-Src) reduces M1 mAChR internalization. Similarly, c-Src inhibitor PP1 as well as the generic tyrosine kinase inhibitor genistein strongly inhibit M1 mAChR internalization. In contrast, M2 mAChR internalization is not (or is only slightly) reduced by expression of these constructs or treatment with PP1 or genistein. Thus, dynamin GTPases are not only essential for M1 mAChR but also for M2 mAChR and AT1AR internalization in HEK-293 cells. Our findings also indicate that dynamin GTPases are differentially regulated by c-Src-mediated tyrosine phosphorylation.

Receptor Subtype-specific Regulation of Muscarinic Acetylcholine Receptor Sequestration by Dynamin DISTINCT SEQUESTRATION OF m2 RECEPTORS

Sustained stimulation of muscarinic acetylcholine receptors (mAChRs) and other G protein-coupled receptors usually leads to a loss of receptor binding sites from the plasma membrane, referred to as receptor sequestration. Receptor sequestration can occur via endocytosis of clathrin-coated vesicles that bud from the plasma membrane into the cell but may also be accomplished by other, as yet ill-defined, mechanisms. Previous work has indicated that the monomeric GTPase dynamin controls the endocytosis of plasma membrane receptors via clathrin-coated vesicles. To investigate whether mAChRs sequester in a receptor subtype-specific manner via dynamin-dependent clathrin-coated vesicles, we tested the effect of overexpressing the dominant-negative dynamin mutant K44A on m1, m2, m3, and m4 mAChR sequestration in HEK-293 cells. The m1, m2, m3, and m4 mAChRs sequestered rapidly in HEK-293 cells following agonist exposure but displayed dissimilar sequestration pathways. Overexpression of dynamin K44A mutant fully blocked m1 and m3 mAChR sequestration, whereas m2 mAChR sequestration was not affected. Also, m4 mAChRs, which like m2 mAChRs preferentially couple to pertussis toxin-sensitive G proteins, sequestered in a completely dynamin-dependent manner. Following agonist removal, sequestered m1 mAChRs fully reappeared on the cell surface, whereas sequestered m2 mAChRs did not. The distinct sequestration of m2 mAChRs was also apparent in COS-7 and Chinese hamster ovary cells. We conclude that the m2 mAChR displays unique subtype-specific sequestration that distinguishes this receptor from the m1, m3, and m4 subtypes. These results are the first to demonstrate that receptor sequestration represents a new type of receptor subtype-specific regulation within the family of mAChRs.

Sphingosine kinase-mediated calcium signaling by muscarinic acetylcholine receptors

Based on the finding that G protein-coupled receptors (GPCRs) can induce Ca2+ mobilization, apparently independent of the phospholipase C (PLC)/inositol-1,4,5-trisphosphate (IP3) pathway, we investigated whether sphingosine kinase, which generates sphingosine-1-phosphate (SPP), is involved in calcium signaling by mAChR and other GPCRs. Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,/N-dimethylsphingosine markedly inhibited [Ca2+]i increases elicited by M2 and M3 mAChRs in HEK-293 cells without affecting PLC activation. Activation of M2 and M3 mAChR rapidly and transiently stimulated production of SPP. Furthermore, microinjection of SPP into HEK-293 cells induced rapid and transient Ca2+ mobilization. Pretreatment of HEK-293 cells with the calcium chelator BAPTA/AM fully blocked mAChR-induced SPP production. On the other hand, incubation of HEK-293 cells with calcium ionophores activated SPP production. Similar findings were obtained for formyl peptide and P2Y2 purinergic receptors in HL-60 cells. On the basis of these studies we propose, that following initial IP3 production by receptor-mediated PLC activation, a local discrete increase in [Ca2+]i induces sphingosine kinase stimulation, which ultimately leads to full calcium mobilization. Thus, sphingosine kinase activation most likely represents an amplification system for calcium signaling by mAChRs and other GPCRs.

CALCIUM SIGNALLING BY G PROTEIN-COUPLED SPHINGOLIPID RECEPTORS IN BOVINE AORTIC ENDOTHELIAL CELLS

Besides its role as a putative second messenger releasing Ca2+ from intracellular stores, sphingosine-1-phosphate (SPP) has recently been identified as an extracellularly acting ligand activating a high affinity G protein-coupled membrane receptor in various cell types. Since SPP can be released from activated platelets, we examined in the present study whether endothelial cells express receptors for SPP and related sphingolipids. In bovine aortic endothelial cells loaded with fura-2, addition of SPP caused a rapid and transient increase in intracellular Ca2+ concentration ([Ca2+]i), amounting to maximally about 230 nM. Removal of extracellular Ca2+ revealed that SPP-induced [Ca2+]i elevations were due to both release of Ca2+ from intracellular stores and influx of extracellular Ca2+. Pretreatment of the cells with pertussis toxin inhibited the SPP-induced increase in [Ca2+]i by 83%, in line with the previously reported involvement of G proteins of the Gi/o family in SPP signalling in other cell types. In contrast to other [Ca2+]i-elevating agonists, e.g., ATP and bradykinin, SPP did not activate phospholipase C in bovine aortic endothelial cells, suggesting the involvement of a novel, unidentified signalling pathway in SPP-induced release of intracellular Ca2+. Furthermore, SPP also did not cause activation of either phospholipase D or A2. Out of various related sphingolipids studied, only sphingosylphosphorylcholine (SPPC) induced a similar maximal increase in [Ca2+i as SPP, and its effect was also fully pertussis toxin-sensitive. However, the potencies of the two sphingolipids to increase [Ca2+]i differed by more than two orders of magnitude, with the EC50 values being 0.8 nM and 260 nM for SPP and SPPC, respectively. These results identify SPP and SPPC as novel and potent endothelial agonists, inducing calcium signalling by activation of a Gi/o protein-coupled receptor(s). Given the recently reported release of SPP from thrombin-activated platelets, SPP may represent a novel mediator of platelet-endothelial cell interactions.

β-Arrestin Recruitment Assay for the Identification of Agonists of the Sphingosine 1-Phosphate Receptor EDG1

β-Arrestin recruitment assays provide a generic assay platform for drug discovery on G-protein-coupled receptors (GPCRs). The PathHunter™ assay technology developed by DiscoveRx (Fremont, CA) uses enzyme fragment complementation of β-galactosidase to measure receptor-β-arrestin proximity by chemiluminescence. This study describes an agonistic screen on the human endothelial differentiation sphingolipid GPCR 1 (EDG1), also known as S1P1, using PathHunter™ β-arrestin recruitment technology. Screening of a collection of 345,052 compounds yielded 2157 agonistic hits. Only 10 of these compounds showed β-arrestin recruitment activity on a nonrelated receptor, indicating high accuracy and specificity of the assay. The authors show that receptor activation with reference agonists can be detected within the same EDG1 PathHunter™ cell line at the level of β-arrestin recruitment, Gi/o protein-mediated inhibition of cyclic adenosine monophosphate (cAMP), and activation of downstream phosphorylation of extracellular signal-regulated protein kinases. The degree of β-arrestin recruitment was largely unaffected upon blockade of Gi/o protein signaling with pertussis toxin, whereas kinetic studies demonstrated a lower rate of β-arrestin-receptor association. In contrast, inhibition of cAMP and phosphorylation of extracellular signal-regulated protein kinases were fully Gi/o protein regulated. The data indicate that the β-arrestin enzyme fragment complementation cell line can be used not only for agonistic screening of GPCRs but also for the identification of “biased ligands” (i.e., compounds that differ in G-protein coupling and β-arrestin-mediated cellular effects). (Journal of Biomolecular Screening 2008:986-998)

Distinct Internalization of M2 Muscarinic Acetylcholine Receptors Confers Selective and Long-Lasting Desensitization of Signaling to Phospholipase C

Abstract: Although M1‐M4 muscarinic acetylcholine receptors (mAChRs) in HEK‐293 cells internalize on agonist stimulation, only M1, M3, and M4 but not M2 mAChRs recycle to the plasma membrane. To investigate the functional consequences of this phenomenon, we compared desensitization and resensitization of M2 versus M4 mAChRs. Treatment with 1 mM carbachol for 1 h at 37°C reduced numbers of cell surface M2 and M4 mAChRs by 40‐50% and M2 and M4 mAChR‐mediated inhibition of adenylyl cyclase, intracellular Ca2+ concentration ([Ca2+]i) increases, and phospholipase C (PLC) activation by 60‐70%. Receptor‐mediated inhibition of adenylyl cyclase and [Ca2+]i increases significantly resensitized within 3 h. However, M4 but not M2 mAChR‐mediated PLC activation resensitized. At 16°C, M2 mAChR‐mediated [Ca2+]i increases and PLC stimulation desensitized to a similar extent as at 37°C. However, at 16°C, where M2 mAChR internalization is negligible, both M2 mAChR responses resensitized, demonstrating that M2 mAChR resensitization proceeds at the plasma membrane. Examination of M2 mAChR responses following inactivation of cell surface mAChRs by quinuclidinyl benzilate revealed substantial receptor reserve for coupling to [Ca2+]i increases but not to PLC. We conclude that M2 mAChR internalization induces long‐lasting PLC desensitization predominantly because receptor loss is not compensated for by receptor recycling or receptor reserve.

Discrimination between plasma membrane and intracellular target sites of sphingosylphosphorylcholine

On the background of the emerging concept of G protein-coupled sphingolipid receptors, Ca2+ mobilization by sphingosylphosphorylcholine (SPPC) in intact cells and SPPC-induced Ca2+ release in permeabilized cells, both occurring at similar, micromolar concentrations, were characterized and compared. In intact human embryonic kidney (HEK-293) cells, SPPC rapidly increased [Ca2+]i by mobilization of Ca2+ from thapsigargin-sensitive stores. In saponin-permeabilized HEK-293 cells, SPPC released stored Ca2+, in a manner similar to but independent of inositol 1,4,5-trisphosphate. Only the action of SPPC on intact cells, but not that in permeabilized cells, was, at least in part, sensitive to pertussis toxin. In addition and most important, Ca2+ release by SPPC in permeabilized cells was not stereoselective, whereas in intact cells only the naturally occurring D-erythro-SPPC, but not L-threo-SPPC, increased [Ca2+]i. Stereoselectivity of SPPC-induced [Ca2+]i increase was also demonstrated in bovine aortic endothelial cells. In conclusion, Ca2+ mobilization by SPPC in intact cells is independent of the previously described SPPC-gated Ca2+ channel on endoplasmic reticulum but probably mediated by a membrane sphingolipid receptor. Thus, SPPC can regulate Ca2+ homeostasis by acting apparently at two cellular targets, which exhibit clearly distinct recognition patterns.

Pharmacological Characterization of Receptor Redistribution and β-Arrestin Recruitment Assays for the Cannabinoid Receptor 1

Receptor redistribution and β-arrestin recruitment assays provide a G-protein-subtype-independent method to measure ligand-stimulated activation of G-protein-coupled receptors. In particular β-arrestin assays are becoming an increasingly popular tool for drug discovery. The authors have compared a high-content-imaging-based Redistribution® assay and 2 nonimaging-based β-arrestin recruitment assays, Tango™ and PathHunter ™, for the cannabinoid receptor 1. Inasmuch as all 3 assays use receptors that are modified at the C-terminus, the authors verified their pharmacology via detection of Gαi coupling of the receptor in cAMP assays using reference ligands. The potencies and efficacies of the cannabinoid receptor agonists CP55,940 and WIN55,212-2 correlated well between the 3 assays, and are comparable with the measured ligand binding affinities. The inverse agonist SR141716 decreased basal signal in all 3 assays, but only in the Tango bla assay a reliable EC50 could be determined for this compound, suggesting that Tango is the most suitable assay for the identification of new inverse agonists. Both the Redistribution and the PathHunter assay could discriminate partial agonists from full agonists, whereas in the Tango assay partial agonists behaved as full agonists. Only the PathHunter cells allowed detection of cannabinoid receptor activation via β-arrestin recruitment and Gαi-protein-mediated inhibition of cAMP, thus enabling the identification of biased ligands that differ in these cellular effects. The characteristics and limitations of the different assays are discussed. (Journal of Biomolecular Screening 2009:811-823)