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Dynamin Mediates Caveolar Sequestration of Muscarinic Cholinergic Receptors and Alteration in NO Signaling

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Dynamin Mediates Caveolar Sequestration of Muscarinic Cholinergic Receptors and Alteration in NO Signaling The EMBO Journal Vol. 19 No. 16 pp. 4272±4280, 2000 Dynamin mediates caveolar sequestration of muscarinic cholinergic receptors and alteration in NO signaling Chantal Dessy, Ralph A.Kelly1, phorylation of the agonist-stimulated receptor increases its Jean-Luc Balligand2 and Olivier Feron af®nity for arrestin, which sterically prevents further coupling with G-proteins, whereas sequestration reduces Department of Medicine, Unit of Pharmacology and Therapeutics, FATH 53.49, Universite Catholique de Louvain, Avenue E.Mounier, the access for membrane-impermeable agonists. 53, B-1200 Brussels, Belgium and 1Cardiovascular Division, Several authors have addressed the existence of a link Department of Medicine, Brigham and Women's Hospital and between GRK-dependent phosphorylation and internaliza- Harvard Medical School, Boston, MA 02115, USA tion of the m2 subtype of mAchR in heterologous 2Corresponding author expression systems (Tsuga et al., 1994; Pals-Rylaarsdam e-mail: [email protected] et al., 1995; Schlador and Nathanson, 1997). Although the extent of overexpression of each signal-transducing com- In cardiac myocytes, agonist binding to muscarinic ponent of the sequestration pathways may confound the acetylcholine receptors (mAchRs) leads to the target- interpretation of some of these studies, discrepancies in the ing of stimulated receptors to plasmalemmal micro- results also suggested the existence of cell type-speci®c domains termed caveolae. Here, we examined whether mechanisms for receptor internalization. Evidence for this translocation leads to mAchR internalization and distinct sequestration pathways was indeed provided alteration in downstream NO signaling. Differential recently by the ®nding that the m2 mAchR could binding of membrane-permeant and -impermeant be internalized by both arrestin/clathrin-dependent and mAchR radioligands on caveolae-enriched mem- -independent pathways (Pals-Rylaarsdam et al., 1997; branes revealed that carbachol stimulation of cardiac VoÈgler et al., 1998). These data underscore the plasticity myocytes induces sequestration of mAchRs through of the mechanisms of m2 mAchR traf®cking and justify caveolae ®ssion. GTP but not its non-hydrolyzable the growing interest in the processes of mAchR seques- analog GTPgS drove the further detachment of caveo- tration/desensitization in primary cells such as cardiac lae from myocyte sarcolemma. Also, incubation of myocytes where the m2 subtype is the predominant extracts of carbachol-stimulated myocytes with isoform. recombinant GTPase dynamin induced mAchR We have reported previously that in rat cardiac sequestration in budded caveolae, while dominant- myocytes, a large fraction of sarcolemmal m2 mAchR is negative K44A dynamin inhibited it. These data were targeted, upon agonist stimulation, to non-clathrin vesicles con®rmed by immuno¯uorescence microscopy on m2 termed caveolae (Feron et al., 1997a). Caveolae are small mAchR-expressing COS cells. Finally, repeated carba- ¯ask-shaped invaginations of the plasma membrane chol stimulations of mAchRs co-expressed in COS characterized by high levels of cholesterol, glycosphingo- cells with endothelial nitric oxide synthase (eNOS) lipids and lipid-anchored proteins (Anderson, 1998). and wild-type, but not mutant, dynamin led to a pro- These specialized lipid microdomains ensure the compart- gressive increase in mAchR sequestration and a mentation of a number of signaling proteins including the concurrent stabilization of the inhibitory eNOS± endothelial nitric oxide synthase (eNOS), various kinases caveolin complex. These ®ndings emphasize the role of and G-proteins (Anderson, 1998; Okamoto et al., 1998). In caveolae in mAchR traf®cking and NO signaling, and addition, a number of GPCRs including the B2 bradykinin, suggest that caveolae ®ssion may contribute to b-adrenergic, cholecystokinin, endothelin, angiotensin II G-protein-coupled receptor desensitization. and calcium-sensing receptors have been shown to be Keywords: caveolae/caveolin/dynamin/eNOS/mAchR located within caveolae in native conditions or upon agonist stimulation (for references see Anderson, 1998; Okamoto et al., 1998). However, these observations do not address the issue of whether the caveolar location of a Introduction speci®c GPCR is required to initiate its downstream Muscarinic acetylcholine receptors (mAchRs) belong to signaling cascade or to mediate the receptor desensitiza- the superfamily of G-protein-coupled receptors (GPCRs) tion/resensitization process, or both. Recently, the latter that couple extracellular stimuli to plasmalemmal and hypothesis gained some ground from the ®nding that intracellular effectors including ion channels and enzymes dynamin, which is known to play a crucial regulatory role such as adenylate cyclase and phospholipases. Agonist in the processes of endocytosis through clathrin-coated stimulation of GPCRs also leads to adaptive changes that pits, is largely enriched in caveolae and promotes cholera serve to facilitate responsiveness of the cell to successive toxin B chain internalization through budded caveolae stimulations. This process is initiated by receptor desensi- (Henley et al., 1998; Oh et al., 1998). tization, which involves receptor phosphorylation by The inotropic and chronotropic state of the heart is GPCR kinases (GRKs) and its sequestration in specialized closely regulated by the parasympathetic system (in part cell compartments (Lefkowitz, 1998). Accordingly, phos- through the activation of eNOS; Balligand et al., 1993; 4272 ã European Molecular Biology Organization Caveolar ®ssion and mAchR sequestration Feron et al., 1998b), and the cardiac function is therefore highly dependent on the responsiveness to mAchR stimu- lation, which varies according to the degree of mAchR sequestration. In this study, we examined the subcellular mechanisms contributing to the sequestration of the m2 mAchR through the caveolar pathway in freshly isolated cardiac myocytes. We took advantage of the existence of membrane-permeant and -impermeant mAchR radio- ligands and of the distinct buoyant density of caveolar vesicles to demonstrate the GTP-driven budding of caveolae in cardiac myocytes exposed to the mAchR agonist carbachol. We also established the role of dynamin in promoting caveolar ®ssion and mAchR sequestration. Finally, using transfected COS-7 cells, we observed that agonist-induced dynamin-dependent sequestration of m2 mAchR diminishes the extent of activatability of eNOS, a downstream signaling target. Results Time-dependent mAchR sequestration in cardiac myocyte caveolae We ®rst examined the potential sequestration of mAchRs targeted to cardiac myocyte caveolae (upon agonist stimulation) by comparing the binding pattern of mem- brane-permeant and -impermeant mAchR radioligands. Following exposure to the muscarinic cholinergic agonist carbachol (100 mM) for 0±60 min, adult rat cardiac myocytes were washed extensively and the intact cells were then exposed for 3 h at 4°C to the radiolabeled lipophilic and hydrophilic mAchR antagonists [3H]QNB (quinuclidinyl benzylate) and [3H]NMS (N-methyl-scopo- lamine), respectively. Figure 1A shows that, whereas the Fig. 1. Carbachol-induced targeting and sequestration of mAchRs in [3H]QNB-speci®c binding did not reveal changes in total cardiac myocyte caveolae. (A) Time course of [3H]NMS (open squares) and [3H]QNB (®lled squares) binding to intact cardiac myocytes mAchR density, the fraction of receptors accessible to exposed to 100 mM carbachol. The data are expressed as a percentage 3 [ H]NMS progressively decreased with the time of agonist (means 6 SEM, n = 3) of the total speci®c binding determined at time exposure; a maximum of 26 6 3% of mAchRs appeared 0(*P <0.01 versus the corresponding [3H]QNB binding); the actual sequestrated after a 1 h exposure to carbachol. It is of note density of [3H]QNB-binding sites is 178 6 12 fmol/mg protein that in experiments performed on corresponding myocyte (n = 3). (B) Fractionation of cardiac myocytes. The distribution of caveolin-3 (cav-3) and eNOS between caveolae (CAV) and non- lysates in the presence of saponin (used to disrupt vesicle caveolae (NON-CAV) membrane-enriched fractions is shown; these membranes), we could completely restore the speci®c data represent the result of a typical fractionation experiment from binding of [3H]NMS (not shown), suggesting that 1 h after resting cardiac myocytes, as described in Materials and methods. carbachol exposure, sequestrated mAchRs were not (C)[3H]NMS (open columns) and [3H]QNB (black columns) binding to the caveolae-enriched fraction obtained from cardiac myocytes degraded and remained susceptible to be recycled. exposed to 100 mM carbachol for the indicated time. The data are In a subsequent series of experiments, following expressed as a percentage (means 6 SEM, n = 3) of the total amount exposure to carbachol (0±60 min), myocytes were lysed of [3H]NMS- or [3H]QNB-speci®c binding determined in the in a carbonate-containing buffer and fractionated by CAV + NON-CAV fractions at time 0 (*P <0.01 versus the isopycnic centrifugation into caveolar and non-caveolar corresponding [3H]QNB binding). membrane fractions (see Materials and methods). The isolation of caveolar membranes from the remaining sarcolemmal and internal membranes was validated by of total cell receptors after 15 min of carbachol exposure; using subcellular compartment-speci®c markers. Figure 1B this effect was observed independently of the radioligand shows that caveolin-3 and >90% of eNOS
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