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• W2014596089 abstract "Article11 May 2010free access CaMKIIα interacts with M4 muscarinic receptors to control receptor and psychomotor function Ming-Lei Guo Ming-Lei Guo Department of Basic Medical Science, University of Missouri-Kansas City, Kansas City, MO, USA Search for more papers by this author Eugene E Fibuch Eugene E Fibuch Department of Anesthesiology, School of Medicine, University of Missouri-Kansas City, Kansas City, MO, USA Search for more papers by this author Xian-Yu Liu Xian-Yu Liu Department of Anesthesiology, Washington University School of Medicine, St Louis, MO, USA Search for more papers by this author Eun Sang Choe Eun Sang Choe Department of Biological Sciences, Pusan National University, Kumjeong-gu, Pusan, Korea Search for more papers by this author Shilpa Buch Shilpa Buch Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, USA Search for more papers by this author Li-Min Mao Corresponding Author Li-Min Mao Department of Basic Medical Science, University of Missouri-Kansas City, Kansas City, MO, USA Search for more papers by this author John Q Wang Corresponding Author John Q Wang Department of Basic Medical Science, University of Missouri-Kansas City, Kansas City, MO, USA Department of Anesthesiology, School of Medicine, University of Missouri-Kansas City, Kansas City, MO, USA Search for more papers by this author Ming-Lei Guo Ming-Lei Guo Department of Basic Medical Science, University of Missouri-Kansas City, Kansas City, MO, USA Search for more papers by this author Eugene E Fibuch Eugene E Fibuch Department of Anesthesiology, School of Medicine, University of Missouri-Kansas City, Kansas City, MO, USA Search for more papers by this author Xian-Yu Liu Xian-Yu Liu Department of Anesthesiology, Washington University School of Medicine, St Louis, MO, USA Search for more papers by this author Eun Sang Choe Eun Sang Choe Department of Biological Sciences, Pusan National University, Kumjeong-gu, Pusan, Korea Search for more papers by this author Shilpa Buch Shilpa Buch Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, USA Search for more papers by this author Li-Min Mao Corresponding Author Li-Min Mao Department of Basic Medical Science, University of Missouri-Kansas City, Kansas City, MO, USA Search for more papers by this author John Q Wang Corresponding Author John Q Wang Department of Basic Medical Science, University of Missouri-Kansas City, Kansas City, MO, USA Department of Anesthesiology, School of Medicine, University of Missouri-Kansas City, Kansas City, MO, USA Search for more papers by this author Author Information Ming-Lei Guo1, Eugene E Fibuch2, Xian-Yu Liu3, Eun Sang Choe4, Shilpa Buch5, Li-Min Mao 1 and John Q Wang 1,2 1Department of Basic Medical Science, University of Missouri-Kansas City, Kansas City, MO, USA 2Department of Anesthesiology, School of Medicine, University of Missouri-Kansas City, Kansas City, MO, USA 3Department of Anesthesiology, Washington University School of Medicine, St Louis, MO, USA 4Department of Biological Sciences, Pusan National University, Kumjeong-gu, Pusan, Korea 5Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, USA *Corresponding authors. Department of Basic Medical Science, University of Missouri-Kansas City, School of Medicine, 2411 Holmes Street, Kansas City, MO 64108, USA. Tel: +1 816 235 1786; Fax: +1 816 235 5574; E-mail: [email protected] or Tel: +1 816 235 1719; Fax: +1 816 235 5574; E-mail: [email protected] The EMBO Journal (2010)29:2070-2081https://doi.org/10.1038/emboj.2010.93 There is a Have you seen ...? (June 2010) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Muscarinic acetylcholine receptors (mAChRs) are widely expressed in the mammalian brain and are essential for neuronal functions. These receptors are believed to be actively regulated by intracellular signals, although the underlying mechanisms are largely unknown. In this study, we show that Ca2+/calmodulin-dependent protein kinase II (CaMKII) binds directly and selectively to one of five mAChR subtypes, M4 receptors (M4Rs), at their C-terminal regions of second intracellular loops. This binding relies on Ca2+ activation of the kinase and leads to the phosphorylation of M4Rs at a specific threonine site (Thr145). Complementary in vivo studies in rat striatal neurons enriched with M4Rs confirm that rising Ca2+ recruits CaMKIIα to M4Rs to potentiate receptor signalling, which controls behavioural sensitivity to dopamine stimulation in an activity-dependent manner. Our data identify a new model of protein–protein interactions. In a Ca2+-sensitive manner, CaMKIIα regulates M4R efficacy and controls the acetylcholine–dopamine balance in the basal ganglia and also the dynamics of movement. Introduction Muscarinic acetylcholine receptors (mAChRs) are widely expressed in neurons, cardiac and smooth muscles, and many other tissues (Nathanson, 2008). As members of the G protein-coupled receptor (GPCR) superfamily, five muscarinic receptor subtypes (M1–M5) are subdivided into two functional groups (Wess, 1996; Nathanson, 2000). In general, the M1, M3, and M5 subtypes are preferentially coupled to Gαq proteins. As such, they activate phospholipase C to trigger a phosphoinositide-dependent signalling pathway. The M2 and M4 subtypes, however, are coupled to Gαi/o proteins. Through them, they inhibit adenylyl cyclase and the downstream formation of cAMP. The broad expression and diverse signalling pathway connections enable mAChRs to regulate various cellular activities. In rodent brains, mAChRs are enriched in the striatum (Nathanson, 2008). Particularly, M4 receptors (M4Rs) are most abundantly present in this region and are preferentially localized at post-synaptic sites (Levey et al, 1991; Yasuda et al, 1993; Hersch et al, 1994). This situates M4Rs well to regulate striatal cellular activity related to learning, memory, cognition, reward, and movement. Moreover, malfunctional M4Rs are frequently linked to the pathogenesis of mental illnesses, such as schizophrenia and substance addiction, and neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases (Langmead et al, 2008; Scarr and Dean, 2008). The G protein-coupled receptors are actively regulated by protein–protein interactions between their intracellular domains and submembranous regulatory proteins. Among all regulatory proteins, protein kinases are of particular importance. Various serine/threonine protein kinases can directly or indirectly interact with GPCRs. Through a phosphorylation mechanism, they drastically modulate the efficacy of receptor signalling. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is among those kinases that regulate GPCR signalling via a mechanism involving interaction and phosphorylation. This enzyme is especially abundant in brain cells and is enriched at synaptic sites (Kelly et al, 1984). It is activated by the binding of Ca2+ and calmodulin (CaM). Activated CaMKII can then access and phosphorylate both exogenous substrates and its own autophosphorylation site (T286 in the α isoform). Such autophosphorylation renders the enzyme a Ca2+/CaM-independent (autonomous) activity even after the initial Ca2+ stimulus subsides (Hudmon and Schulman, 2002; Colbran and Brown, 2004; Griffith, 2004). As a result, CaMKII can transmit a transient Ca2+ signal into the relatively prolonged regulation of its downstream targets (for review, see Hudmon and Schulman, 2002; Lisman et al, 2002; Colbran and Brown, 2004). Although CaMKII is believed to interact with and regulate many targets (Colbran, 2004), a limited number of such interacting partners have been identified. In this study, we identify that CaMKIIα interacts directly with the C-terminal region of the second intracellular loop of M4Rs in vitro. The interaction is dependent on Ca2+ level and autophosphorylation. The inducible interaction enables CaMKIIα to phosphorylate M4Rs at a selective threonine residue within the binding motif. In striatal neurons in vivo, Ca2+ stimulates the association of CaMKIIα with M4Rs and increases threonine phosphorylation of M4Rs, which thereby potentiates M4R efficacy. The CaMKII-mediated potentiation of M4Rs has a significant role in regulating behavioural responsiveness to dopamine stimulation with cocaine. Together, our data reveal a new model of protein–protein interactions. Through this interaction, CaMKII participates in maintaining a proper acetylcholine–dopamine balance in the striatum. Results CaMKIIα selectively binds to M4Rs Rat M4Rs possess four intracellular domains: three intracellular loops and one C-terminus (Supplementary Figure S1). To explore whether cytoplasmic CaMKIIα interacts with any of these domains, we synthesized a panel of glutathione-S-transferase (GST) fusion proteins containing full, or fragments of, individual domains (Figure 1A). Using these GST-immobilized baits in pull-down assays with rat soluble striatal lysates, we observed that the GST-tagged second intracellular loop of M4Rs (GST–M4RIL2) pulled down CaMKIIα (Figure 1B), whereas ther GST fusion proteins and GST alone did not. The GST fusion protein containing the second intracellular loop of muscarinic M2 receptors (M2Rs) also did not pull down CaMIIα. None of the GST fusion proteins pulled down CaMKIV, another isoform of CaMK. Blots that were probed in parallel with an anti-GST antibody ensured equivalent protein loading (Supplementary Figure S2A). To identify the region within CaMKIIα involved in the interaction with M4Rs, we prepared GST fusion proteins containing each key structure of the kinase (Figure 1C). We observed that the N-terminal catalytic domain of CaMKIIα (GST–CaMKIIαCD), similar to the full-length GST–CaMKIIα, precipitated M4Rs in pull-down experiments with striatal lysates (Figure 1D). The GST fusion proteins containing the regulatory domain (GST–CaMKIIαRD) or the C-terminal association domain (GST–CaMKIIαAD) produced no precipitation. We did not observe that GST–CaMKIV pulled down M4Rs. Furthermore, none of the GST fusion proteins pulled down M2Rs (Figure 1D). Thus, a restricted region of the enzyme, that is, CaMKIIαCD, is responsible for the M4R interaction. Figure 1.Interactions of CaMKIIα with M4Rs. (A) GST fusion proteins containing the second intracellular loop (IL2), third intracellular loop (IL3) fragments, and C-terminus (CT) of rat M4Rs. (B) Pull-down assays with immobilized GST fusion proteins and rat striatal lysates. (C) GST fusion proteins derived from rat CaMKIIα. (D) Pull-down assays showing that GST–CaMKIIα and GST–CaMKIIαCD pulled down M4Rs from rat striatal lysates. (E, F) In vitro binding assays with immobilized GST fusion proteins and purified CaMKIIα or CaMKIV. (G, H) In vitro binding assays with immobilized biotinylated-M4RIL2 or -M2RIL2 and purified GST–CaMKIIα fragments or GST–CaMKIV. Binding assays in (E) were performed in the presence or absence of CaCl2 (0.5 mM), CaM (1 μM), or EGTA (1 mM). Other binding assays (F–H) were conducted in the presence of CaCl2 (0.5 mM) and CaM (1 μM). Proteins bound to GST fusion proteins (B, D–F) or biotinylated proteins (G, H) in either pull-down or binding assays were visualized with immunoblots (IBs) using the specific antibodies as indicated. Download figure Download PowerPoint We next examined whether CaMKIIα directly interacts with M4Rs in binding assays with purified proteins. CaMKIIα rather than CaMKIV bound to immobilized GST–M4RIL2, but not to other GST–M4RIL3 fragments, GST–M4RCT, or GST alone (Figure 1E). Interestingly, CaMKIIα exhibited the binding only in the presence of Ca2+/CaM. In the absence of Ca2+/CaM, wherein the enzyme remains inactive, no binding was detected. Thus, activation of the enzyme by Ca2+/CaM is required for its binding. CaMKIIα also bound to full-length M4Rs (Supplementary Figure S2B). However, CaMKIIα did not bind to GST–M2RIL2 (Figure 1F). This is noteworthy given that only two residues are different between M2RIL2 and M4RIL2 (Figure 1F). Further assays aimed at screening the binding of CaMKIIα to the second intracellular loops of all five muscarinic subtypes revealed that the kinase bound only to M4RIL2 (Supplementary Figure S2C), a domain highly conserved among mammals (Supplementary Figure S2D). We also tried to immobilize biotinylated M4RIL2 and subsequently tested the binding of GST fusion CaMKIIα fragments to M4RIL2. The bound GST fusion proteins were visualized by immunoblots with an anti-GST antibody. We observed that the immobilized M4RIL2 was bound by GST–CaMKIIα and GST–CaMKIIαCD, but not by other GST–CaMKIIα fragments or GST–CaMKIV (Figure 1G). The immobilized M2RIL2 was not bound by any GST fusion proteins (Figure 1H). These results indicate that CaMKIIα and M4Rs bind directly to each other through their defined subdomains. Ca2+ and autophosphorylation regulate CaMKIIα binding to M4RIL2 The Ca2+-dependent nature of CaMKIIα–M4R binding was further investigated. Similar to the results described above, the addition of Ca2+ (0.5 mM) and CaM (1 μM) enabled CaMKIIα to bind to M4RIL2 (L2 versus L1 in Figure 2A). In contrast, the Ca2+/CaM binding-defective CaMKIIα mutant, T305/306D (Colbran and Soderling, 1990), showed no binding regardless of the presence of Ca2+/CaM (Figure 2A). Adding a peptide (L290–A309), which corresponds to the CaM-binding domain of CaMKII and thereby antagonizes the CaM–CaMKII association, blocked the Ca2+/CaM-induced binding between CaMKIIα and M4Rs (Figure 2B). Similar effect was observed with a Ca2+ chelator EGTA (1 mM; data not shown). These results substantiate the model that activation of CaMKIIα by Ca2+/CaM is essential to transform the kinase to a state preferred for physical interaction with M4Rs. Notably, CaMKIIα can bind to M4Rs in a form bearing no autophosphorylation at T286. This was evidenced by the lack of detectable T286-autophosphorylated CaMKIIα (pCaMKIIα) in the binding assays containing no ATP (a preferred phosphate donor) (middle panel of Figure 2A). Thus, activation of CaMKIIα by Ca2+/CaM is sufficient to promote the binding of the enzyme to M4Rs. Figure 2.Ca2+/CaM- and autophosphorylation-regulated CaMKIIα binding to M4RIL2. (A) CaMKIIα T305/306D mutant lacks the binding to M4RIL2. In the middle panel, CaMKIIα was not autophosphorylated at T286 in binding assays lacking ATP (L1–L4) as opposed to T286-autophosphorylated CaMKIIα detected in the synaptosomal fraction (P2) from the rat striatum (L7) with a phospho-specific antibody. (B) The inhibitory peptide (L290–A309) prevented the Ca2+/CaM-induced CaMKIIα-M4RIL2 binding. (C, D) Binding of WT CaMKIIα/pCaMKIIα (C), mutant T286A (D), or mutant T286D (D) to GST–M4RIL2. (E) A graph of the data from (C, D). (F) Effects of five peptides derived from M4RIL2 on the binding of CaMKIIα to M4RIL2. Bold letters (in red) indicate the potential CaMKIIα-binding motif. Binding assays were performed between His-tagged WT CaMKIIα (∼57 kDa), WT pCaMKIIα (∼57 kDa), T305/306D mutant (∼50 kDa), T286A mutant (∼50 kDa), or T286D mutant (∼50 kDa) proteins and immobilized GST or GST–M4RIL2 in the presence or absence of CaCl2 (0.5 mM), CaM (1 μM), or L290–A309 (5 μM) as indicated. EGTA (1 mM) was added in the assays lacking CaCl2. Bound CaMKIIα, pCaMKIIα, or mutants were visualized by immunoblots. Data are presented as means±s.e.m. for 4–6 experiments per group. *P<0.05 and **P<0.01 versus L2. Download figure Download PowerPoint The autophosphorylation of T286 may further impact on binding properties of the kinase. To examine this, we tested the binding capacity of autophosphorylated CaMKIIα. We found that CaMKIIα that was activated by Ca2+/CaM and autophosphorylated at T286 before the binding assays exhibited a higher level of binding to GST–M4RIL2 compared with unphosphorylated CaMKIIα (L4 versus L2 in Figure 2C and E). In the absence of Ca2+/CaM in binding reactions, autophosphorylated rather than unphosphorylated CaMKIIα bound to the target (L3 versus L1 in Figure 2C). These data indicate that the autophosphorylation further enhances the binding and that, once autophosphorylated, the kinase no longer relies on Ca2+/CaM to bind to M4Rs. In support of this, an autophosphorylation-defective mutant, T286A, showed no further enhancement of the binding (L4 versus L2 in Figure 2D and E). A constitutively active mutant, T286D, that has the same property as autophosphorylated CaMKIIα, showed a high level of binding, regardless of the presence or absence of Ca2+/CaM (Figure 2D and E). To identify a core binding motif within the 20 amino acids of M4RIL2, we used a panel of peptides truncated from M4RIL2 to compete with full-length M4RIL2 for binding to CaMKIIα. Four peptides (a–d) completely prevented GST–M4RIL2 from precipitating Ca2+/CaM-activated CaMKIIα (Figure 2F) or autophosphorylated CaMKIIα (Supplementary Figure S3). These findings seem to suggest a 9-residue sequence (YPARRTTKM) as a core binding motif for CaMKIIα. Phosphorylation of M4RIL2 by CaMKII The biochemical binding of CaMKIIα to M4RIL2 suggests the latter to be a potential phosphorylation substrate of the kinase. To determine this, we assayed the CaMKIIα-mediated incorporation of 32P into M4RIL2 by sensitive autoradiography. We observed that activated CaMKIIα in the presence of Ca2+/CaM phosphorylated GST–M4RIL2 but not GST, whereas the inactive CaMKIIα in the absence of Ca2+/CaM did not (Figure 3A). In separate assays, active CaMKIIα did not phosphorylate three GST–M4RIL3 fragments and GST–M4RCT (Figure 3B). Consistent with the fact that CaMKIIα did not bind to M2RIL2, the kinase induced little or no phosphorylation of GST–M2RIL2 (Figure 3C). In fact, among all the five muscarinic receptor subtypes, CaMKIIα induced a strong phosphorylation only in M4RIL2 (Supplementary Figure S4). These data identify M4RIL2 as a preferred substrate for selective phosphorylation by CaMKIIα. To identify accurate phosphorylation site(s) within M4RIL2, we screened phosphorylation of a series of synthetic M4RIL2 peptides bearing mutations of four known threonine sites (Figure 3D). CaMKIIα readily phosphorylated a wild-type (WT) M4RIL2 peptide substrate to a substantial stoichiometry (0.62 mol of phosphate per mol of substrate, Figure 3D). The kinase also phosphorylated the peptide with mutation of T134 to alanine (T134A), T138 to alanine (T138A), or T144 to alanine (T144A) to an extent comparable with the WT peptide. Remarkably, mutation of T145 to alanine (T145A) completely abolished the phosphorylation. So did mutation of all four threonine sites to alanine. These data support T145 to be the site of phosphorylation within M4RIL2. Notably, the CaMKIIα-mediated phosphorylation at this site is comparable with that at a threonine site in a classical CaMKII substrate, autocamtide-2 (KKALRRQETVDAL, Figure 3E). To determine whether this Ca2+-dependent phosphorylation was physiologically relevant, we tested the phosphorylation of M4RIL2 at physiological Ca2+ concentrations. At two physiological concentrations, CaMKIIα reliably phosphorylated M4RIL2 (Figure 3F), but not M2RIL2 (Figure 3G). Figure 3.Phosphorylation of M4RIL2 by CaMKIIα. (A) Autoradiographs illustrating phosphorylation of GST–M4RIL2 (upper) but not GST (lower) in the presence of Ca2+/CaM. (B) An autoradiograph illustrating phosphorylation of GST–M4RIL2 but not other GST fusion proteins. (C) An autoradiograph illustrating phosphorylation of GST–M4RIL2 but not GST–M2RIL2. (D) Phosphorylation of synthetic peptides (WT or mutants). An autoradiograph shows phosphorylation of these peptides. Phosphorylation stoichiometry was calculated from the radioactivity measured on Coomassie-stained bands by liquid scintillation counting. (E) Phosphorylation of M4RIL2 and autocamtide-2. 32P densitometry of two phosphorylated peptides (2 μM) was directly quantified on the films. (F, G) Phosphorylation of M4RIL2 but not M2RIL2 at physiological concentrations of Ca2+. Phosphorylation reactions were carried out at 30°C for 10 min with [γ-32P]ATP in the presence of Ca2+ (0.5 mM)/CaM (1 μM) or as indicated. The reactions were then subjected to gel electrophoresis followed by autoradiograph. The solid arrows indicate autophosphorylated CaMKIIα, whereas open arrows indicate phosphorylated GST–M4RIL2. Data are presented as means±s.e.m. for 3–5 experiments per group. Download figure Download PowerPoint In vivo interactions of CaMKIIα with M4Rs We next wanted to define whether the protein–protein interaction between native CaMKIIα and M4Rs occurs in neurons in vivo. We targeted the striatum because it exhibits the highest level of M4Rs in the brain (Levey et al, 1991; Yasuda et al, 1993; Hersch et al, 1994). In addition, M4Rs are a predominant subtype in this region as they account for 45% of total mAChRs, whereas M1Rs and M2Rs account for 35 and 12%, respectively. To assess the interaction, we performed a series of co-immunoprecipitation experiments with the solubilized synaptosomal fraction (P2) from the rat striatum. We observed that a CaMKIIα-immunoreactive band was consistently seen in the proteins precipitated by an anti-M4R antibody (Figure 4A). No immunoreactivity of CaMKIV was detected in the M4R precipitates. In reverse co-immunoprecipitation assays, an M4R-immunoreactive band was displayed in the CaMKIIα precipitates, whereas the M2R immunoreactivity was not (Figure 4B). These data demonstrate an evident interaction between CaMKIIα and M4Rs in striatal neurons in vivo. This interaction and its specificity were further confirmed by experiments in mutant mice. In M4R-deficient mice, co-immunoprecipitation of the two proteins was not seen in the striatum, whereas it was evident in the WT mice (Figure 4C). Figure 4.Interactions of CaMKIIα with M4Rs in striatal neurons. (A, B) Co-immunoprecipitation (IP) of CaMKIIα and M4Rs in the rat striatum. Lanes 3 and 4 showed no specific bands due to the lack of any antibody (L3) and the use of an irrelevant IgG (L4). (C) Co-immunoprecipitation of CaMKIIα and M4Rs in the striatum of wild-type (WT) and M4R mutant mice. (D) Effects of ionomycin (10 min) on the association of CaMKIIα and pCaMKIIα(T286) with M4Rs. (E, F) Effects of KN93 and KN92 on the ionomycin-stimulated association of CaMKIIα and pCaMKIIα(T286) with M4Rs. Ionomycin (10 μM) was co-treated with KN93 or KN92 (20 μM) for 10 min. (G) M4Ri disrupted the binding between CaMKIIα and M4RIL2 in vitro. Bound CaMKIIα was visualized by immunoblots (IB). (H) Effects of Tat peptides on the ionomycin-induced association of CaMKIIα with M4Rs. (I) Effects of Tat peptides on the ionomycin-stimulated threonine phosphorylation of M4Rs. Tat peptides (2 μM) were applied 45 min before ionomycin treatment(10 μM, 10 min). Immunoblots of CaMKIIα, pCaMKIIα(T286), phosphothreonine (pThr), or M4Rs were performed on M4R precipitates from drug-treated striatal slices (D–F, H, I). The ratio of the optical density (OD) of CaMKIIα, pCaMKIIα, and pThr bands over M4R bands was calculated. Data are presented as means±s.e.m. for 4–5 experiments per group. *P<0.05 versus vehicle or vehicle+vehicle. +P<0.05 versus vehicle+ionomycin. Download figure Download PowerPoint Ca2+ regulates CaMKIIα–M4R interactions in vivo To determine whether Ca2+ regulates CaMKIIα–M4R interactions in vivo as seen in vitro, we subjected rat striatal slices to a Ca2+ ionophore, ionomycin. We then assayed changes in interaction levels of two proteins using co-immunoprecipitation. Ionomycin (0.1–10 μM, 10 min) produced a concentration-dependent elevation in the amounts of both CaMKIIα and pCaMKIIα proteins bound to M4Rs (Figure 4D). This elevation was blocked by the CaMKII inhibitor KN93 (20 μM), which is able to inhibit the kinase by preventing the CaM binding (Figure 4E and F). The inactive analogue of KN93, KN92, had no effect. These results establish a Ca2+-sensitive nature of CaMKIIα–M4R interactions in striatal neurons in vivo. To directly confirm the accurate receptor site at which CaMKIIα binds, we developed an assay with Tat fusion peptides. We first synthesized an M4R interfering peptide (M4Ri) based on the core CaMKIIα-binding sequence on M4RIL2 (YPARRTTKM). We assured that this peptide efficiently competed with M4RIL2 for binding to CaMKIIα in vitro (Figure 4G). A sequence-scrambled control (MRRKPYATT, M4Rc), however, did not (Figure 4G). We then wanted to test the effect of these peptides in striatal neurons in vivo. To do so, we synthesized M4Ri or M4Rc together with an N-terminal Tat domain (YGRKKRRQRRR) derived from the human immunodeficiency virus-type 1 (HIV-1). This arginine-enriched Tat domain renders Tat fusion peptides a cell permeability in living neurons (Aarts et al, 2002; Liu et al, 2009). Using these cell-permeable peptides, we observed that Tat–M4Ri (2 μM, 45 min before ionomycin treatment) blocked the ionomycin-stimulated interactions between CaMKIIα and M4Rs in rat striatal slices, whereas Tat–M4Rc did not (Figure 4H). This indicates that Ca2+ rises in response to ionomycin can directly recruit CaMKIIα to an M4Ri-sensitive site on M4RIL2. Moreover, ionomycin increased threonine phosphorylation of M4Rs, as detected by an antibody selective for phosphothreonine (Figure 4I). This was blocked by pretreatment with Tat–M4Ri, but not Tat–M4Rc (Figure 4I). Thus, the inducible CaMKIIα interaction with M4RIL2 constitutes an initial required step leading to the phosphorylation of the receptor. The selectivity of Tat–M4Ri in disrupting the CaMKIIα–M4R interaction was confirmed by the ineffectiveness of this peptide in interfering with the ionomycin-stimulated interaction between CaMKIIα and another substrate, NR2B (Gardoni et al, 1998; Leonard et al, 1999; Bayer et al, 2001; Supplementary Figure S5A). This selectivity is further supported by the finding that CaMKIIα I205K, an NR2B binding-defective mutant, could sufficiently bind to M4RIL2 (Supplementary Figure S5B). CaMKII potentiates M4R signalling The next important question was what functional roles this Ca2+-regulated interaction might have. We therefore investigated whether the CaMKII–M4R interaction regulates the M4R-coupled signalling pathway. The activation of Gαi/o-coupled M4Rs has been well documented to principally inhibit adenylyl cyclase, thereby lowering the rate of cAMP production (Wess, 1996; Nathanson, 2000). We thus measured changes in cAMP levels as the signalling efficacy of M4Rs. In rat striatal slices, pharmacological activation of adenylyl cyclase with the selective activator, forskolin (1 or 10 μM, 10 min), substantially elevated the basal levels of cAMP (Supplementary Figure S6). The co-application of the mAChR-selective agonist, oxotremorine-M (10 μM), significantly reduced the forskolin-stimulated cAMP accumulation (Figure 5A), establishing a prevalent inhibition of the cAMP response by mAChRs in striatal neurons. Remarkably, co-adding ionomycin augmented the inhibition of cAMP responses in a concentration-dependent manner (Figure 5A). Oxotremorine-M or ionomycin alone did not alter basal cAMP levels (Figure 5B). Nor did ionomycin affect the forskolin-stimulated cAMP response (Figure 5C). These data demonstrate a Ca2+-dependent potentiation of the inhibitory mAChR linkage to cAMP. The role of CaMKII in this linkage was supported by the following data. KN93 but not KN92 (20 μM) blocked the effect of ionomycin (Figure 5A). Tat–CaMKIINtide, a highly selective and cell-permeable inhibitory peptide of CaMKII (Chang et al, 1998; Vest et al, 2007), produced the same blocking effect when applied at 2 but not 0.2 μM (Figure 5D). To directly determine whether CaMKII acts through its interactions with M4Rs, we evaluated the importance of the CaMKII–M4R association. We observed that the CaMKII–M4R association interfering peptide Tat–M4Ri (2 but not 0.2 μM, 45 min before ionomycin/oxotremorine-M treatment) reversed the effect of ionomycin, whereas Tat–M4Rc did not (Figure 5E). Neither Tat–M4Ri nor Tat–M4Rc significantly altered the forskolin-stimulated cAMP formation or the oxotremorine-M-induced inhibition of cAMP responses to forskolin (data not shown). Thus, Ca2+-activated CaMKII associates with M4Rs, thereby potentiating M4R efficacy in suppressing cAMP production. Figure 5.Regulation of M4R signalling by CaMKII. (A) Ionomycin potentiated the oxotremorine-M-induced inhibition of the forskolin-stimulated cAMP accumulation. (B) Oxotremorine-M (Oxo) or ionomycin (Iono) at 10 μM (10 min) did not alter basal cAMP levels. (C) Ionomycin (10 μM, 10 min) had no effect on the forskolin-stimulated cAMP formation. (D) Tat–CaMKIINtide reversed the effect of ionomycin. (E) Tat–M4Ri but not Tat–M4Rc blocked the effect of ionomycin. (F) MT3 blocked the oxotremorine-M-induced inhibition of the forskolin-stimulated cAMP accumulation. (G) MT3 did not alter the effect of forskolin. (H) Effects of MT3 (0.5 μM, 30 min) on basal cAMP concentrations. (I) Effects of oxotremorine-M on the forskolin-stimulated cAMP accumulation in wild-type (WT) and M4R mutant mice. (J) Ionomycin augmented the effect of oxotremorine-M in HEK293 cells transfected with WT M4Rs but not T145A mutants. Experiments were conducted on striatal slices from rats (A–H) or mice (I) or on HEK293 cells (J). Oxotremorine-M (10 μM), ionomycin (10 μM with 1 mM CaCl2), KN93 (20 μM), and/or KN92 (20 μM) were co-incubated" @default.
• W2014596089 created "2016-06-24" @default.
• W2014596089 creator A5006168167 @default.
• W2014596089 creator A5009297589 @default.
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• W2014596089 date "2010-05-11" @default.
• W2014596089 modified "2023-10-08" @default.
• W2014596089 title "CaMKIIα interacts with M4 muscarinic receptors to control receptor and psychomotor function" @default.
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• W2014596089 doi "https://doi.org/10.1038/emboj.2010.93" @default.
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