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Neurochem. Int. Vol. 31, No. 5, pp. 651-658, 1997

Pergamon

PII: S0197-0186(97)00023-5

© 1997ElsevierScienceLtd Printed in Great Britain. All rights reserved 0197-0186/'97$17.00+0.00

CRITIQUE PHOSPHORYLATION OF RECEPTORS A N D ION CHANNELS A N D THEIR INTERACTION WITH STRUCTURAL PROTEINS Johannes W. Hell Department of Pharmacology, University of Wisconsin, 3770 MSC, 1300 University Avenue, Madison, WI 53706-1532, U.S.A. (Received for publication 21 March 1997)

INTRODUCTION

control the activity of different ion channels and neurotransmitter receptors. N-methyl-D-aspartate The role of protein tyrosine phosphorylation in the (NMDA)-type glutamate receptors are associated regulation of cell growth and cell cycle as well as cell with the non-receptor PTK Src, which increases the differentiation has been investigated intensively within activity of N M D A receptors by tyrosine phosthe last two decades (Hunter, 1996). Receptor-type phorylation (Yu et al., 1997). Pyk2, another nonprotein tyrosine kinases (RTK) possess an extra- receptor PTK highly expressed in the nervous system, cellular ligand binding domain, a single trans- becomes tyrosine-phosphorylated and thereby actimembrane region and a cytosolic catalytic domain. vated upon Ca 2÷ influx, muscarinic stimulation or They are usually activated by ligand-induced dimer- stimulation of protein kinase C (PKC) by phorbol ization and subsequent tyrosine autophosphorylation. esters (Lev et al., 1995; Siciliano et al., 1996). Pyk2 Specific phosphotyrosine residues bind SH2 or PTP has been shown to contribute to a suppression of domains of various proteins, which participate in the activity of the delayed-rectifier K ÷ channel Kv1.2 different signaling pathways thereby recruiting vari- when coexpressed in oocytes (Lev et al., 1995). In vitro ous components of these pathways to the plasma experiments using tyrosine-phosphorylated, activated membrane (Schlessinger, 1994; Kavanaugh et al., Pyk2 immunoisolated from angiotensin II stimulated 1995; Hunter, 1996). Non-receptor-type protein tyro- rat liver epithelial cells (Earp et al., 1995) and immusine kinases (PTK) do not possess a transmembrane nopurified N M D A receptors did not reveal phosregion, although several non-receptor PTKs are phorylation of N M D A receptors by Pyk2, nor did involved in transmembrane signaling. For example, these experiments indicate that Pyk2 binds to N M D A focal adhesion kinase (Fak) is localized at focal receptors (Leonard and Hell, unpublished results). adhesions, where it is associated with the cytoplasmic However, stimulation of different G protein-coupled tail of integrins which interact through their extra- receptors by lysophosphatidic acid or bradykinin in cellular domains with extracellular matrix proteins the pheochromocytoma cell line PC12 induces both such as fibronectin and with their cytosolic regions tyrosine phosphorylation of Pyk2 and association of with the cytoskeleton. Fak is activated upon integrin Pyk2 with Src, which is stimulated by Pyk2 overbinding to fibronectin and may subsequently phos- expression (Dikic et al., 1996). It is possible that Pyk2 phorylate cytoskeletal proteins such as paxillin (Mere- regulates N M D A receptors in intact neurons by actidith et al., 1996; Otey, 1996). vating Src, although this hypothesis has not been An increasing body of evidence now suggests that tested yet. Regulation of N M D A receptor activity and tyrosine phosphorylation may have functions in of other synaptic functions by PTKs are summarized addition to assembling various signal-transducing in the preceding review article (Gurd, 1997). In the components around PTKs localized in or at the following sections I will focus on a particular aspect plasma membrane and to concomitantly activating of protein phosphorylation with relevance to synaptic signaling cascades. In neurons, for example, PTKs functions, the specific localization of neurotransmitter 651

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receptors and ion channels at postsynaptic sites. I will discuss how this localization might be regulated by phosphorylation and how interaction with structural proteins might regulate the activity of postsynaptic ion channels. THE ACETYLCHOLINE RECEPTOR AT THE NEUROMUSCULAR J U N C T I O N

The RTK prototype is the epidermal growth factor (EGF) receptor, which when activated stimulates cell growth and proliferation through a pathway involving Ras, Raf, MEK, and MAP kinases (Bonfini e/ al., 1996). ErbB2/Neu, ErbB3 and ErbB4, all members of the EGF receptor family, are concentrated at synaptic sites (Zhu et al., 1995) together with their ligands, the neuregulins (Chu et al., 1995; Jo et al., 1995). Neuregulins are secreted proteins of about 45 kDa, which may be associated with the extracellular matrix in the synaptic cleft (Carraway III and Burden, 1995). They are involved in regulating differentiation of the neuromuscular junction. They stimulate the expression of acetylcholine receptors (AChR) and Na + channels as these postsynaptic channels are transcribed in myofiber nuclei located close to the synapse (Corfas and Fischbach, 1993; Carraway III and Burden, 1995; Chu et al., 1995; Jo et al., 1995). MUSK, another RTK, is activated by agrin, a proteoglycan of the synaptic basal lamina which causes AChRs to cluster (Tsim et al., 1992; Denzer et al., 1995; Tsen et al., 1995). MuSK may, therefore, mediate agrin-induced aggregation and coincident tyrosine phosphorylation of AChR (Gillespie et al., 1996; Glass et al., 1996). As mentioned above, phosphorylation of individual tyrosine residues of RTKs or RTK-associated proteins creates docking sites for proteins involved in the downstream signaling of RTKs (Bonfini et al., 1996; Hunter, 1996). It is, therefore, tempting to speculate that phosphorylated, but not unphosphorylated, tyrosine residues of AChR subunits constitute sites for specific interactions with proteins which regulate the subcellular localization of these receptors, possibly by mediating interactions between AChR and the cytoskeleton. In support of such a model are findings that precisely correlate agrin-induced, staurosporine-sensitive tyrosine phosphorylation and clustering of AChR with an increase in the resistance of AChR to detergent extraction (Wallace, 1994). Similarly, the tyrosine-phosphatase inhibitor pervanadate increases tyrosine phosphorylation and decreases detergent solubility and agrin-stimulated clustering with identical time courses, and also diminishes the lateral mobility of

AChRs, possibly by strengthening the interaction of diffusely distributed AChRs and the cytoskeleton (Meier et al., 1995; Wallace, 1995). These findings support a diffusion-trap model predicting that AChR are tyrosine-phosphorylated by relevant kinases which could specifically be localized at postsynaptic sites. AChRs are then trapped in the vicinity of these kinases by phosphorylation-dependent interaction with structural proteins. Such a diffusion-trap model implicates that AChRs are not permanently fixed to postsynaptic structures but can disperse upon dephosphorylation. In fact, removal of agrin from muscle cell cultures causes de-aggregation of AChRs. This disintegration of AChR clusters is inhibited by blocking tyrosine phosphatases with pervanadate (Wallace, 1995) and promoted by inhibiting protein kinases with staurosporin (Wallace, 1994), suggesting that constitutive, ongoing phosphorylation is required for the maintenance of agrin-induced AChR aggregation. The adapter protein Grb2, which is known to bind with its SH2 domain to specific phosphotyrosine residues of various RTKs and with its SH3 domain to other proteins involved in downstream signaling of RTKs, has recently been shown to associate with the tyrosine-phosphorylated but not with non-phosphorylated 6 subunit of AChRs (Colledge and Froehner, 1997). Because Grb2 also associates with fl-dystroglycan (Yang et al., 1995), a protein necessary for the structural integrity of muscle cells and thought to be involved in AChR clustering (Falton and Hall, 1994; Hoch et al., 1994), it is quite possible that Grb2 links tyrosine-phosphorylated AChRs to fl-dystroglycan, fl-dystroglycan possesses a transmembrane region and is associated with extracellular ~-dystroglycan. Both proteins are derived from a common precursor (dystroglycan) by posttranslational proteolyric processing. A 43 kDa protein named rapsyn coclusters AChRs and dystroglycan when all three proteins are co-expressed in fibroblast cell lines (Apel et al., 1995). Rapsyn is, therefore, an alternative candidate linking AChR with the dystroglycane complex. Although ~-dystroglycan binds agrin (Bowe et al., t994; Campanelli et al., 1994; Gee et al., 1994; Sugiyama et al., 1994), it does not appear to be the agrin receptor mediating agrin-induced tyrosine phosphorylation and clustering of AChRs (Meier et al., 1996). Dystroglycan may, therefore, not function as a signaling element but as a structural protein. Accordingly, the interaction between ~-dystroglycan and agrin may contribute to the aggregation of proteins at neuromuscular junctions by direct protein-protein interactions rather than by inducing tyrosine phosphorylation, which depends on MUSK.

Critique It has been reported that tyrosine phosphorylation of AChR has also a more direct functional effect: it increases the rate of a decline in channel openings over a time period of about 1 min, which was interpreted as desensitization (Hopfield et al., 1988). However, the physiological relevance of this effect is unclear and run-down effects of the isolated AChRs have not been ruled out in this study. INTERACTIONS BETWEEN POSTSYNAPTIC ION CHANNELS AND T H E CYTOSKELETON IN T H E BRAIN

As discussed in the previous section for AChRs at neuromuscular junctions, ligand- or voltage-gated ion channels may become clustered at postsynaptic sites through their specific interaction with postsynaptic structural proteins. A single report based on fluorescent photobleach recovery of cultured cortical neurons labeled with a fluorescent ligand for N M D A receptors suggested that N M D A receptors which are not clustered at postsynaptic sites diffuse freely within the plasma membrane (Benke et al., 1993). Similar findings have been reported from this group for the glycine receptor (Srinivasan et aL, 1990). Postsynaptic aggregation of the glycine receptor depends on its interaction with the structural protein gephyrin and on an intact cytoskeleton (Kirsch et aL, 1993; Kirsch and Betz, 1995; Kuhse et aL, 1995). These observations support the idea that neurotransmitter receptors are clustered at postsynaptic sites of central synapses through their interaction with structural proteins. Within the last two years several structural postsynaptic proteins have been shown to interact with N M D A receptors (reviewed by Garner and Kindler, 1996; Gomperts, 1996; Sheng and Kim, 1996). A yeast two-hybrid screen with N M D A receptor sequences as baits indicated that PSD-95 (Cho et aL, 1992) also known as SAP90 (Kistner et al., 1993) interacts with most of the N M D A receptor subunits which are homologous to each other (Kornau et aL, 1995). Other members of the P S D - 9 5 / S A P 9 0 family, collectively known as synapse-associated proteins (SAPs), include SAP97 (MfJller et aL, 1995), SAP102(MtIIIer et al., 1996), and PSD-93/chapsyn-l10 (Brenman et al., 1996; Kim et aL, 1996). The interaction of N M D A receptor subunits and SAPs is mediated by the three amino acids at the very C-terminal end of each N M D A receptor subunit (S/TXV; S/T" serine or threonine; X: any residue; V: valine). SAPs also interact with other ion channels, especially voltage-gated K + channels including Kvl.1, 1.2, 1.3, and 1.4 (Kim et al., 1995) and Kit2.1 and 2.3 (Cohen et al., 1996),

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which possess the same ( K v l . l - 4 ) or a very similar (Kir2.1 and 3: SXI; I: isoleucine) C-terminal motif. Important from a functional point of view is the finding that N M D A receptors and K ÷ channels, when overexpressed alone in heterologous cell lines, are evenly distributed in the plasma membrane, but form clusters when coexpressed with either PSD-95/SAP90 or PSD-93/chapsyn-110 (Kim et al., 1995, 1996; Sheng and Kim, 1996). More recently it has been shown that a-actinin, which interacts with actin in a Ca 2+ dependent manner, binds to the CO domain in the C-terminal tail of N M D A receptor subunit NR1 (Wyszynski et al., 1997). In the presence of Ca 2÷, this interaction was disrupted by calmodulin (Wyszynski et al., 1997), which binds to the C 1 domain and, like ~-actinin, also to the CO domain, thereby inhibiting the activity of N M D A receptors (Ehlers et al., 1996). The integrity of actin filaments is crucial for the functional properties of N M D A receptors (Rosenmund and Westbrook, 1993). Ca 2+, which depolymerizes actin, causes a fast run-down of the N M D A receptor activity. Actin filaments can be stabilized by the alkaloid phaUoidine and by ATP, and both compounds prevented the Ca2+-induced run-down (Rosenmund and Westbrook, 1993). Because calmodulin inhibits N M D A receptors and disrupts the interaction of N M D A receptors with ~-actinin in the presence of Ca 2+, it is conceivable that ~-actinin links N M D A receptors to actin filaments. Accordingly, both Ca 2+dependent direct binding of calmodulin and a disturbance of the actin cytoskeleton may inhibit N M D A receptors. It is quite possible that the binding of a structural protein to N M D A receptors modulates the activity of the latter directly. Such a mechanism would keep N M D A receptors and other ion channels active mainly at sites where their activity is desired, the postsynaptic sites in the case of neurotransmitter receptors. Another ion channel that is specifically clustered at postsynaptic sites is the class C L-type Ca 2+ channel (Hell et al., 1996). Similar to N M D A receptors, the activity of different types of Ca 2+ channels (Johnson and Byerly, 1993, 1994) including class C L-type channels as measured in ventricular cells (Galli and DeFelice, 1994) is decreased when microtubules or actin filaments are depolymerized and stabilized when microtubules or actin filament depolymerization is inhibited. However, interaction with structural proteins could also promote phosphorylation of postsynaptic N M D A receptors or class C L-type Ca 2+ channels which then causes an increase in activity. As discussed above, the N M D A receptor is associated

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with Src and phosphorylation by Src increases its activity (Yu et al., 1997). Tyrosine phosphatases reduce N M D A receptor currents (Wang and Salter, 1994). PTKs also upregulate L-type Ca 2÷ channels in pituitary GH3 cells and this effect is reversed by tyrosine phosphatases (Cataldi et al., 1996). PTKs are, therefore, potential candidates for the upregulation of ion channel activity upon interaction with structural proteins. N M D A receptor subunits are also phosphorylated by second messenger-activated serine/threonine kinases including Ca 2÷ and calmodulin-dependent protein kinase II (CaMKII) (Omkumar et al., 1996), cAMP-dependent protein kinase (PKA) (Leonard and Hell, 1997; Tingley et al., 1997) and PKC (Tingley et al., 1993; Hall and Soderling, 1997; Leonard and Hell, 1997). N M D A receptor responses are potentiated upon PKC activation in trigeminal (Chen and Huang, 1991; Chen and Huang, 1992), spinal cord dorsal horn (Gerber et al., 1989), and hippocampal neurons (Aniksztejn et al., 1992; Wang et al., 1994a,b; but see Markram and Segal, 1992), as well as in oocytes injected with total m R N A (Kelso et al., 1992; Urushihara et al., 1992) or with R N A encoding different subunits of the N M D A receptor (Kutsuwada et al., 1992; Durand et al., 1993; Mori et al., 1993). PKA enhances N M D A receptor activity in spinal dorsal horn neurons (Cerne et al., 1993) and in neuronal microcultures from the hippocampus (Raman et al., 1996). Furthermore, phosphatase 1 and 2A are involved in downregulation of N M D A receptor activity (Lieberman and Mody, 1994; Wang et al., 1994b; Tong et al., 1995). Similarly, the class C L-type Ca 2+ channel is a substrate for PKA, PKC, CaMKII and cGMP-dependent kinase (PKG) in vitro (Hell et al., 1993, 1994) and for P K A and PKC in intact hippocampal neurons (Hell et al., 1995; Hell, unpublished results). The activity of L-type Ca 2÷ channels is increased upon P K A stimulation in GH3 cells (Armstrong and Eckert, 1987), guinea-pig hippocampal neurons (Gray and Johnston, 1987; Fisher and Johnston, 1990), rat nodose ganglion neurons (Gross et al., 1990), chromatiin cells (Artalejo et al., 1990), and rat neostriatal neurons (Surmeier et al., 1995). More specifically, the current through class C L-type channels is significantly potentiated by PKA injection when expressed in CHO cells (Sculptoreanu et al., 1993). PKC increases neuronal L-type Ca 2÷ channel currents in frog sympathetic ganglion neurons (Yang and Tsien, 1993). Taken together the findings summarized in the preceding paragraphs demonstrate that N M D A receptors and class C L-type Ca 2+ channels can be

phosphorylated by PTKs as well as by the major arginine/lysine-directed serine/threonine kinases. Several of these phosphorylation sites are tonically phosphorylated as predicted by a model that assumes that the activity of postsynaptic ion channels is constitutively upregulated upon binding to cytoskeletal proteins. Tyrosine residues are phosphorylated tonically on N M D A receptors (Moon et al., 1994; Wang and Salter, 1994) and on L-type Ca 2+ channels (Cataldi et al., 1996). Serine/threonine residues corresponding to PKA, PKC or C a M K I I phosphorylation sites are constitutively phosphorylated on N M D A receptors (Omkumar et al., 1996; Raman et al., 1996; Leonard and Hell, 1997; Tingley et al., 1997), and on class C L-type Ca 2+ channels (Hell, unpublished results; see also De Jongh et al., 1996). In this context it is interesting that protein-protein interactions between P K A or PKC with proteins known as A-kinase anchoring proteins (AKAPs) are necessary for the tonic upregclation of AMPA-type glutamate receptors by constitutive phosphorylation by P K A (Rosenmund et al., 1994). Similar to N M D A receptors, the activity of A M P A receptors is upregulated in hippocampal cells by PKA (Greengard et al., 1991; Blackstone et al., 1994) as well as by PKC (Wang et al., 1994a) and C a M K I I (McGlade-McCulloh et al., 1993). Because interactions between P K A and AKAPs in muscle cells is also important for PKA-mediated potentiation of skeletal muscle L-type Ca 2÷ channels (Johnson et al., 1994, 1997), it is possible that P K A is localized next to postsynaptic class C L-type Ca 2÷ channels by a postsynaptic AKAP. A K A P 79 and A K A P 150 are potential candidates as they are associated with postsynaptic structures ( C a r r e t al., 1992). Interestingly, A K A P 79 can also bind to PKC (Klauck et al., 1996) and to the Ca 2+ dependent phosphatase 2B or calcineurin (Coghlan et al., 1995), thereby serving as a scaffold protein for these enzymes at locations crucial for signaling through serine/threonine phosphorylation (Faux and Scott, 1996). Finally, phosphorylation of some sites of the ion channels discussed above may disrupt rather than foster the interaction with structural proteins such as the SAPs. Accordingly, it has been shown recently that PKA-mediated phosphorylation of the K + channel Kit2.3 prevents this channel from binding to PSD95/SAP90 in vitro (Cohen et al., 1996). Such a disruption may be necessary to allow reconstruction of synapse when they undergo long-lasting plastic changes such as long-term potentiation (LTP) (Bear and Malenka, 1994), which requires the activity of serine/threonine protein kinases (Madison et aL, 1991) and of PTKs (Grant et al., 1992).

Critique CONCLUSION Phosphorylation of specific amino acids of postsynaptic ion channels such as A C h R , N M D A receptors or class C L-type Ca 2÷ channels may control both the subcellular localization and the activity of these ion channels. Conversely, binding to structural proteins may change the activity o f ion channels either directly or indirectly by inducing further phosphorylation by P T K s or serine/threonine kinases. D a t a supporting these models are very limited. However, methods to study the interactions between postsynaptic ion channels and structural proteins are now available and the proposed models will be tested within the next few years. Acknowledgements--The author would like to thank Dr

Peter Lipton, University of Wisconsin at Madison, for critically reading the manuscript and Dr Stanley C. Froehner, University of North Carolina at Chapel Hill, for sharing unpublished results. This work was supported in part by Faculty Scholar Award FSA-94-033 from the Alzeimer's Association. REFERENCES

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