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CREB transcription activity. This implies that induction of I-1* permits a step involved in formation of long-term memory to proceed in short-interval-trained mice that would normally only occur in long-interval-trained mice. However, it seems unlikely that new gene expression contributes to enhancing memory only 5 min after training. In addition, because CREB activation appeared to be widespread across the cortex, it would be informative to know which of these brain areas are relevant for long-term memory in the object recognition task employed. I-1* transgenic mice apparently remember for longer Genoux et al. [7] also tested I-1* transgenic mice in the Morris water maze, in which mice learn the location of an escape platform in a tank of opaque water, aided by spatial cues [13]. I-1* transgenic mice learned faster than control mice, needing fewer training trials before they reached their quickest escape time, consistent with their enhanced memory for objects. This improved performance in I-1* transgenic mice correlated with a modest increase in the phosphorylation of CaMKII and the GluR1 subunit of the AMPA-type glutamate receptor – two factors more likely to be involved in the immediate effects of training. There was no difference between I-1*-transgenic and control mice when they were trained repetitively with a spaced protocol in the water maze. However, there was a startling effect on the persistence of memory. I-1* mice displayed a transgene-expression-dependent reduction in memory decay – that is, they remember for longer or forget less. This reduced decay was also evident if I-1* was induced only after training, suggesting that PP1 plays a role in maintenance of memory. However, because the memory decay is monitored by testing the same mice over and over again for the platform location (without it being there) it is also plausible that I-1* transgenic mice are impaired in extinction of the spatial memory. Put differently, perhaps they are unable to learn that the platform is no longer where it used to be. Older animals were also tested for water-maze learning and memory. Aged I-1* mice learned slightly faster than similar-age-control mice. Furthermore, aged control mice had no apparent memory for the platform location one week after training, whereas aged I-1* mice remembered for a month. The authors suggested that this is further evidence that PP1 promotes forgetting. If this role of PP1

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were normally relevant to memory decline, aged control mice would be expected to exhibit higher PP1 activity. The authors mention that I-1 might affect PP1 independent processes, which is certainly a caveat of the study. Concluding that all observed effects are PP1dependent is a small leap of faith. Nevertheless, the results provide good evidence for a role of I-1 itself in memory, which is supported by previous data showing that PP1 regulates LTD [14]. It will be interesting to determine whether improved memory of I-1* transgenic mice also correlates with altered neuronal plasticity. Perhaps physiology will further enlighten us as to why practice makes perfect. References 1 Spear, N.E. (1978) The Processing of Memories: Forgetting and Retention, Erlbaum 2 Dash, P.K. (1990) Injection of the cAMP-responsive element into the nucleus of Aplysia sensory neurons blocks long-term facilitation. Nature 345, 718 – 721 3 Tully, T. et al. (1994) Genetic dissection of consolidated memory in Drosophila. Cell 79, 35– 47 4 Muller, U. (2000) Prolonged activation of cAMP-dependent protein kinase during conditioning induces long-term memory in honeybees. Neuron 27, 159 – 168 5 Mayford, M. and Kandel, E.R. (1999) Genetic approaches to memory storage. Trends Genet. 15, 463– 470 6 McGaugh, J.L. (2000) Memory – a century of consolidation. Science 287, 248 – 251 7 Genoux, D. et al. (2002) Protein phosphatase 1 is a molecular constraint on learning and memory. Nature 418, 970– 975 8 Malleret, G. et al. (2001) Inducible and reversible enhancement of learning, memory, and long-term potentiation by genetic inhibition of calcineurin. Cell 104, 675 – 686 9 Oliver, C.J. and Shenolikar, S. (1998) Physiologic importance of protein phosphatase inhibitors. Front Biosci. 3, D961 – D972 10 Zeng, H. et al. (2001) Forebrain-specific calcineurin knockout selectively impairs bidirectional synaptic plasticity and working/ episodic-like memory. Cell 107, 617– 629 11 Save, E. et al. (1992) Object exploration and reactions to spatial and nonspatial changes in hooded rats following damage to parietal cortex or hippocampal formation. Behav. Neurosci. 106, 447 – 456 12 Impey, S. et al. (1996) Induction of CRE-mediated gene expression by stimuli that generate long-lasting LTP in area CA1 of the hippocampus. Neuron 16, 973– 982 13 Morris, R.G. et al. (1982) Place navigation impaired in rats with hippocampal lesions. Nature 297, 681– 683 14 Morishita, W. et al. (2001) Regulation of synaptic strength by protein phosphatase 1. Neuron 32, 1133– 1148 0166-2236/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0166-2236(03)00029-8

Receptor tyrosine kinase transactivation: fine-tuning synaptic transmission Stephen S.G. Ferguson Cell Biology Research Group, Robarts Research Institute, Department of Physiology and Pharmacology, University of Western Ontario, 100 Perth Drive, PO Box 5015, London, Ontario, Canada, N6A 5K8

G-protein-coupled receptors generate signals that promote gene transcription through the ‘transactivation’ of Corresponding author: Stephen S.G. Ferguson ([email protected]). http://tins.trends.com

receptor tyrosine kinases (RTKs) and activation of the mitogen-activated protein kinase (MAPK) cascade – a process that involves RTK autophosphorylation and endocytosis. Pioneering work now suggests that

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D4-dopamine-receptor-mediated transactivation of the platelet-derived growth factor b receptor has immediate effects on synaptic neurotransmission via Ca21-dependent inactivation of NMDA receptors. The demonstration of a physiological role for RTK transactivation in the CNS provides novel opportunities for understanding how aberrant dopamine signalling might contribute to cognitive and attention deficits associated with schizophrenia and attentiondeficit hyperactivity disorder. G-protein-coupled receptors (GPCRs) constitute the largest family of integral membrane proteins and are found in organisms ranging from slime mould and yeast to mammals. GPCRs transduce information provided by extracellular signals (e.g. neurotransmitters) to the cell interior by coupling to heterotrimeric guanine-nucleotide binding proteins (i.e. G proteins) [1]. In turn, G proteins positively and/or negatively regulate the activity of effector enzymes and ion channels [1]. The role of GPCR-stimulated second messenger signals in modulating synaptic activity and neurotransmission in the CNS is well established. However, it is now recognized that, in addition to classical second messenger-regulated mechanisms, GPCRs activate growth factor signalling cascades via additional mechanisms involving focal adhesion complexes, intermediary scaffolding proteins (e.g. b-arrestins) and the transactivation of receptor tyrosine kinases (RTKs) [2– 9]. Focal adhesion complexes and synaptic transmission One mechanism by which GPCRs modulate NMDA receptor signalling is via focal adhesion complexes. Focal adhesions are points of contact between cells and the extracellular matrix that can be utilized as scaffolds for the assembly of protein complexes required for GPCR-dependent activation of the Ras – mitogenactivated-protein kinase (MAPK) signalling cascade [10]. GPCR activation in fibroblasts leads to the rapid tyrosine phosphorylation and activation of p125 focal adhesion kinase (FAK), a process that is inhibited by the disruption of the actin cytoskeleton [11]. In neurons, activation of the FAK family kinase that is regulated by Ca2þ and protein kinase C (PKC), cell adhesion kinaseb/proline-rich tyrosine kinase (CAKb/Pyk2), provides a link between GPCR signaling and MAPK activation [12]. The activation of CAKb/Pyk2 in neurons involves the convergence of cell adhesion with Ca2þ and PKC signals [11,12]. The Ca2þ- and PKC-dependent autophosphorylation of CAKb/Pyk2 creates an SH2 ligand that binds to the SH2 domain of Src, thereby relieving Src autoinhibition and leading to activation of the MAPK cascade [13]. In CA1 hippocampal neurons, the activation of CAKb/Pyk2 results in Src-dependent upregulation of NMDA receptor activity, indicating that GPCR signalling via focal adhesion complexes could function to increase synaptic excitability [14]. RTK transactivation and GPCR signaling GPCRs and RTKs regulate many of the same signals and molecular intermediates involved in the MAPK signalling http://tins.trends.com

cascade and this convergence involves the transactivation of RTKs by GPCRs [3,7 – 9]. It is now appreciated that the receptors for epidermal growth factor (EGF), plateletderived growth factor (PDGF), brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) can be transactivated in response to GPCR activation [3,9,15,16]. The transactivation of RTKs by GPCRs appears to involve RTK autophosphorylation, recruitment of non-receptor tyrosine kinases and RTK endocytosis [7,15 – 21]. The bestcharacterized mechanisms by which GPCRs transactivate EGF receptors involve the autocrine and/or paracrine release of soluble cell-surface heparin-binding (HB) EGF [7]. Although the effectors mediating activation of ADAM-family metalloproteinases remain undefined, Src-family kinases and phosphatidylinositol-30 -kinases (PI3-kinase) are thought to be required for EGF receptor transactivation [19– 21]. Pierce and colleagues [21] utilized an elegant co-culture system to demonstrate that a2A-adrenoceptor-mediated activation of the MAPK cascade requires Gbg subunits upstream of HB-EGF shedding, and requires the dynamin-dependent endocytosis of the EGF receptor downstream of HB-EGF shedding. By contrast, Src activity was required for both HB-EGF shedding and downstream activation of ERK by activated EGF receptors. Additional intracellular mechanisms might also contribute to the transactivation of RTKs by GPCRs, because transactivation of the EGF receptor by 5HT2A receptors is not affected by metalloproteinase inhibitors and no ectodomain-shedding model for PDGFb receptor activation has been described [3,22]. It has recently been suggested that the high-affinity NGF receptor, Trk A, and the PDGF receptor form tethered complexes with GPCRs that contain both G protein-coupled receptor kinase 2 (GRK2) and b-arrestin1 [23,24]. b-Arrestins are multifunctional proteins that contribute to the regulation of GPCR desensitization, endocytosis and signalling [6,25 –28]. They not only function as molecular scaffolds, coupling desensitized GPCRs to activation of the MAPK cascade, but also contribute to GPCR-dependent reorganization of the actin cytoskeleton by regulating the activation of small G proteins [26– 28]. b-Arrestins regulate GPCR-dependent MAPK signalling via their interaction with multiple components of the MAPK signalling pathway, including Src, Raf, MAPK and extracellular-signal-regulated kinases (ERKs) [6,17,27,29]. Owing to their role as endocytic adaptor proteins, b-arrestins regulate not only the formation of signal transduction complexes, but also the intracellular compartmentalization of these complexes. Consequently, GPCRs that internalize as a stable complex with b-arrestins direct the redistribution of components of the MAPK cascade to endosomes, rather than facilitating the activation and translocation of ERK1/2 into the nucleus [29]. Thus, in addition to regulating gene transcription associated with cell proliferation and differentiation, GPCR signalling via the MAPK cascade might also regulate dynamic cellular processes, such as neutrophil granule release [30]. Despite the fact that b-arrestins are concentrated at synapses [31] and, as such, are ideally localized to modulate synaptic activity, the role

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of b-arrestin-mediated signalling at the synapse has not yet been examined. In the case of the Trk A receptor, NGF stimulated recruitment of b-arrestin1 appears necessary for Trk A endocytosis, MAPK signalling and differentiation of PC12 cells [23]. Thus, depending upon the cell type being studied, cell proliferation and differentiation could involve the coordinated convergence of b-arrestin-mediated and RTK-transactivation-mediated activation of MAPK signalling by GPCRs. This could be particularly relevant to the transactivation of PDGFb receptors by D4 dopamine receptors [32]. RTK transactivation and synaptic transmission RTK transactivation by GPCRs leading to the activation of MAPK signalling is thought to regulate cell proliferation and differentiation, rather than short-term regulatory events [3]. This is what is unique and exciting about the recent study published in the journal Neuron by Kotecha and co-workers [32]. In their study, Kotecha et al. [32] demonstrate that D4 dopamine receptor transactivation of the PDGFb receptor depresses excitatory neurotransmission mediated by the NMDA subtype of glutamate receptor. It is well appreciated that PDGF has neurotrophic effects on both GABAergic and dopaminergic neurons in the CNS and that PDGF has long-lasting effects on NMDA-receptor-mediated synaptic transmission in the hippocampus [33,34]. Whereas EGF-receptor and BDNF-receptor activation leads to enhanced synaptic transmission, activation of PDGFb receptors depresses NMDA receptor activity [23,33 – 35]. However, until now, the molecular mechanism(s) underlying PDGF-mediated

depression of NMDA receptors had not been delineated. Kotecha and colleagues [32] show that the selective activation of D4 dopamine receptors, both in acutely dissociated CA1 neurons and in the CA1 region of the hippocampus but in not cultured neurons, results in the depression of NMDA currents. In heterologous cell systems, D4 dopamine receptors transactivate PDGFb receptors [36] and, consistent with this observation, PDGF tyrosine kinase inhibitors (WIN41662 and tryphostin A9) added to the patch pipette block both PDGFand quinpirole-induced depression of NMDA currents. Furthermore, quinpirole treatment induces the internalization of PDGFb receptors in acutely dissociated cultures, and both PDGFb receptor phosphorylation and MAPK activation in CA1 region hippocampal slices [32]. The autophosphorylation of tyrosine residues on the PDGFb receptor provides intracellular docking sites for the recruitment and activation of effector enzymes such as phospholipase Cg (PLCg), PI3-kinase and Src [37]. Accordingly, Kotecha et al. [32] report that D4 dopamine receptor transactivation of the PDGFb receptor stimulates PLCg-dependent release of intracellular Ca2þ stores to inactivate NMDA receptors, as well as resulting in ERK1/2 activation and phosphorylation of the MAPK substrate Elk-1 (Fig.1). The Ca2þ-dependent inactivation of NMDA currents is dependent upon both calmodulin activity and the polymerization-state of filamentous actin, but is independent of PI3-kinase, calmodulin kinase II and calcineurin activity (Fig. 1). The study by Kotecha and colleagues [32] provides convincing evidence that RTK transactivation might not only contribute to the survival and differentiation of neurons, but also dynamically and acutely modulate synaptic transmission. Although the mechanism(s) by

PDGFβ receptor D4 Dopamine receptor

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Fig. 1. D4 dopamine receptor transactivation of the platelet-derived growth factor (PDGF) b receptor leads to the Ca2þ and calmodulin-dependent inactivation of NMDA receptors and mitogen-activated protein kinase (MAPK) signalling. The activation of D4 dopamine receptors in CA1 hippocampal neurons results in the pertussis toxin- and G-protein bg-subunit-dependent autophosphorylation of the PDGFb receptor on tyrosine residues (Y). PDGFb receptor autophosphorylation leads to the recruitment of phospholipase C-g (PLCg), but not phosphatidylinositol-30 -kinases (PI3-K). PLCg-dependent increases in intracellular levels of inositol (1,4,5)-triphosphate (IP3) results in the IP3 receptor (IP3R)-mediated release of intracellular Ca2þ stores from the endoplasmic reticulum (ER). The Ca2þ-dependent inactivation of NMDA currents is dependent upon both calmodulin (CaM) and the state of polymerization of filamentous actin. Dopamine receptor-mediated transactivation of the PDGFb receptor also results in ERK1/2 activation and the phosphorylation of the MAPK substrate Elk-1. The molecular determinants leading to synaptic MAPK signalling remain undetermined but could be dependent upon either the recruitment of complexes of b-arrestin (bArr) and Src, or on other components of the MAPK signalling pathway. http://tins.trends.com

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which dopamine receptors transactivate PDGF receptors remains to be determined, these findings highlight the under-appreciated but pivotal role for RTKs in regulating the communication between two major neurotransmitter systems in the CNS. D4 dopamine receptors and reduced glutamate-mediated signalling have been implicated in neurological disorders that affect cognition and attention, such as schizophrenia and attention-deficit hyperactive disorder. Thus, RTK transactivation could provide a novel mechanism explaining how D2/D4 receptors might modulate attention and cognition via NMDA receptors.

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Acknowledgements I am the recipient of a Heart and Stroke Foundation of Canada MacDonald Scholarship, Premier’s Research Excellence Award and Canada Research Chair in Molecular Neuroscience. This work was supported by CIHR grant MA-15506.

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