The PSD-95/SAP90 family of scaffold proteins organizes the postsynaptic density (PSD) and regulates NMDA receptor signaling at excitatory synapses. We report that SPAR, a Rap-specific GTPase-activating protein (RapGAP), interacts with the guanylate kinase-like domain of PSD-95 and forms a complex with PSD-95 and NMDA receptors in brain. In heterologous cells, SPAR reorganizes the actin cytoskeleton and recruits PSD-95 to F-actin. In hippocampal neurons, SPAR localizes to dendritic spines and causes enlargement of spine heads, many of which adopt an irregular appearance with putative multiple synapses. Dominant negative SPAR constructs cause narrowing and elongation of spines. The effects of SPAR on spine morphology depend on the RapGAP and actin-interacting domains, implicating Rap signaling in the regulation of postsynaptic structure.

Developmental changes in the signaling properties of NMDA receptors have been proposed to underlie the loss of plasticity that accompanies brain maturation. Calcium influx through postsynaptic NMDA receptors can stimulate neuronal gene expression via signaling pathways such as the Ras-MAP kinase (MAPK) pathway and the transcription factor cAMP response element-binding protein (CREB). We analyzed MAPK (Erk1/2) and CREB activation in response to NMDA receptor stimulation during the development of hippocampal neurons in culture. At all stages of development NMDA stimulation induced a rapid phosphorylation of CREB on Ser-133 (phospho-CREB). However, the time course of decline in phospho-CREB changed dramatically with neuronal maturation. At 7 d in vitro (7 DIV) phospho-CREB remained elevated 2 hr after strong NMDA stimulation, whereas at 14 DIV phospho-CREB rose only transiently and fell back to below basal levels within 30 min. Moreover, at 14 DIV, but not at 7 DIV, NMDA receptor stimulation induced a dephosphorylation of CREB that previously had been phosphorylated by KCl depolarization or forskolin, suggesting an NMDA receptor-dependent activation of a CREB phosphatase. There was no developmental change in the time course of phospho-CREB induction that followed KCl depolarization or PKA activation, nor was there a developmental change in the time course of phospho-Erk1/2 induced by NMDA receptor activation. We suggest that, during neuronal maturation, NMDA receptor activation becomes linked specifically to protein phosphatases that act on Ser-133 of CREB. Such a developmentally regulated switch in the mode of NMDA receptor coupling to intracellular signaling pathways may contribute to the changes in neural plasticity observed during brain development.