Rapid modulation of hippocampal synaptic plasticity by estrogen has long been a hot topic, but analysis of molecular mechanisms via synaptic estrogen receptors has been seriously difficult. Here, two types of independent synaptic plasticity, long-term depression (LTD) and spinogenesis, were investigated, in response to 17b-estradiol and agonists of estrogen receptors using hippocampal slices from adult male rats. Multi-electrode investigations demonstrated that estradiol rapidly enhanced LTD not only in CA1 but also in CA3 and dentate gyrus. Dendritic spine morphology analysis demonstrated that the density of thin type spines was selectively increased in CA1 pyramidal neurons within 2 h after application of 1 nM estradiol. This enhancement of spinogenesis was completely suppressed by mitogen-activated protein (MAP) kinase inhibitor. Only the estrogen receptor (ER) alpha agonist, (propyl-pyrazole-trinyl)tris-phenol (PPT), induced the same enhancing effect as estradiol on both LTD and spinogenesis in the CA1. The ERbeta agonist, (4-hydroxyphenyl)-propionitrile (DPN), suppressed LTD and did not affect spinogenesis. Because the mode of synaptic modulations by estradiol was mostly the same as that by the

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Receptor tyrosine kinases (RTKs) control a host of biological functions by phosphorylating tyrosine residues of intracellular proteins upon extracellular ligand binding. The phosphotyrosines (p-Tyr) then recruit a subset of ∼100 Src homology 2 (SH2) domaincontaining proteins to the cell membrane. The in vivo kinetics of this process are not well understood. Here we use total internal reflection (TIR) microscopy and single-molecule imaging to monitor interactions between SH2 modules and p-Tyr sites near the cell membrane. We found that the dwell time of SH2 modules within the TIR illumination field is significantly longer than predictions based on chemical dissociation rate constants, suggesting that SH2 modules quickly rebind to nearby p-Tyr sites after dissociation. We also found that, consistent with the rebinding model, the effective diffusion constant is negatively correlated with the respective dwell time for different SH2 domains and the dwell time is positively correlated with the local density of RTK phosphorylation. These results suggest a mechanism whereby signal output can be regulated through the spatial organization of multiple binding sites, which will prompt reevaluation of many aspects of RTK signaling, such as signaling specificity, mechanisms of spatial control, and noise suppression.reaction | epidermal growth factor receptor | diffusion-limited dissociation T he Src homology 2 (SH2) domain (1) controls many cellular activities by binding specifically to tyrosine-phosphorylated peptides (2). The tyrosine phosphorylation signal often originates from the cell surface through activations of receptor tyrosine kinases (RTKs). Humans, for example, have 58 known RTKs (3), each of which could generate diverse phosphorylation patterns at their cytosolic tails as well as at other associated proteins. These phosphorylation patterns are "read" mainly by a repertoire of effector proteins containing the SH2 domain (2, 4, 5), via recruitment of these effector molecules to the phosphotyrosine (pTyr) sites. For example, the epidermal growth factor receptor (EGFR), a well-known model system for studying phosphotyrosine signaling, is activated by epidermal growth factor (EGF), a small protein ligand. The binding of EGF turns on the kinase activity of EGFR molecules, which then autophosphorylate themselves at more than 20 tyrosine sites on the cytoplasmic tail. The phosphotyrosines are thought to serve as docking sites for SH2-containing molecules, such as Grb2, which bind to these sites with varying affinity. Downstream signals are propagated by the SH2 proteins, which in many cases are enzymes whose substrates are localized to the plasma membrane. In other cases such as Grb2, the SH2 protein serves to recruit additional downstream effector proteins such as the Ras activator Sos. EGFR family members also play critical roles in cancer development and progression and are frequently disregulated in tumors (6).During the last decade, an expanding assembly of quantitative datasets has accumulated regarding tyrosine p...