HHMI-NIH Research Scholars
Learn about the HHMI-NIH Research Scholars Program, also known as the Cloister Program. More

Janelia Farm Research Campus
Learn about the new HHMI research campus located in Virginia. More

Summary: Edward Ziff's laboratory studies the regulation of AMPA receptors, glutamate-regulated cation channels that are found at excitatory synapses in the central nervous system. The lab has cloned a group of proteins that bind to the cytoplasmic domains of AMPA receptor subunits and is studying their contributions to receptor transport and the regulation of synapse strength.

Our lab studies the mechanisms that underlie the novel ability of the nervous system to modify the strength of signaling between neurons at synapses in response to synaptic activity. The strength of synapses is thought to define functional circuits that connect neurons in the central nervous system. Changes in synaptic strength can modify this circuitry and contribute to nervous system function, including the formation of memory. At excitatory synapses, the major neurotransmitter is glutamate, which is released from the nerve terminal of the presynaptic neuron and stimulates glutamate receptors in the postsynaptic cell. Our lab studies the mechanisms that control the function of glutamate-regulated ion channels at excitatory synapses. Several types of ionotropic glutamate receptors have been identified. One, the NMDA (N-methyl-D-aspartate) receptor, provides synaptic plasticity by inducing changes in synaptic architecture and Ca²⁺-dependent modifications of the synapse. A second, the focus of our lab, the AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazole-proprionate) receptor, is the major source of fast excitatory Na⁺ currents that depolarize the postsynaptic cell.

Models for the mechanism of long-lasting changes in synaptic strength rest on the identification of a class of "silent synapses." Such synapses are initially nontransmitting but may be activated by synaptic stimulation. This activation is thought to result from increases in the number of functional AMPA receptors in the postsynaptic membrane. The mechanisms that control the levels of these receptors in the postsynaptic membrane are thus of great interest. We are studying the control of receptor trafficking. The relative rate of receptor trafficking to and from the synapse is thought to determine receptor synaptic abundance.

	A dendritic shaft of a hippocampal pyramidal cell...

Intracellular domains of glutamate receptors bind to cytoplasmic proteins located in a subsynaptic protein matrix called the postsynaptic density (PSD). NMDA receptors cluster at synapses by binding to a PSD protein scaffold composed of members of the PSD-95 protein family. AMPA receptors also cluster at synapses, but we have determined that AMPA receptor–binding proteins differ from those employed by the NMDA receptor. AMPA receptors are multimers, most likely tetramers, of four subunit types, GluR1–4. Using the yeast two-hybrid approach, we have cloned several proteins that bind the carboxyl-terminal cytoplasmic domain of the GluR2 AMPA receptor subunit. These include AMPA receptor–binding protein (ABP), the GRIP protein (first identified by the lab of Richard Huganir, HHMI, Johns Hopkins University), and PICK1, a protein that also binds protein kinase C-a (PKCa). These three factors bind to the extreme carboxyl terminus of GluR2 via PDZ domains, which are 80– to 90–amino acid structures that bind peptide carboxyl termini.

ABP and GRIP appear to be distant counterparts to the PSD-95 family of proteins that bind NMDA receptors. ABP is expressed in pyramidal cells and interneurons in cortex and hippocampus and also in neurons of the cerebellum and spinal cord. Using Sindbis virus vectors to express wild-type and mutant forms of the GluR2 AMPA receptor subunit in cultured primary neurons, we have shown that binding of GluR2 to ABP anchors GluR2 in the synaptic membrane. This stabilizes GluR2 at the synapse and increases its synaptic abundance. ABP is found at the cytoplasmic face of the postsynaptic membrane of excitatory synapses, where it is juxtaposed to synaptic AMPA receptors. ABP is also found associated with membranes of vesicles in the cytoplasm. We have cloned several splice variant forms of ABP. Two of these variants differ only in their amino-terminal 18 amino acids. One, which is palmitoylated (a lipid modification) on cysteine in the unique 18–amino acid amino-terminal region, is concentrated at synapses. The other variant, which lacks the lipid modification, is located predominantly on vesicles in the cytoplasm. This demonstrates that different forms of an AMPA receptor–anchoring protein can be targeted to different intracellular locations in neurons. We are testing a hypothesis that GluR2 trafficks between these locations through regulated association with synaptic and cytoplasmic ABP anchors. The control of such trafficking is a potential mechanism for regulating receptor synaptic abundance.

The third PDZ protein that binds to GluR2 is the PICK1 protein. PICK1 has a novel PDZ domain that can bind to the carboxyl terminus of PKCa as well as to GluR2. PICK1 can form dimers, a property that may enable PICK1 to juxtapose PKCa to the GluR2 carboxyl-terminal domain. Phosphorylation of GluR2 by PKCa within the PDZ binding site at the subunit carboxyl terminus has been shown to block the ABP-GluR2 interaction. We have shown that upon activation of PKCa, the kinase binds to PICK1, and that the PICK1-PKCa complex is targeted to synapses within dendritic spines. Thus PICK1 may induce the phosphorylation of GluR2 by targeting PKCa to the receptor at synapses, leading to receptor release from its ABP anchor.

The fourth GluR2-binding protein we have cloned is the N-ethylmaleimide fusion protein, NSF. This specialized chaperone is best known for its role in synaptic vesicle recycling. In this role, NSF dissociates SNARE complexes that form during the fusion of synaptic vesicles with the presynaptic nerve terminal membrane. We have shown that NSF makes complexes with GluR2 through sequence-specific contacts at the juxtamembrane region of the GluR2 subunit carboxyl-terminal domain. We have isolated these complexes from rat brain extracts. The complexes were stabilized by nonhydrolyzable ATP analogs and disrupted by ATP hydrolysis. This demonstrated that a component of the cellular membrane trafficking machinery binds directly to GluR2. Recently we have assembled the NSF-GluR2 carboxyl-terminus complex in a fully defined system from purified protein components, and we are analyzing the contributions of NSF to GluR2 trafficking.

The sites of binding of ABP and NSF to GluR2 appear to define two functional regions within the receptor subunit carboxyl-terminal domain. We hypothesize that protein interactions within these two regions of the GluR2 carboxyl terminus contribute to molecular mechanisms that potentiate and depotentiate excitatory synapses. The NMDA receptor has been shown to regulate synaptic strength through inducing changes in AMPA receptor trafficking. One objective of our lab is to determine how signals from the NMDA receptor control functional properties of the AMPA receptor.

A grant from the National Institues of Health supported the work on ABP.

Last updated June 04, 2001