Homer is a neuronal immediate early gene (IEG) that is enriched at excitatory synapses and binds group 1 metabotropic glutamate receptors (mGluRs). Here, we characterize a family of Homer-related proteins derived from three distinct genes. Like Homer IEG (now termed Homer 1a), all new members bind group 1 mGluRs. In contrast to Homer 1a, new members are constitutively expressed and encode a C-terminal coiled-coil (CC) domain that mediates self-multimerization. CC-Homers form natural complexes that cross-link mGluRs and are enriched at the postsynaptic density. Homer 1a does not multimerize and blocks the association of mGluRs with CC-Homer complexes. These observations support a model in which the dynamic expression of Homer 1a competes with constitutively expressed CC-Homers to modify synaptic mGluR properties.

Excitatory amino-acid carrier 1 (EAAC1) is a high-affinity Na+-dependent L-glutamate/D,L-aspartate cell-membrane transport protein. It is expressed in brain as well as several non-nervous tissues. In brain, EAAC1 is the primary neuronal glutamate transporter. It has a polarized distribution in cells and mainly functions perisynaptically to transport glutamate from the extracellular environment. In the kidney it is involved in renal acidic amino-acid re-absorption and amino-acid metabolism. Here we describe the identification and characterization of an EAAC1-associated protein, GTRAP3-18. Like EAAC1, GTRAP3-18 is expressed in numerous tissues. It localizes to the cell membrane and cytoplasm, and specifically interacts with carboxy-terminal intracellular domain of EAAC1. Increasing the expression of GTRAP3-18 in cells reduces EAAC1-mediated glutamate transport by lowering substrate affinity. The expression of GTRAP3-18 can be upregulated by retinoic acid, which results in a specific reduction of EAAC1-mediated glutamate transport. These studies show that glutamate transport proteins can be regulated potently and that GTRAP can modulate the transport functions ascribed to EAAC1. GTRAP3-18 may be important in regulating the metabolic function of EAAC1.

Exocytosis of synaptic vesicles is calcium-dependent, with synaptotagmin serving as the calcium sensor. Endocytosis of synaptic vesicles has also been postulated as a calcium-dependent process; however, an endocytic calcium sensor has not been found. We now report a physical association between the calcium-dependent phosphatase calcineurin and dynamin 1, a component of the synaptic endocytic machinery. The calcineurin-dynamin 1 interaction is calcium-dependent, with an EC 50 for calcium in the range of 0.1-0.4 M. Disruption of the calcineurin-dynamin 1 interaction inhibits clathrin-mediated endocytosis. Thus, the calcium-dependent formation of the calcineurin-dynamin 1 complex, delivered to the other endocytic coat proteins, provides a calciumsensing mechanism that facilitates endocytosis.Neurotransmitter release occurs by calcium-dependent exocytosis of synaptic vesicles from nerve terminals mediated by a complex of proteins whose interactions are calcium-dependent (1, 2). Endocytic recycling of released synaptic vesicles is initiated at the same time as exocytosis and involves a complex of endocytic proteins including clathrin, clathrin adapters, dynamin 1, amphiphysin, and synaptojanin (3-5). Dephosphorylation of endocytic proteins is required for their assembly into a functional complex (6). A possible link between calcium and dephosphorylation is suggested by evidence that the calciumsensitive phosphatase calcineurin (Cn) 1 can dephosphorylate endocytic proteins (7-9) and that the drugs cyclosporin A and FK506, which inhibit Cn, impair endocytosis (10, 11). However, the evidence for calcium-dependence in endocytosis has been conflicting (11-17). We now provide direct evidence that Cn is physically linked to the endocytic machinery. We show that Cn binds to dynamin 1 independent of its catalytic activity and that the Cn-dynamin 1 complex combines with amphiphysin 1, the anchor protein of the endocytic complex. Moreover, we demonstrate that the Cn-dynamin 1 interaction is calcium-dependent, allowing this complex to act as a calcium sensor. Finally, we show that disruption of the Cn-dynamin 1 interaction leads to inhibition of clathrin-mediated endocytosis. EXPERIMENTAL PROCEDURESAffinity Purification of the Cn-Dynamin 1 Complex-Adult rat brain was homogenized in lysis buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM CaCl 2 , 2 mM MgCl 2 , 0.2% Triton X-100, 0.5 mM ␤-mercaptoethanol, 5 g/ml aprotinin, 1 g/ml leupeptin, 6 g/ml chymostatin, 0.7 g/ml pepstatin, 1 mM PMSF) and centrifuged at 20,000 ϫ g for 20 min to remove insoluble materials. The resulting lysate was divided into equal aliquots and incubated with GST-FKBP12-Sepharose beads for 2 h at 4°C. Where indicated, GST-FKBP12-Sepharose beads were preabsorbed with FK506 for 1 h at 4°C and then washed twice in lysis buffer prior to adding to the cell lysate. Following the incubation, the Sepharose beads were washed three times with lysis buffer and boiled in SDS sample buffer. Eluted proteins were separated by SDS-PAGE and stained with Coomassie Blue or tra...