The basis for differences in activity-dependent trafficking of AMPA receptors (AMPARs) and NMDA receptors (NMDARs) remains unclear. Using single-molecule tracking, we found different lateral mobilities for AMPARs and NMDARs: changes in neuronal activity modified AMPAR but not NMDAR mobility, whereas protein kinase C activation modified both. Differences in mobility were mainly detected for extrasynaptic AMPARs, suggesting that receptor diffusion between synaptic and extrasynaptic domains is involved in plasticity processes.

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Many integrin adhesion receptors bind ligands containing the Arg-Gly-Asp (RGD) peptide motif. Most integrins exhibit considerable specificity for particular ligands and can distinguish among the many conformations of RGD. In this study we identify the domain of the integrin ␤ subunit involved in determining ligand binding specificity. Chimeras of ␤3 and ␤5, the most homologous integrin ␤ subunits, were expressed with ␣v on the surface of human 293 cells. The ligand binding phenotype of each chimera was assessed using the ligands Fab-9 and fibrinogen, both of which have a binding preference for ␣v␤3. The results of the study show that when exons C and D of the ␤3 subunit (residues 95-233) are substituted into ␤5, the chimera gained the ability to bind Fab-9 with an affinity close to that of wild-type ␣v␤3. This chimera was able to mediate cell adhesion to fibrinogen. Furthermore, the swap of only a 39-residue segment of this larger domain, ␤3 residues 164 -202, into the backbone of ␤5 enabled the chimeric integrin to bind soluble Fab-9. This small domain is highly divergent among the integrin ␤ subunits, suggesting that it may play a role in determining ligand selection by all integrins.Integrins are transmembrane ␣␤ heterodimers that are responsible for most of the physical contacts between cells and the extracellular matrix. Integrins are involved in a number of tissue remodeling events including embryogenesis, angiogenesis, wound repair, and bone resorption (1-5). Integrins are also causally linked to pathological conditions such as tumor progression, thrombosis, inflammation, and osteoporosis (6 -8).The integrin protein family contains 13 ␣ and 9 ␤ subunits. These subunits can cross-pair to form heterodimers with different expression patterns and distinct ligand binding profiles (9, 10). Each integrin binds to only a limited series of ligands, ensuring that cell adhesion and migration are precisely regulated. Such precision is key to tissue remodeling.Based on their ligand recognition specificity, integrins can be divided into two broad sub-families. About one-half of the integrins bind to ligands that lack any consensus binding motif.The remaining integrins bind to the RGD 1 consensus sequence. Importantly though, even the integrins that bind to RGD display a great deal of specificity for matrix ligands (11,12). Some of this specificity is determined by contacts that are ancillary to the RGD sequence (13,14), but much of the specificity is determined by the shape of the RGD motif and the residues in its immediate vicinity. Thus, to understand the structural basis of matrix selection, it is important to understand how integrins distinguish the many conformations of RGD.The goal of the present study was to identify domain(s) within the integrin ␤ subunit that confer ligand binding specificity. As a model system, we compared the ligand binding domains of the two most homologous integrins, ␣v␤3 and ␣v␤5. The ␤3 and ␤5 subunits have 56% identity at the amino acid level (15, 16). Although both ␣v␤3 and ␣v␤5 recogn...

The plasma membrane expression of the rat brain calcium channel subunits alpha1A, alpha2-delta and the beta subunits beta1b, beta2a, beta3b and beta4 was examined by transient expression in COS-7 cells. Neither alpha1A nor alpha2-delta localized to the plasma membrane, either alone or when coexpressed. However, coexpression of alpha1A or alpha2-delta/alpha1A with any of the beta subunits caused alpha1A and alpha2 to be targetted to the plasma membrane. The alpha1A antibody is directed against an exofacial epitope at the mouth of the pore, which is not exposed unless cells are depolarized, both for native alpha1A channels in dorsal root ganglion neurons and for alpha1A expressed with a beta subunit. This subsidiary result provides evidence that either channel opening or inactivation causes a conformational change at the mouth of the pore of alpha1A. Immunostaining for alpha1A was obtained in depolarized non-permeabilized cells, indicating correct orientation in the membrane only when it was coexpressed with a beta subunit. In contrast, beta1b and beta2a were associated with the plasma membrane when expressed alone. However, this is not a prerequisite to target alpha1A to the membrane since beta3 and beta4 alone showed no differential localization, but did direct the translocation of alpha1A to the plasma membrane, suggesting a chaperone role for the beta subunits.

In neurons, the proper distribution of mitochondria is essential because of a requirement for high energy and calcium buffering during synaptic neurotransmission. The efficient, regulated transport of mitochondria along axons to synapses is therefore crucial for maintaining function. The trafficking kinesin protein (TRAK)/Milton family of proteins comprises kinesin adaptors that have been implicated in the neuronal trafficking of mitochondria via their association with the mitochondrial protein Miro and kinesin motors. In this study, we used gene silencing by targeted shRNAi and dominant negative approaches in conjunction with live imaging to investigate the contribution of endogenous TRAKs, TRAK1 and TRAK2, to the transport of mitochondria in axons of hippocampal pyramidal neurons. We report that both strategies resulted in impairing mitochondrial mobility in axonal processes. Differences were apparent in terms of the contribution of TRAK1 and TRAK2 to this transport because knockdown of TRAK1 but not TRAK2 impaired mitochondrial mobility, yet both TRAK1 and TRAK2 were shown to rescue transport impaired by TRAK1 gene knock-out. Thus, we demonstrate for the first time the pivotal contribution of the endogenous TRAK family of kinesin adaptors to the regulation of mitochondrial mobility.Mitochondria serve several functions in cells. These functions include the generation of energy in the form of ATP, the buffering of calcium ions, and the regulation of apoptosis. Thus, within cells, mitochondria need to move so they can respond to local needs. In the nervous system, mitochondria and mitochondrial transport are particularly important because of a requirement for high energy and calcium buffering during synaptic neurotransmission. The mitochondrial population in neurons is therefore highly mobile, and the dynamics of their transport are tightly regulated to satisfy these demands. Mitochondria can move in both anterograde and retrograde directions, utilizing motor proteins and the microtubule network (for reviews, see Refs. 1-3). Furthermore, they can be anchored at defined sites; one example is mitochondrial immobilization by a Ca 2ϩ -dependent mechanism at synaptic sites (4 -7). Recently, there has been significant progress in the understanding of mitochondrial transport processes with the identification of several proteins implicated in their trafficking mechanisms.The best characterized of these include the trafficking kinesin protein (TRAK) 2 /Milton family of kinesin adaptors; Miro1 and Miro2, atypical Rho GTPases that reside in the mitochondrial outer membrane that are purported receptors for TRAKs; syntabulin, also a kinesin adaptor protein; and syntaphilin, an axonal mitochondrial docking protein (for reviews, see Refs. 3 and 8).There are two mammalian TRAKs, TRAK1 and TRAK2, that share ϳ58% amino acid homology (9, 10). The TRAKs, like their Drosophila orthologue Milton (11), have been shown to function as kinesin adaptors linking kinesin heavy chain (KHC) to mitochondria by their association with Miro1/2. T...

A novel 913-amino acid protein, ␥-aminobutyric acid type A (GABA A ) receptor interacting factor-1 (GRIF-1), has been cloned and identified as a GABA A receptorassociated protein by virtue of its specific interaction with the GABA A receptor ␤2 subunit intracellular loop in a yeast two-hybrid assay. GRIF-1 has no homology with proteins of known function, but it is the rat orthologue of the human ALS2CR3/KIAA0549 gene. GRIF-1 is expressed as two alternative splice forms, GRIF-1a and a C-terminally truncated form, GRIF-1b. GRIF-1 mRNA has a wide distribution with a major transcript size of 6.2 kb. GRIF-1a protein is only expressed in excitable tissues, i.e. brain, heart, and skeletal muscle major immunoreactive bands of M r ϳ 115 and 106 kDa and, in muscle and heart only, an additional 88-kDa species. When expressed in human embryonic kidney 293 cells, GRIF-1a yielded three immunoreactive bands with M r ϳ 115, 106, and 98 kDa. Co-expression of GRIF-1a and ␣1␤2␥2 GABA A receptors in mammalian cells revealed some co-localization in the cell cytoplasm. Anti-FLAGagarose specifically precipitated GRIF-1 FLAG and GABA A receptor ␤2 subunits from human embryonic kidney 293 cells co-transfected with GRIF-1a FLAG and ␤2 subunit clones. Further, immobilized GRIF-1-(8 -633) specifically precipitated in vitro GABA A receptor ␣1 and ␤2 subunit immunoreactivities from detergent extracts of adult rat brain. The respective GABA A receptor ␤2 subunit/GRIF-1 binding domains were mapped using the yeast two-hybrid reporter gene assays. A possible role for GRIF-1 as a GABA A receptor ␤2 subunit trafficking factor is proposed.

1. The beta‐subunit has marked effects on the biophysical and pharmacological properties of voltage‐dependent calcium channels. In the present study we examined the ability of the GABAB agonist (‐) ‐baclofen to inhibit calcium channel currents in cultured rat dorsal root ganglion neurones following depletion of beta‐subunit immunoreactivity, 108‐116 h after microinjection of a beta‐subunit antisense oligonucleotide. 2.We observed that, although the calcium channel current was markedly reduced in amplitude following beta‐subunit depletion, the residual current (comprising both N‐ and L‐type calcium channel currents) showed an enhanced response to application of (‐) ‐baclofen. Therefore, it is possible that there is normally competition between activated G protein G(o) and the calcium channel beta‐subunit for binding to the calcium channel alpha 1‐subunit; and this competition shifts in favour of the binding of activated G(o) following depletion of the beta‐subunit, resulting in increased inhibition. 3. This hypothesis is supported by evidence that an antibody against the calcium channel beta‐subunit completely abolishes stimulation of the GTPase activity of G(o) by the dihydropyridine agonist S‐(‐) ‐Bay K 8644 in brain membranes. This stimulation of GTPase is thought to result from an interaction of G(o) alpha‐subunit (G alpha o) with its calcium channel effector which may operate as a GTPase‐activating protein. 4. These data suggest that the calcium channel beta‐subunit when complexed with the beta 1‐subunit normally inhibits its association with activated G(o). It may function as a GTPase‐activating protein to reduce the ability of activated G(o) to associate with the calcium channel, and thus limit the efficacy of agonists such as (‐) ‐baclofen.

␥-Aminobutyric acid, type A (GABA A ) receptor interacting factor-1 (GRIF-1) and N-acetylglucosamine transferase interacting protein (OIP) 106 are both members of a newly identified coiled-coil family of proteins. They are kinesin-associated proteins proposed to function as adaptors in the anterograde trafficking of organelles to synapses. Here we have studied in more detail the interaction between the prototypic kinesin heavy chain, KIF5C, kinesin light chain, and GRIF-1. The GRIF-1 binding site of KIF5C was mapped using truncation constructs in yeast two-hybrid interaction assays, co-immunoprecipitations, and co-localization studies following expression in mammalian cells. Using these approaches, it was shown that GRIF-1 and the KIF5C binding domain of GRIF-1, GRIF-1-(124 -283), associated with the KIF5C non-motor domain. Refined studies using yeast two-hybrid interactions and co-immunoprecipitations showed that GRIF-1 and GRIF-1-(124 -283) associated with the cargo binding region within the KIF5C non-motor domain. Substantiation that the GRIF-1-KIF5C interaction was direct was shown by fluorescence resonance energy transfer analyses using fluorescently tagged GRIF-1 and KIF5C constructs. A significant fluorescence resonance energy transfer value was found between the C-terminal EYFP-tagged KIF5C and ECFP-GRIF-1, the C-terminal EYFP-tagged KIF5C nonmotor domain and ECFP-GRIF-1, but not between the N-terminal EYFP-tagged KIF5C nor the EYFP-KIF5C motor domain and ECFP-GRIF-1, thus confirming direct association between the two proteins at the KIF5C C-terminal and GRIF-1 N-terminal regions. Co-immunoprecipitation and confocal imaging strategies further showed that GRIF-1 can bind to the tetrameric kinesin light-chain/kinesin heavy-chain complex. These findings support a role for GRIF-1 as a kinesin adaptor molecule requisite for the anterograde delivery of defined cargoes such as mitochondria and/or vesicles incorporating ␤2 subunit-containing GABA A receptors, in the brain.