Kieran Brickley

GRIF-1 and OIP106, Members of a Novel Gene Family of Coiled-Coil Domain Proteins ASSOCIATION IN VIVO AND IN VITRO WITH KINESIN

γ-Aminobutyric acidA receptor-interacting factor (GRIF-1) is a 913-amino acid protein proposed to function as a GABAA receptor β2 subunit-interacting, trafficking protein. GRIF-1 shares ∼44% amino acid sequence identity with O-linked N-acetylglucosamine transferase interacting protein 106, OIP106. Both proteins contain predicted coiled-coil domains and probably constitute a novel gene family. The Drosophila orthologue of this family of proteins may be Milton. Milton shares ∼44% amino acid homology with GRIF-1. Milton is proposed to function in kinesin-mediated transport of mitochondria to nerve terminals. We report here that GRIF-1 and OIP106 also associate with kinesin and mitochondria. Following expression in human embryonic kidney 293 cells, both GRIF-1 and OIP106 were shown by co-immunoprecipitation to be specifically associated with an endogenous kinesin heavy chain species of 115 kDa and exogenous KIF5C. Association of GRIF-1 with kinesin was also evident in native brain and heart tissue. In the brain, anti-GRIF-1-(8–633) antibodies specifically co-immunoprecipitated two kinesin-immunoreactive species with molecular masses of 118 and 115 kDa, and in the heart, one kinesin-immunoreactive species, 115 kDa, was immunoprecipitated. Further studies revealed that GRIF-1 was predominantly associated with KIF5A in the brain and with KIF5B in both the heart and in HEK 293 cells. Yeast two-hybrid interaction assays and immunoprecipitations showed that GRIF-1 associated directly with KIF5C with the GRIF-1/KIF5C interaction domain localized to GRIF-1-(124–283). These results further support a role for GRIF-1 and OIP106 in protein and/or organelle transport in excitable cells in a manner analogous to glutamate receptor-interacting-protein 1, in the motor-dependent transport of α-amino-3-hydroxy-5-methylisoxazole-4-propionate glutamate excitatory neurotransmitter receptors to dendrites.

Importance of the Different |β Subunits in the Membrane Expression of the α1A and α2 Calcium Channel Subunits: Studies Using a Depolarization-sensitive α1A Antibody

The plasma membrane expression of the rat brain calcium channel subunits α1A, α2‐Δ and the β subunits β1b, β2a, β3b and β4 was examined by transient expression in COS‐7 cells. Neither α1A nor α2‐Δ localized to the plasma membrane, either alone or when coexpressed. However, coexpression of α1A or α2‐Δ/α1A with any of the p subunits caused α1A and α2 to be targetted to the plasma membrane. The α1A antibody is directed against an exofacial epitope at the mouth of the pore, which is not exposed unless cells are depolarized, both for native α1A channels in dorsal root ganglion neurons and for α1A expressed with a β subunit. This subsidiary result provides evidence that either channel opening or inactivation causes a conformational change at the mouth of the pore of α1A. Immunostaining for α1A was obtained in depolarized non‐permeabilized cells, indicating correct orientation in the membrane only when it was coexpressed with a subunit. In contrast, β1b and β2a were associated with the plasma membrane when expressed alone. However, this is not a prerequisite to target α1A to the membrane since β3 and β4 alone showed no differential localization, but did direct the translocation of α1A to the plasma membrane, suggesting a chaperone role for the β subunits

Identification, Molecular Cloning, and Characterization of a Novel GABAA Receptor-associated Protein, GRIF-1

A novel 913-amino acid protein, γ-aminobutyric acid type A (GABAA) receptor interacting factor-1 (GRIF-1), has been cloned and identified as a GABAAreceptor-associated protein by virtue of its specific interaction with the GABAA receptor β2 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 ofM r ∼ 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 withM r ∼ 115, 106, and 98 kDa. Co-expression of GRIF-1a and α1β2γ2 GABAA receptors in mammalian cells revealed some co-localization in the cell cytoplasm. Anti-FLAG-agarose specifically precipitated GRIF-1FLAG and GABAAreceptor β2 subunits from human embryonic kidney 293 cells co-transfected with GRIF-1aFLAG and β2 subunit clones. Further, immobilized GRIF-1-(8–633) specifically precipitated in vitro GABAA receptor α1 and β2 subunit immunoreactivities from detergent extracts of adult rat brain. The respective GABAA receptor β2 subunit/GRIF-1 binding domains were mapped using the yeast two-hybrid reporter gene assays. A possible role for GRIF-1 as a GABAA receptor β2 subunit trafficking factor is proposed.

GTPase dependent recruitment of Grif-1 by Miro1 regulates mitochondrial trafficking in hippocampal neurons

The transport of mitochondria to specific neuronal locations is critical to meet local cellular energy demands and for buffering intracellular calcium. A critical role for kinesin motor proteins in mitochondrial transport in neurons has been demonstrated. Currently however the molecular mechanisms that underlie the recruitment of motor proteins to mitochondria, and how this recruitment is regulated remain unclear. Here we show that a protein trafficking complex comprising the adaptor protein Grif-1 and the atypical GTPase Miro1 can be detected in mammalian brain where it is localised to neuronal mitochondria. Increasing Miro1 expression levels recruits Grif-1 to mitochondria. This results in an enhanced transport of mitochondria towards the distal ends of neuronal processes. Uncoupling Grif-1 recruitment to mitochondria by expressing a Grif-1/Miro1 binding fragment dramatically reduces mitochondrial transport into neuronal processes. Altering Miro1 function by mutating its first GTPase domain affects Miros ability to recruit Grif-1 to mitochondria and in addition alters mitochondrial distribution and shape along neuronal processes. These data suggest that Miro1 and the kinesin adaptor Grif-1 play an important role in regulating mitochondrial transport in neurons.

Inhibition of the interaction of G protein G(o) with calcium channels by the calcium channel beta‐subunit in rat neurones.

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.

Identification, molecular cloning and characterization of a novel GABA-A receptor associated protein, GRIF-1

A novel 913-amino acid protein, γ-aminobutyric acid type A (GABAA) receptor interacting factor-1 (GRIF-1), has been cloned and identified as a GABAAreceptor-associated protein by virtue of its specific interaction with the GABAA receptor β2 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 ofM r ∼ 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 withM r ∼ 115, 106, and 98 kDa. Co-expression of GRIF-1a and α1β2γ2 GABAA receptors in mammalian cells revealed some co-localization in the cell cytoplasm. Anti-FLAG-agarose specifically precipitated GRIF-1FLAG and GABAAreceptor β2 subunits from human embryonic kidney 293 cells co-transfected with GRIF-1aFLAG and β2 subunit clones. Further, immobilized GRIF-1-(8–633) specifically precipitated in vitro GABAA receptor α1 and β2 subunit immunoreactivities from detergent extracts of adult rat brain. The respective GABAA receptor β2 subunit/GRIF-1 binding domains were mapped using the yeast two-hybrid reporter gene assays. A possible role for GRIF-1 as a GABAA receptor β2 subunit trafficking factor is proposed.

Trafficking Kinesin Protein (TRAK)-mediated Transport of Mitochondria in Axons of Hippocampal Neurons

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.

Antisense depletion of beta-subunits modulates the biophysical and pharmacological properties of neuronal calcium channels.

1. The role of the voltage‐dependent calcium channel (VDCC) beta‐subunit has been examined in cultured rat dorsal root ganglion neurones (DRGs). An antipeptide antibody was raised and this recognized proteins corresponding to beta‐subunits in a number of preparations. Immunoreactivity for the VDCC beta‐subunit in DRGs was concentrated on the internal side of the plasma membrane but was also present in the cytoplasm. 2. A twenty‐six‐mer antisense oligonucleotide with homology to all published VDCC beta‐subunit sequences was microinjected into individual cells, and maximal depletion of VDCC beta‐subunit immunoreactivity was observed after 108 h suggesting a half‐life for the turnover of the beta‐subunit greater than 50 h. No depletion was obtained with nonsense oligonucleotide. 3. The effect of depletion of VDCC beta‐subunit immunoreactivity on calcium channel currents in these cells was a reduction in amplitude of the maximum current of about 47%, and a shift in the voltage dependence of current activation of about +7 mV. These effects are the converse of those observed following co‐expression of cloned beta‐ with alpha 1‐subunits in oocytes and other expression systems. 4. The ability of the 1,4‐dihydropyridine (DHP) agonist Bay K 8644 to enhance calcium channel currents was greatly reduced following depletion of beta‐subunit immunoreactivity. This result is in agreement with the finding in several systems that co‐expression of the beta‐subunit with alpha 1‐subunits results in an increased number of DHP binding sites. 5. These results show that calcium channel beta‐subunits form part of native neuronal calcium channels and modify their biophysical and pharmacological properties.

Voltage‐dependent calcium channel β‐subunits in combination with α 1 subunits, have a GTPase activating effect to promote the hydrolysis of GTP by Gα o in rat frontal cortex

The dihydropyridine‐sensitive calcium channel agonist (−)‐BayK 8644 was found to produce an enhancement of the intrinsic hydrolysis of GTP by Go in rat frontal cortex membranes. An anti‐calcium channel β‐subunit antiserum abolished the (−)‐BayK 8644‐stimulated hydrolysis of GTP by Go and reduced the dihydropyridine binding capacity of the cortical membranes. A peptide which mimics the β‐subunit binding domain of the calcium channel complex, also attenuated (−)‐BayK 8644 activation of GTPase. This study suggests that the calcium channel β‐subunit is the principal component of the channel complex involved in linking dihydropyridine agonist binding to enhanced hydrolysis of GTP by Go. This may be a mechanism by which calcium channels can normally act to limit the duration of a G‐protein modulatory signal.

Use of site-directed antibodies to probe the topography of the alpha 2 subunit of voltage-gated Ca2+ channels.

Polyclonal antibodies were raised against peptides corresponding to residues 1–15, 469–483 and 933–951 of the rabbit skeletal muscle L‐type calcium channelα2/δ primary translation product, for use as topological probes. Immunocytochemical comparison of the abilities of the antibodies to bind to theα2 and δ subunits in intact and detergent‐permeabilised rat dorsal root ganglion cells enabled the membrane orientation of these regions to be established. The resultant data indicate that the regions containing residues 1–15 and 469–483 of theα2 subunit, and residues 1–17 of the δ subunit, are exposed on the extracellular surface of the membrane, findings consistent with a model that proposesα2 to be entirely extracellular.