Andrés Couve

INTRACELLULAR RETENTION OF RECOMBINANT GABAB RECEPTORS

γ-Aminobutyric acid type B (GABAB) receptors mediate the transmission of slow and prolonged inhibitory signals in the central nervous system. Two splice variants of GABAB receptors, GABABR1a and GABABR1b, were recently cloned from a mouse cortical and cerebellar cDNA library. As predicted, these receptors belong to the G protein-coupled receptor superfamily. We have used epitope-tagged versions of GABABR1a receptors to study the cellular distribution of these proteins in a variety of non-neuronal and neuronal cell types. Here we report that recombinant GABAB receptors fail to reach the cell surface when expressed in heterologous systems and are retained in the endoplasmic reticulum when introduced into COS cells. In addition, we prove that recombinant GABAB receptors are excluded from the cell surface when overexpressed in ganglion neurons and we further demonstrate that they fail to activate in superior cervical ganglion neurons. Together our observations suggest that recombinant GABAB receptors require additional information for functional targeting to the plasma membrane.

The expression of GABAB1 and GABAB2 receptor subunits in the CNS differs from that in peripheral tissues

GABA(B) receptors are G-protein-coupled receptors that mediate the slow and prolonged synaptic actions of GABA in the CNS via the modulation of ion channels. Unusually, GABA(B) receptors form functional heterodimers composed of GABA(B1) and GABA(B2) subunits. The GABA(B1) subunit is essential for ligand binding, whereas the GABA(B2) subunit is essential for functional expression of the receptor dimer at the cell surface. We have used real-time reverse transcriptase-polymerase chain reaction to analyse expression levels of these subunits, and their associated splice variants, in the CNS and peripheral tissues of human and rat. GABA(B1) subunit splice variants were expressed throughout the CNS and peripheral tissues, whereas surprisingly GABA(B2) subunit splice variants were neural specific. Using novel antisera specific to individual GABA(B) receptor subunits, we have confirmed these findings at the protein level. Analysis by immunoblotting demonstrated the presence of the GABA(B1) subunit, but not the GABA(B2) subunit, in uterus and spleen. Furthermore, we have shown the first immunocytochemical analysis of the GABA(B2) subunit in the brain and spinal cord using a GABA(B2)-specific antibody. We have, therefore, identified areas of non-overlap between GABA(B1) and GABA(B2) subunit expression in tissues known to contain functional GABA(B) receptors. Such areas are of interest as they may well contain novel GABA(B) receptor subunit isoforms, expression of which would enable the GABA(B1) subunit to reach the cell surface and form functional GABA(B) receptors.

Association of GABAB Receptors and Members of the 14-3-3 Family of Signaling Proteins

Two GABA(B) receptors, GABA(B)R1 and GABA(B)R2, have been cloned recently. Unlike other G protein-coupled receptors, the formation of a heterodimer between GABA(B)R1 and GABA(B)R2 is required for functional expression. We have used the yeast two hybrid system to identify proteins that interact with the C-terminus of GABA(B)R1. We report a direct association between GABA(B) receptors and two members of the 14-3-3 protein family, 14-3-3eta and 14-3-3zeta. We demonstrate that the C-terminus of GABA(B)R1 associates with 14-3-3zeta in rat brain preparations and tissue cultured cells, that they codistribute after rat brain fractionation, colocalize in neurons, and that the binding site overlaps partially with the coiled-coil domain of GABA(B)R1. Furthermore we show a reduced interaction between the C-terminal domains of GABA(B)R1 and GABA(B)R2 in the presence of 14-3-3. The results strongly suggest that GABA(B)R1 and 14-3-3 associate in the nervous system and begin to reveal the signaling complexities of the GABA(B)R1/GABA(B)R2 receptor heterodimer.

Cyclic AMP–dependent protein kinase phosphorylation facilitates GABA B receptor–effector coupling

GABA (γ-aminobutyric acid)B receptors are heterodimeric G protein–coupled receptors that mediate slow synaptic inhibition in the central nervous system. Here we show that the functional coupling of GABABR1/GABABR2 receptors to inwardly rectifying K+ channels rapidly desensitizes. This effect is alleviated after direct phosphorylation of a single serine residue (Ser892) in the cytoplasmic tail of GABABR2 by cyclic AMP (cAMP)–dependent protein kinase (PKA). Basal phosphorylation of this residue is evident in rat brain membranes and in cultured neurons. Phosphorylation of Ser892 is modulated positively by pathways that elevate cAMP concentration, such as those involving forskolin and β-adrenergic receptors. GABAB receptor agonists reduce receptor phosphorylation, which is consistent with PKA functioning in the control of GABAB-activated currents. Mechanistically, phosphorylation of Ser892 specifically enhances the membrane stability of GABAB receptors. We conclude that signaling pathways that activate PKA may have profound effects on GABAB receptor–mediated synaptic inhibition. These results also challenge the accepted view that phosphorylation is a universal negative modulator of G protein–coupled receptors.

Phosphorylation and Chronic Agonist Treatment Atypically Modulate GABAB Receptor Cell Surface Stability

GABAB receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. The dynamic control of the cell surface stability of GABAB receptors is likely to be of fundamental importance in the modulation of receptor signaling. Presently, however, this process is poorly understood. Here we demonstrate that GABAB receptors are remarkably stable at the plasma membrane showing little basal endocytosis in cultured cortical and hippocampal neurons. In addition, we show that exposure to baclofen, a well characterized GABAB receptor agonist, fails to enhance GABAB receptor endocytosis. Lack of receptor internalization in neurons correlates with an absence of agonist-induced phosphorylation and lack of arrestin recruitment in heterologous systems. We also demonstrate that chronic exposure to baclofen selectively promotes endocytosis-independent GABAB receptor degradation. The effect of baclofen can be attenuated by activation of cAMP-dependent protein kinase or co-stimulation of β-adrenergic receptors. Furthermore, we show that increased degradation rates are correlated with reduced receptor phosphorylation at serine 892 in GABABR2. Our results support a model in which GABABR2 phosphorylation specifically stabilizes surface GABAB receptors in neurons. We propose that signaling pathways that regulate cAMP levels in neurons may have profound effects on the tonic synaptic inhibition by modulating the availability of GABAB receptors.

Wnt-5a Modulates Recycling of Functional GABAA Receptors on Hippocampal Neurons

GABAA receptors (GABAA-Rs) play a significant role in mediating fast synaptic inhibition and it is the main inhibitory receptor in the CNS. The role of Wnt signaling in coordinating synapse structure and function in the mature CNS is poorly understood. In previous studies we found that Wnt ligands can modulate excitatory synapses through remodeling both presynaptic and postsynaptic regions. In this current study we provide evidence for the effect of Wnt-5a on postsynaptic GABAA-Rs. We observed that Wnt-5a induces surface expression and maintenance of this receptor in the neuronal membrane. The evoked IPSC recordings in rat hippocampal slice indicate that Wnt-5a can regulates postsynaptically the hippocampal inhibitory synapses. We found also that Wnt-5a: (a) induces the insertion and clustering of GABAA-Rs in the membrane; (b) increases the amplitude of GABA-currents due exclusively to postsynaptic mechanisms; (c) does not affect the endocytic process, but increases the receptor recycling. Finally, all these effects on the GABAA-Rs are mediated by the activation of calcium/calmodulin-dependent kinase II (CaMKII). Therefore, we postulate that Wnt-5a, by activation of CaMKII, induces the recycling of functional GABAA-Rs on the mature hippocampal neurons.

The endoplasmic reticulum and protein trafficking in dendrites and axons

Neurons are highly polarized cells whose dendrites and axons extend long distances from the cell body to form synapses that mediate neuronal communication. The trafficking of membrane lipids and proteins throughout the neuron is essential for the establishment and maintenance of cell morphology and synaptic function. However, the dynamic shape and spatial organization of secretory organelles, and their role in defining neuronal polarity and the composition of synapses, are not well delineated. In particular, the structure and function of the continuous and intricate network of the endoplasmic reticulum (ER) in neurons remain largely unknown. Here we review our current understanding of the ER in dendrites and axons, its contribution to local trafficking of neurotransmitter receptors, and the implications for synaptic plasticity and pathology.

BDNF Regulates Rab11-Mediated Recycling Endosome Dynamics to Induce Dendritic Branching

Dendritic arborization of neurons is regulated by brain-derived neurotrophic factor (BDNF) together with its receptor, TrkB. Endocytosis is required for dendritic branching and regulates TrkB signaling, but how postendocytic trafficking determines the neuronal response to BDNF is not well understood. The monomeric GTPase Rab11 regulates the dynamics of recycling endosomes and local delivery of receptors to specific dendritic compartments. We investigated whether Rab11-dependent trafficking of TrkB in dendrites regulates BDNF-induced dendritic branching in rat hippocampal neurons. We report that TrkB in dendrites is a cargo for Rab11 endosomes and that both Rab11 and its effector, MyoVb, are required for BDNF/TrkB-induced dendritic branching. In addition, BDNF induces the accumulation of Rab11-positive endosomes and GTP-bound Rab11 in dendrites and the expression of a constitutively active mutant of Rab11 is sufficient to increase dendritic branching by increasing TrkB localization in dendrites and enhancing sensitization to endogenous BDNF. We propose that Rab11-dependent dendritic recycling provides a mechanism to retain TrkB in dendrites and to increase local signaling to regulate arborization.

Prolonged activation of NMDA receptors promotes dephosphorylation and alters postendocytic sorting of GABAB receptors

Slow and persistent synaptic inhibition is mediated by metabotropic GABAB receptors (GABABRs). GABABRs are responsible for the modulation of neurotransmitter release from presynaptic terminals and for hyperpolarization at postsynaptic sites. Postsynaptic GABABRs are predominantly found on dendritic spines, adjacent to excitatory synapses, but the control of their plasma membrane availability is still controversial. Here, we explore the role of glutamate receptor activation in regulating the function and surface availability of GABABRs in central neurons. We demonstrate that prolonged activation of NMDA receptors (NMDA-Rs) leads to endocytosis, a diversion from a recycling route, and subsequent lysosomal degradation of GABABRs. These sorting events are paralleled by a reduction in GABABR-dependent activation of inwardly rectifying K+ channel currents. Postendocytic sorting is critically dependent on phosphorylation of serine 783 (S783) within the GABABR2 subunit, an established substrate of AMP-dependent protein kinase (AMPK). NMDA-R activation leads to a rapid increase in phosphorylation of S783, followed by a slower dephosphorylation, which results from the activity of AMPK and protein phosphatase 2A, respectively. Agonist activation of GABABRs counters the effects of NMDA. Thus, NMDA-R activation alters the phosphorylation state of S783 and acts as a molecular switch to decrease the abundance of GABABRs at the neuronal plasma membrane. Such a mechanism may be of significance during synaptic plasticity or pathological conditions, such as ischemia or epilepsy, which lead to prolonged activation of glutamate receptors.

The Availability of Surface GABAB Receptors Is Independent of γ-Aminobutyric Acid but Controlled by Glutamate in Central Neurons

The efficacy of synaptic transmission depends on the availability of ionotropic and metabotropic neurotransmitter receptors at the plasma membrane, but the contribution of the endocytic and recycling pathways in the regulation of γ-aminobutyric acid type B (GABAB) receptors remains controversial. To understand the mechanisms that regulate the abundance of GABAB receptors, we have studied their turnover combining surface biotin labeling and a microscopic immunoendocytosis assay in hippocampal and cortical neurons. We report that internalization of GABAB receptors is agonist-independent. We also demonstrate that receptors endocytose in the cell body and dendrites but not in axons. Additionally, we show that GABAB receptors endocytose as heterodimers via clathrin- and dynamin-1-dependent mechanisms and that they recycle to the plasma membrane after endocytosis. More importantly, we show that glutamate decreases the levels of cell surface receptors in a manner dependent on an intact proteasome pathway. These observations indicate that glutamate and not GABA controls the abundance of surface GABAB receptors in central neurons, consistent with their enrichment at glutamatergic synapses.