Search

Synaptic transmission depends on the regulated surface expression of neurotransmitter receptors, but many of the cellular processes required to achieve this remain poorly understood. To better define specific mechanisms for the GABAB receptor (GABABR) trafficking, we screened for proteins that bind to the carboxy-terminus of the GABAB1 subunit. We report the identification and characterization of a novel 130-kDa protein, GPCR interacting scaffolding protein (GISP), that interacts directly with the GABAB1 subunit via a coiled-coil domain. GISP co-fractionates with GABABR and with the postsynaptic density and co-immunoprecipitates with GABAB1 and GABAB2 from rat brain. In cultured hippocampal neurons, GISP displays a punctate dendritic distribution and has an overlapping localization with GABABRs. When co-expressed with GABABRs in human embryonic kidney cells, GISP promotes GABABR surface expression and enhances both baclofen-evoked extracellular signal-regulated kinase (ERK) phosphorylation and G-protein inwardly rectifying potassium channel (GIRK) currents. These results suggest that GISP is involved in the forward trafficking and stabilization of functional GABABRs. [ABSTRACT FROM AUTHOR]

Metabotropic γ-aminobutyric acid receptors (GABABRs) play a critical role in inhibitory synaptic transmission in the hippocampus but the ontogeny of their subunit synthesis and synaptic localisation has not been determined. Here we report the distributions and developmental profiles of GABAB1 and GABAB2 subunits in cultured rat embryonic hippocampal neurons. Limited levels of GABAB1 and GABAB2 immunoreactivity were present at 3 days in vitro (DIV). At 7 DIV, when baclofen-evoked inwardly rectifying K+ channel-mediated responses first appear in the cells, there was a more widespread expression within the soma and proximal dendrites. Levels of the K+ channel GIRK 1 were relatively constant at all time points suggesting channel availability does not limit the appearance of functional GABABRs. At 14 DIV the staining displayed a punctate dendritic distribution and near maximal GABABR-mediated electrophysiological responses were obtained. About half of the puncta for each GABABR subunit in dendrites co-localised with the synaptic marker SV2a suggesting that these subunits are at or very near to synapses. Interestingly, at all ages strong GABABR immunoreactivity was also present in the nuclei of neurons. These results provide an important developmental baseline for future studies aimed at investigating, for example, the trafficking and functional regulation of these receptors. [Copyright &y& Elsevier]

Abstract: GPCR interacting scaffold protein (GISP) is a multi-domain brain-specific scaffold protein that can regulate GABAB receptor complexes by both enhancing their surface expression and by inhibiting their lysosomal degradation. GISP retards degradation of GABAB receptors through its interaction with tumour susceptibility gene 101 (TSG101), a member of the endosomal sorting complex required for transport (ESCRT) lysosomal sorting machinery. We show that in addition to GABAB, GISP exerts a more general role to increase the steady-state levels of several neurotransmitter receptors. Further, GISP delays TSG101-dependent agonist-induced EGFR down-regulation in human embryonic kidney (HEK) 293 cells whereas a mutant GISP lacking the TSG101 binding domain has no effect. These data suggest that GISP acts as a negative regulator of TSG101-dependent lysosomal degradation and plays an important role in determining the availability of neurotransmitter receptors. [Copyright &y& Elsevier]