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J. Neurochem. (2011) 118, 958-967. Abstract Signaling by muscarinic agonists is thought to result from the activation of cell surface acetylcholine receptors (mAChRs) that transmit extracellular signals to intracellular systems. In N1E-115 neuroblastoma cells, we detected both plasma membrane and intracellular M1-mAChRs using both biochemical and pharmacological methods. In intact cells, both plasma membrane and intracellular M1-mAChRs were detected by the hydrophobic ligand probe, 1-quinuclidinyl-[phenyl-4-3H]-benzilate ([3H]-QNB) whereas the hydrophilic probe, 1-[ N-methyl-3H] scopolamine ([3H]-NMS), detected only cell surface receptors. These probes detected comparable numbers of receptors in isolated membrane preparations. Immunohistochemical studies with M1-mAChR antibody also detected both cell-surface and intracellular M1-mAChRs. Carbachol-stimulated phosphatidylinositol hydrolysis and Ca2+ mobilization were completely inhibited by a cell-impermeable M1 antagonist, muscarinic toxin -7 and the Gq/11 inhibitor YM-254890. However, carbachol-stimulated extracellular-regulated kinase 1/2 activation was unaffected by muscarinic toxin-7, but was blocked by the cell-permeable antagonist, pirenzepine. extracellular regulated kinase 1/2 phosphorylation was resistant to blockade of Gq/11 (YM-254890) and protein kinase C (bisindolylmaleimide I). Our data suggest that the geographically distinct M1-mAChRs (cell surface versus intracellular) can signal via unique signaling pathways that are differentially sensitive to cell-impermeable versus cell-permeable antagonists. Our data are of potential physiological relevance to signaling that affects both cognitive and neurodegenerative processes. [ABSTRACT FROM AUTHOR]

The neuron-specific G protein-coupled receptor interacting scaffold protein (GISP) is a multidomain, brain-specific protein derived from the A-kinase anchoring protein-9 gene. We originally isolated GISP as an interacting partner for the GABAB receptor subunit GABAB1. Here, we show that the protein tumour susceptibility gene 101 (TSG101), an integral component of the endosomal sorting machinery that targets membrane proteins for lysosomal degradation, also interacts with GISP. TSG101 co-immunoprecipitates with GISP from adult rat brain, and using GST pull-downs, we identified that the eighth coiled-coiled region of GISP is critical for TSG101 association. Intriguingly, although there is no direct interaction between GISP and the GABAB2 subunit, their co-expression in HEK293 cells increases levels of GABAB2. GISP also inhibits TSG101-dependent GABAB2 down-regulation in human embryonic kidney 293 cells whereas over-expression of a mutant GISP lacking the TSG101 binding domain has no effect on GABAB2 degradation. These data suggest that GISP can function as a negative regulator of TSG101-dependent lysosomal degradation of transmembrane proteins in neurons to promote receptor stability. [ABSTRACT FROM AUTHOR]

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]