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GABA receptors (GABA(A)) are the major sites of fast synaptic inhibition in the brain and can be assembled from five subunit classes: alpha, beta, gamma, delta, and epsilon. Receptor function can be regulated by direct phosphorylation of beta and gamma2 subunits, but how kinases are targeted to GABA(A) receptors is unknown. Here we show that protein kinase C-betaII (PKC-betaII) is capable of directly binding to the intracellular domain of the receptor beta1 and beta3 subunits, but not to those of the alpha1 or gamma2 subunits. Moreover, associating PKC-betaII is capable of specifically phosphorylating serine 409 in beta1 subunit and serines 408/409 within the beta3 subunit, key residues for modulating GABA(A) receptor function. The receptor for activated C kinase (RACK-1) was found also to bind to the beta1 subunit intracellular domain, but PKC binding appeared to be independent of this protein. Using immunoprecipitation, the association of PKC isoforms and RACK-1 with neuronal GABA(A) receptors was seen. Furthermore, PKC isoforms associating with neuronal receptors were capable of phosphorylating the receptor beta3 subunit. Together, these observations suggest GABA(A) receptors are intimately associated with PKC isoforms via a direct interaction with receptor beta subunits. This interaction may serve to localize PKC activity to GABA(A) receptors in neurons allowing the rapid regulation of receptor activity by cell-signaling pathways that modify PKC activity.

GABA A receptor-associated protein (GABARAP) was isolated on the basis of its interaction with the ␥2 subunit of GABA A receptors. It has sequence similarity to light chain 3 (LC3) of microtubule-associated proteins 1A and 1B. This suggests that GABARAP may link GABA A receptors to the cytoskeleton. GABARAP associates with tubulin in vitro. However, little is known about the mechanism for the interaction, and it is not clear whether the interaction occurs in vivo. Here, we report that GABARAP interacts directly with both tubulin and microtubules in a salt-sensitive manner, indicating the association is mediated by ionic interactions. GABARAP coimmunoprecipitates with tubulin and associates with both microtubules and microfilaments in intact cells. The cellular distribution is altered by treatment with taxol, nocodazole, and cytochalasin D. The tubulin binding domain was located at the N terminus of GABARAP by using synthetic peptides and deletion constructs and is marked by a specific arrangement of basic amino acids. The interaction between GABARAP and actin might be mediated by other proteins. These results demonstrate that GABARAP interacts with the cytoskeleton both in vitro and in cells and suggest a role of GABARAP in the interaction between GABA A receptors and the cytoskeleton. Such interactions are presumably needed for receptor trafficking, anchoring, and/or synaptic clustering. The structural arrangement of the basic amino acids present in the tubulin binding domain of GABARAP may aid in recognition of the potential of tubulin binding activity in other known proteins. Key Words: GABA A receptor-GABA A -receptor-associated protein-Clustering-MicrotubulesMicrofilaments-Cytoskeleton-Microtubule-associated proteins-Light chain 3.

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The so-called xenobiotic receptors (XRs) have functionally evolved into cellular sensors for both endogenous and exogenous stimuli by regulating the transcription of genes encoding drug-metabolizing enzymes and transporters, as well as those involving energy homeostasis, cell proliferation, and/or immune responses. Unlike prototypical steroid hormone receptors, XRs are activated through both direct ligand-binding and ligand-independent (indirect) mechanisms by a plethora of structurally unrelated chemicals. This review covers research literature that discusses direct vs. indirect activation of XRs. A particular focus is centered on the signaling control of the constitutive androstane receptor (CAR), the pregnane X receptor (PXR) and the aryl hydrocarbon receptor (AhR). We expect that this review will shed light on both the common and distinct mechanisms associated with activation of these three XRs.

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The constitutive androstane receptor (CAR, NR1I3) plays a crucial role in the regulation of drug metabolism, energy homeostasis, and cancer development through modulating the transcription of its numerous target genes. Different from prototypical nuclear receptors, CAR can be activated by either direct ligand binding or ligand-independent (indirect) mechanisms both initiated with nuclear translocation of CAR from the cytoplasm. In comparison to the well-defined ligand-based activation, indirect activation of CAR appears to be exclusively involved in the nuclear translocation through mechanisms yet to be fully understood. Accumulating evidence reveals that without activation, CAR forms a protein complex in the cytoplasm where it can be functionally affected by multiple signaling pathways. In this review, we discuss recent progresses in our understanding of the signaling regulation of CAR nuclear accumulation and activation. We expect that this review will also provide greater insight into the similarity and difference between the mechanisms of direct vs. indirect human CAR activation.

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