The human 5-HT(6) receptor (5-HT(6)R) is one of the latest cloned receptors among the known 5-HT receptors. Its abundant distribution in the limbic region, which participates in the control of mood and emotion and is involved in nervous system diseases such as depression and Alzheimer disease, has caused it to generate much interest. However, the cellular mechanisms of 5-HT(6)R are poorly understood. In the present study we found, using a yeast two-hybrid assay, that the carboxyl-terminal region of 5-HT(6)R interacts with the Fyn-tyrosine kinase. We also determined using a glutathione S-transferase pulldown assay that this interaction was mediated through the SH3 domain of Fyn and confirmed this by co-immunoprecipitation assays in two different transfected cell lines as well as in adult rat brains. Immunocyto(histo)chemistry also showed prominent co-localization between 5-HT(6)R and Fyn in transfected cells and a similar distribution between 5-HT(6)R and Fyn in the rat brain. Based on this interaction, we further examined the modulation of 5-HT(6)R by Fyn and vice versa. In addition, we demonstrated that the activation of 5-HT(6)R activated the extracellular signal-regulated kinase1/2 via an Fyn-dependent pathway. These findings suggest that Fyn may play an important role in 5-HT(6)R- mediated signaling pathways in the central nervous system.

A series of small compounds acting at the orphan G protein-coupled receptor GPR92 were screened using a signaling pathway-specific reporter assay system. Lipid-derived molecules including farnesyl pyrophosphate (FPP), N-arachidonylglycine (NAG), and lysophosphatidic acid were found to activate GPR92. FPP and lysophosphatidic acid were able to activate both G_q/11- and G_s-mediated signaling pathways, whereas NAG activated only the G_q/11-mediated signaling pathway. Computer-simulated modeling combined with site-directed mutagenesis of GPR92 indicated that Thr⁹⁷, Gly⁹⁸, Phe¹⁰¹, and Arg²⁶⁷ of GPR92 are responsible for the interaction of GPR92 with FPP and NAG. Reverse transcription-PCR analysis revealed that GPR92 mRNA is highly expressed in the dorsal root ganglia (DRG) but faint in other brain regions. Peripheral tissues including, spleen, stomach, small intestine, and kidney also expressed GPR92 mRNA. Immunohistochemical analysis revealed that GPR92 is largely co-localized with TRPV1, a nonspecific cation channel that responds to noxious heat, in mouse and human DRG. FPP and NAG increased intracellular Ca²⁺ levels in cultured DRG neurons. These results suggest that FPP and NAG play a role in the sensory nervous system through activation of GPR92.

Neuronal L-type Ca²⁺ channels do not support synaptic transmission, but they play an essential role in synaptic activity-dependent gene expression. Ca_v1.2 and Ca_v1.3 are the two most widely expressed L-type Ca²⁺ channels in neurons and have different biophysical and subcellular distributions. The function of the Ca_v 1.3 L-type Ca²⁺ channel and its cellular mechanisms in the central nervous system are poorly understood. In this study, using a yeast two-hybrid assay, we found that the N terminus of the rat Ca_v1.3 α₁ subunit interacts with a partial N-terminal amino acid sequence of ryanodine receptor type 2 (RyR2). Reverse transcription-PCR and Western blot assays revealed high expression of both Ca_v1.3 and RyR2 in the rat hippocampus. We also demonstrate a physical association of Ca_v1.3 with RyR2 using co-immunoprecipitation assays. Moreover, immunocytochemistry revealed prominent co-localization between Ca_v1.3 and RyR2 in hippocampal neurons. Depolarizing cells by an acute treatment of a high concentration of KCl (high-K, 60 mm) showed that the activation of L-type Ca²⁺ channels induced RyR opening and led to RyR-dependent Ca²⁺ release, even in the absence of extracellular Ca²⁺. Furthermore, we found that RyR2 mRNA itself is increased by long term treatment of high-K via activation of L-type Ca²⁺ channels. These acute and long term effects of high-K on RyRs were selectively blocked by small interfering RNA-mediated silencing of Ca_v1.3. These results suggest a physical and functional interaction between Ca_v1.3 and RyR2 and important implications of Ca_v1.3/RyR2 clusters in translating synaptic activity into alterations in gene expression.