The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) is an intracellular IP(3)-gated Ca(2+) channel that is located on intracellular Ca(2+) stores and modulates Ca(2+) signalling. Using the yeast two-hybrid system, we screened a mouse brain cDNA library with bait constructs for mouse IP(3)R type 1 (IP(3)R1) to identify IP(3)R1-associated proteins. In this way, we found that carbonic anhydrase-related protein (CARP) is a novel IP(3)R1-binding protein. Western blot analysis revealed that CARP is expressed exclusively in Purkinje cells of the cerebellum, in which IP(3)R1 is abundantly expressed. Immunohistochemical analysis showed that the subcellular localization of CARP in Purkinje cells is coincident with that of IP(3)R1. Biochemical analysis also showed that CARP is co-precipitated with IP(3)R1. Using deletion mutagenesis, we established that amino acids 45-291 of CARP are essential for its association with IP(3)R1, and that the CARP-binding site is located within the modulatory domain of IP(3)R1 amino acids 1387-1647. CARP inhibits IP(3) binding to IP(3)R1 by reducing the affinity of the receptor for IP(3). As reported previously, sensitivity to IP(3) for IP(3)-induced Ca(2+) release in Purkinje cells is low compared with that in other tissues. This could be due to co-expression of CARP with IP(3)R in Purkinje cells and its inhibitory effects on IP(3) binding.

The vomeronasal organ (VNO) is a chemosensory organ specialized in the detection of pheromones in higher vertebrates. In mouse and rat, two gene superfamilies, V1r and V2r vomeronasal receptor genes, are expressed in sensory neurons whose cell bodies are located in, respectively, the apical and basal layers of the VNO epithelium. Here, we report that neurons of the basal layer express another multigene family, termed H2-Mv, representing nonclassical class I genes of the major histocompatibility complex. The nine H2-Mv genes are expressed differentially in subsets of neurons. More than one H2-Mv gene can be expressed in an individual neuron. In situ hybridization with probes for H2-Mv and V2r genes reveals complex and nonrandom combinations of coexpression. While neural expression of Mhc class I molecules is increasingly being appreciated, the H2-Mv family is distinguished by variegated expression across seemingly similar neurons and coexpression with a distinct multigene family encoding neural receptors. Our findings suggest that basal vomeronasal sensory neurons may consist of multiple lineages or compartments, defined by particular combinations of V2r and H2-Mv gene expression.

In mice, Ϸ1,000 odorant receptor (OR) genes are expressed in olfactory sensory neurons (OSNs). Homeodomain sites can be recognized in the promoter and upstream regions of several OR genes. Here, using the yeast one-hybrid system and electrophoretic mobility shift assay, we report that Lhx2, a LIM-homeodomain protein, binds to the homeodomain site in the mouse M71 OR promoter region. In Lhx2-deficient mice, the morphology of the olfactory epithelium is grossly normal. However, expression of OMP is abolished and that of GAP43 is severely reduced, indicating that no mature and few immature OSNs are produced. M71 and other OR genes also are not expressed. OSN development appears to be arrested between the terminal differentiation into neurons and the transition to immature neurons. Thus, Lhx2 is required for complete development of OSNs in mice.I n the mammalian olfactory epithelium (OE), olfactory sensory neurons (OSNs) detect chemical stimuli in the external environment by expressing odorant receptor (OR) genes (1, 2). These genes encode proteins with a putative seven-transmembrane domain structure, a defining characteristic of G proteincoupled receptors. It is believed that an individual OSN expresses a single OR gene (3) from the Ϸ1,000 OR genes in the mouse genome (4), but the strength of the evidence for this one-neuron-one-receptor hypothesis has been questioned (5). An OR gene is expressed from a single allele in a given cell (6). The OE is thus a complex mosaic of Ϸ2,000 OSN populations, each defined as expressing one allele from a choice of 2 ϫ Ϸ1,000 genes. OSNs expressing the same OR gene typically reside within one of at least four zones in the OE (7,8), where they are interspersed with OSNs expressing other OR genes. By targeted mutagenesis, all OSNs expressing the same OR were shown to project their axons to the same glomeruli in a remarkably precise manner, and these OR-specific projections to the olfactory bulb are influenced by the specificity of the expressed OR (9, 10). The onset of OR expression occurs before the first contact between OSN axons and the forebrain in embryos, and OSNs express OR genes in the absence of the axonal target, the olfactory bulb (11).The mechanisms underlying OR gene choice and expression are not understood, but irreversible DNA rearrangements have been excluded (12, 13). Sequence analyses of human, mouse, and rat OR genes have revealed a variety of motifs upstream of the transcription start site, including O͞E-like, E-box, Ikaros, homeodomain, and methylation-sensitive vMYB motifs (14). However, an involvement in OR gene regulation has not been shown for any of these motifs. Short (9-kb) transgenes of two OR genes, MOR23 and M71, replicate most OR expression features (15). A region of 405 bp upstream of the MOR23 transcription start site is required for transgene expression. In this and other upstream OR regions, common sequence motifs of homeodomain and O͞E-like sites have been recognized (15).Here, we have conducted yeast one-hybrid screening (16) to identify transcr...

The dependency of purified mouse cerebellar type 1 inositol 1,4,5-trisphosphate receptor (IP3R1)/Ca2+ channel function on cytoplasmic Ca2+ was examined. In contrast to the channels in crude systems, the purified IP3R1 reconstituted into planar lipid bilayers did not show the bell-shaped dependence on Ca2+. It was activated with increasing Ca2+ sublinearly without inhibition even up to 200 microM. The addition of calmodulin to the cytoplasmic side inhibited the channel at high Ca2+ concentrations. Calmodulin antagonists reversed the Ca2+-dependent inactivation of the native channels in cerebellar microsomes. These results indicate that the bell-shaped dependence on cytoplasmic Ca2+ is not an intrinsic property of the IP3R1, and the Ca2+-dependent inactivation is directly mediated by calmodulin.