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Complex neuronal functions rely upon the precise sorting, targeting, and restriction of receptors to specific synaptic microdomains. Little is known, however, of the molecular signals responsible for mediating these selective distributions. Here we report that metabotropic glutamate receptor subtype 7a (mGluR7a) is polarized at the basolateral surface when expressed in Madin-Darby canine kidney (MDCK) epithelial cells but is not polarized when expressed in cultured hippocampal neurons. Truncation of the mGluR7 cytoplasmic tail produces a protein that is restricted to a perinuclear intracellular compartment in both neurons and MDCK cells, where this protein colocalizes with a trans-Golgi network antigen. The mGluR7 cytoplasmic domain appended to the transmembrane portion of the vesicular stomatitis virus G protein and the ectodomain of human placental alkaline phosphatase is distributed over the entire cell surface in cultured neurons. When expressed in MDCK cells, this construct remains in an intracellular compartment distinct from endosomes or lysosomes. Thus, the cytoplasmic tail domain of mGluR7 is necessary but not sufficient for polarized targeting in MDCK monolayers, whereas in neurons the cytoplasmic tail is sufficient for cell surface expression but not polarization. Additional mechanisms are likely required to mediate mGluR7 neuronal polarization and synaptic clustering.

Subtypes of nicotinic receptors previously reported to be unaffected by neuronal bungarotoxin (NBT), including alpha 3 beta 4-containing and muscle type (alpha 1 beta 1 gamma delta) receptors, are shown to be inhibited by this toxin, but with rapid kinetics of onset and recovery. This inhibition is in contrast to the slow and prolonged inhibition of alpha 3 beta 2-containing receptors, suggesting that the beta subunits determine the kinetics of NBT inhibition of alpha 3 receptors. We have coexpressed chimeric beta subunits with alpha 3, and our results show that the first 121 amino acids of the beta subunit extracellular domain are sufficient to regulate the kinetics of NBT inhibition. This domain is also an important determinant of whether cytisine will act as a full agonist or a partial agonist of nicotinic receptors formed with alpha 3.

1. In situ hybridization histochemistry has revealed that neuronal nicotinic acetylcholine receptor subunits are widely expressed in unique although overlapping patterns in the central and peripheral nervous systems. In addition, an intricate regulation of expression is apparent during development. This raises the question of the transcription control of these complex distributions. 2. An analysis of the transcription control elements of one nicotinic receptor subunit, alpha 3, which is expressed both in the central and peripheral nervous systems as well as in PC12 cells, is presented here. 3. Overlapping genomic clones containing the rat beta 4, alpha 3, and alpha 5 gene cluster were isolated and a restriction map of this region was constructed. 4. The transcription start sites of the alpha 3 gene were mapped by RNase protection experiments. Surprisingly, there is no consensus TATA box upstream of these sites. This is consistent with the finding of multiple initiation sites. 5. Functional assays were done in PC12 cells using the bacterial chloramphenicol acetyltransferase gene as a reporter gene. Several restriction fragments from the region located 5' and including the transcription start sites were shown to promote transcription of the reporter gene and thus contain at least part of the alpha 3 promoter. In addition, a region further upstream, within the beta 4 transcription unit, appears to reduce transcriptional activity and thus could be a silencer. 6. It is proposed that this putative silencer is a selective force in maintaining the tight linkage of these three genes.

We have identified two additional members of the neuronal nicotinic acetylcholine receptor (nAChR)-related gene family. cDNA clones for one new gene, designated alpha 5, were isolated from rat hippocampus and rat PC12 cell line cDNA libraries. The alpha 5 gene encodes a protein of 48,800 daltons (424 amino acids) which exhibits significant overall amino acid sequence identity with the previously cloned rat nAChR subunits alpha 1 (49%), alpha 2 (55%), alpha 3 (52%), and alpha 4 (49%). Features characteristic of other nAChR alpha-subunits are present such as conserved cysteine residues at positions 127, 141, 191, and 192, and four strongly hydrophobic domains. A second addition to the nAChR-related gene family, designated beta 4, is encoded in overlapping rat genomic clones lambda DD15 and lambda RG518A. The beta 4 gene, encoding a mature protein of 53,300 daltons (475 amino acids), consists of 6 exons and has a transcription unit length of approximately 18 kilobase pairs. The beta 4 gene encoded protein shows considerable amino acid sequence identity with nAChR beta 1 (43%), beta 2 (64%), and beta 3 (44%) subunits. Northern blots showed that, along with alpha 3 and beta 2, transcripts for both the alpha 5 and beta 4 genes are present in the PC12 cell line, while in situ hybridization experiments demonstrated expression of the alpha 5 and beta 4 genes in a small number of nuclei in the central nervous system. Finally, the genes that encode the beta 4, alpha 3, and alpha 5 proteins are transcribed with convergent polarities and form a tightly linked gene cluster spanning approximately 60 kilobase pairs.

A new nicotinic acetylcholine receptor (nAChR) subunit, beta 4, was identified by screening a rat genomic library. In situ hybridization histochemistry revealed expression of the beta 4 gene in the medial habenula of adult rat brains. The primary structure of this subunit was deduced from a cDNA clone isolated from a PC12 cDNA library. Functional nAChRs were detected in Xenopus oocytes injected in pairwise combinations with in vitro synthesized RNAs encoding beta 4 and either the alpha 2, alpha 3, or alpha 4 subunit. Unlike the alpha 3 beta 2 receptor, the alpha 3 beta 4 receptor is not blocked by bungarotoxin 3.1, indicating that the beta subunit can affect the sensitivity of neuronal nAChRs to this toxin. These results extend the functional diversity of nicotinic receptors in the nervous system.