Eckart D. Gundelfinger

VILIP, a cognate protein of the retinal calcium binding proteins visinin and recoverin, is expressed in the developing chicken brain

Using a selective cloning approach we previously isolated a number of cDNAs of transcripts that are newly expressed during terminal differentiation of the chicken optic tectum. Here, we have characterized one of these cDNAs (OZ1) by Northern analysis and in situ hybridization. The OZ1 cDNA hybridizes to two transcripts of 1.6 kb and 2.9 kb which are widely expressed in the brain but not detectable in liver, heart or skeletal muscle. Cloning of overlapping cDNAs revealed that both transcripts encode the same open reading frame for a polypeptide of 191 amino acids. The deduced protein contains 4 EF-hand consensus motifs characteristic of calmodulin-like Ca(2+)-binding proteins. It displays 40% and 46% sequence identity with the retinal photoreceptor-specific Ca(2+)-binding proteins visinin and recoverin, respectively, and was termed VILIP (visinin-like protein). VILIP transcripts are also expressed in the retina. However, the expression pattern does not overlap with that of visinin or recoverin. The possible functional implications of the similarity to recoverin, which regulates guanylate cyclase activity of retinal rod cells in a Ca(2+)-dependent manner, are discussed.

How complex is the nicotinic receptor system of insects

In insects, nicotinic acetylcholine receptors (nAChRs) are confined to the nervous system. It is a long-standing open question whether the insect nicotinic cholinergic receptor system is less complex than that of the vertebrate nervous system. Simplicity can be conceived in two ways. (1) Fewer receptor subtypes may exist. (2) Single receptors may have a more primitive (homo-oligomeric) quaternary structure. Recent approaches to the molecular cloning of insect nAChRs may contribute valuable new information to this issue. Thus, the identification of multiple genes encoding proteins similar to vertebrate nAChR subunits implicates a remarkable heterogeneity for these receptors. The discovery of putatively non-ligand-binding subunits hints to the existence of vertebrate-like hetero-oligomeric nAChRs. However, the simultaneous occurrence of homo-oligomeric receptors must still be considered.

The Calcium-independent Receptor for α-Latrotoxin from Human and Rodent Brains Interacts with Members of the ProSAP/SSTRIP/Shank Family of Multidomain Proteins

Subtypes of the calcium-independent receptors for α−latrotoxin (CIRL1–3) define a distinct subgroup within the large family of the seven-transmembrane region cell surface receptors. The physiological function of CIRLs is unknown because neither extracellular ligands nor intracellular coupling proteins (G-proteins) have been identified. Using yeast two-hybrid screening, we identified a novel interaction between the C termini of CIRL1 and -2 and the PSD-95/discs large/ZO-1 (PDZ) domain of a recently discovered multidomain protein family (ProSAP/SSTRIP/Shank) present in human and rat brain. In vitro, CIRL1 and CIRL2 interacted strongly with the PDZ domain of ProSAP1. The specificity of this interaction has been verified by in vivo experiments using solubilized rat brain membrane fractions and ProSAP1 antibodies; only CIRL1, but not CIRL2, was co-immunoprecipitated with ProSAP1. In situhybridization revealed that ProSAP1 and CIRL1 are co-expressed in the cortex, hippocampus, and cerebellum. Colocalization was also observed at the subcellular level, as both CIRL1 and ProSAP1 are enriched in the postsynaptic density fraction from rat brain. Expression of all three CIRL isoforms is highly regulated during postnatal brain development, with CIRL3 exhibiting its highest expression levels immediately after birth, followed by CIRL2 and finally CIRL1 in aged rats.

Protein components of a rat brain synaptic junctional protein preparation

Antisera against a rat brain synaptic protein preparation, the postsynaptic density (PSD) fraction, were used to isolate cDNA clones by expression screening of a rat brain cDNA library. About one fifth of more than 200 analyzed cDNAs encoding potential synapse-associated proteins were previously unknown. Identifiable proteins include, among others, components of the pre- and postsynaptic cytoskeleton, synaptic vesicle proteins and several protein kinases and kinase substrates. This demonstrates that both pre- and postsynaptic elements purify with the PSD fraction.

Structure and developmental expression of the Dα2 gene encoding a novel nicotinic acetylcholine receptor protein of Drosophila melanogaster

Nicotinic acetylcholine receptors (nAChRs) represent a heterogeneous group of excitatory neurotransmitter receptors in the insect brain. We have characterized the Dα2 gene of Drosophila melanogaster, a new member of the nAChR gene family. The protein coding region is interrupted by six introns. The positions of three of these introns are shared with all other nAChR genes. The deduced Dα2 protein shows the structural features of ligand‐binding nAChR α‐subunits. Cytogenetically, the Dα2 gene maps at position 96A of the 3rd chromosome, close to the ALS gene which also encodes an α‐like nAChR subunit. Dα2 transcripts are predominantly expressed in late embryos and in fly heads.

Distribution pattern of three neural calcium-binding proteins (NCS-1, VILIP and recoverin) in chicken, bovine and rat retina

SummaryNeural Ca2+-binding proteins (NCaPs) constitute a subfamily of 4-EF-hand proteins, and display a histological and structural dichotomy: the A-type NCaPs are selectively expressed by the retina and pineal organ and display two canonical EF-hands, whereas the B-type NCaPs are found in the entire brain and present three regular EF-hands. In this study, antisera were raised against the A-type NCaP recoverin (26 kDa) and the B-type NCaPs VILIP and NCS-1 (22 kDa). Since the sequence identity among NCaPs is high, specific polyclonal antibodies were purified by double cross-immunoaffinity chromatography; both ELISA and immunoblot analyses determined that the resulting antibodies showed selectivity ratios inferior to 1/363 for the two other related NCaPs. Besides, the anti-VILIP antibodies displayed some affinity toward neurocalcin δ, and the antirecoverin antibodies recognized a 24 kDa protein, which is most likely visinin. Thus, immunohistochemical studies on the chicken, rat and cow retina revealed that anti-recoverin antibodies recognized the vertebrate photoreceptors and a small number of mammalian bipolar cells. Anti-VILIP antibodies exclusively labelled the inner Retina, I.e. the amacrine and ganglion cells. NCS-1 was mainly present in the photoreceptor inner segments, the inner plexiform layer and the ganglion cells. NCS-1 showed the highest species disparity. The retinal localization of NCS-1 and VILIP offered an important morphological basis for the understanding of their function. Furthermore, specific antibodies against the NCaPs may enable the identification of cell populations in more complex neural tissues, such as the brain.

Acetylcholine receptors of theDrosophila brain: A 900 bp promoter fragment contains the essential information for specific expression of theard gene in vivo

Theard gene encodes a β‐subunit ofDrosophila nicotinic acetylcholine receptors specifically expressed in a subset of neurons. To identify thecis‐regulatory region responsible for this cell‐specific expression, various 5′ fragments of theard gene were fused to alacZ reporter gene and introduced into theDrosophila genome. A DNA fragment spanning ∼760 bp upstream and ∼140 bp downstream of a cluster of putative transcription start sites produced a pattern of β‐galactosidase activity that resembles the distribution of ARD transcripts. Both in embryos and adults the levels oflacZ RNA were similar to those of endogenous ARD transcripts, suggesting that the 900 bp fragment harbors all essential elements for proper expression of theard gene.