Abstract
Subtypes of the calcium-independent receptors for alpha-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 situ hybridization 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.
Highlights
From the ‡Institut fur Zellbiochemie und Klinische Neurobiologie, Universitat Hamburg, Martinistrasse 52, 20246 Hamburg, ¶Leibnitz Institut fur Neurobiologie, Brenneckestrasse 6, 39118 Magdeburg, and **Institut fur Anatomie, Westfalische Wilhelms-Universitat Munster, Vesaliusweg 2-4, 48149 Munster, Germany
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
Interaction between the PDZ Domain of Human SSTRIP/ Rat ProSAP1 and the C Terminus of calcium-independent receptor for ␣-latrotoxin (CIRL)—To identify potential candidates interacting with members of the SSTRIP/ ProSAP family, the PDZ domain of human SSTRIP was used as bait in the yeast two-hybrid system
Summary
The Calcium-independent Receptor for ␣-Latrotoxin from Human and Rodent Brains Interacts with Members of the ProSAP/SSTRIP/Shank Family of Multidomain Proteins*. 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. We show that the intracellular C terminus of CIRLs binds with high affinity to the PDZ domains of the ProSAP/ SSTRIP/Shank family of multidomain proteins. The data reported here suggest that members of the ProSAP/SSTRIP/Shank protein family are involved in the synaptic targeting of CIRLs and may contribute to signal transduction events mediated by the so far unknown physiological ligands of these receptors
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