2 during synaptogenesis has fundamentally different effects on the nuclear localization of CASK and CASK/TBR-1 mediated gene expression. We therefore asked next what might be the underlying mechanism and reasoned that it might a differential shedding of extracellular domain of SDC-2 as compared to SDC-3. Previous work has shown that SDC-3 is susceptible to intramembranous cleavage by c-secretase and that Y-P30 has proteolytical activity on its own. We therefore overexpressed PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19651758 SDC-2 and -3 that harbor a myc-tag in their extracellular domain in HEK-293 cells and examined cleavage of the extracellular domain after application of Y-P30. It turned out that Y-P30 induced cleavage of the extracellular domain of SDC-2 but not of SDC-3. An inhibitor of Matrix-metalloproteinase 9 blocked the effects of Y-P30 on shedding of the extracellular domain of SDC-2, suggesting that proteolytical activity of Y-P30 alone is not sufficient to induce cleavage. In previous work we found that the neuritogenic effects of YP30 are based on 212141-51-0 price binding of the peptide to PTN and SDC-2 and 3. In the present study we have analyzed potential mechanisms by which the interaction of Y-P30/PTN with SDC can regulate neurite outgrowth. We found that during early development Y-P30 application in primary neurons reduces the nuclear localization of CASK whereas the opposite was found in older neurons. This effect of Y-P30 needs the association with SDC-3 in young and SDC-2 in older neurons. The underlying signaling mechanism probably involves the nuclear localization of 9 Y-P30 and Nuclear CASK CASK and a differential effect of Y-P30 on ectodomain cleavage of SDC-2 and -3. In young cultures Y-P30 binding appears to reduce ectodomain shedding of SDC-3 and thereby probably stabilizes a SDC-3/CASK complex that shifts the distribution of CASK from the nucleus to the plasma membrane. In older neurons Y-P30 binding to SDC-2 has the opposite effect and intramembranous cleavage of SDC-2 might release CASK for nuclear import. One interesting question regarding these actions of Y-P30 concerns the mechanism of SDC cleavage. Ecodomain shedding of SDC has been described for all family members in different tissues and is considered to be important for SDC signalling. In many cases matrix metalloproteases are involved in ectodomain shedding but only few co-factors like insulin have been shown to be involved in this cleavage event. It is tempting to speculate that following shedding intramembranous cleavage of SDC-3 by c-secretase might release the intracellular domain. In turn this would allow for nuclear transport of CASK. Unfortunately very little is known 
about the mechanisms of transport to the nucleus and whether transport is regulated by ligand binding to SDC. Nuclear CASK associates with TBR-1 and this transcription factor has been shown to regulate the expression of genes important during neuronal development. In line with this role we found increased expression of reelin and GluN2B mRNA at cortical neurons at DIV 18. In summary, we propose that in development the neuritogenic effects of the peptide as well as its effect on gene expression might be related to SDC-binding and the control of nuclear import of CASK. Apart from Y-P30 the only other known soluble ligand of SDC is the ubiquitously expressed trophic factor PTN. It was shown that the SDC interaction with CASK depends on SDC homodimerization. This is interesting since Y-P30/PTN profoundly oligomerize and subsequent SDC binding mig