Untersuchungen zu der Entstehung und der Bedeutung löslicher Fragmente der neuralen Zelladhäsionsmoleküle NCAM und L1 im Zentralen Nervensystem der Maus

A variety of different membrane proteins also occur as a circulating, soluble form. These soluble forms are often derived from the membrane forms by proteolysis. Different soluble fragments of the transmembrane and multidomain neural cell adhesion molecules NCAM and L1 playing important functional roles in the developing and adult nervous system have been detected. The mechanism of generation of soluble NCAM fragments with a molecular range of 110 kDa to 190 kDa is poorly understood. In this study, evidence was presented that a metalloprotease is able to solubilize NCAM fragments via proteolytic processing. A member of the ADAM family of metalloproteases, the TNFa converting enzyme (TACE) cleaves the NCAM isoforms NCAM140 and NCAM180 near their membrane anchors resulting in the release of soluble NCAM fragments with a molecular weight of 110 kDa and higher depending on the glycosylation state of the extracellular domain of NCAM. The extracellular domain of the GPI-linked isoform NCAM120 is liberated from the membrane via a different mechanism. The metalloprotease inhibitor GM 6001 reduces the release of soluble NCAM fragments. Its inhibitory effect on NCAM dependent neurite outgrowth of cerebellar explants indicates that the proteolytic processing of NCAM plays an important role for NCAM dependent neurite outgrowth. Further evidence for an essential role of the ectodomain shedding of NCAM for the process of NCAM dependent neurite outgrowth was obtained in experiments showing that the calmodulin inhibitor CGS 9343 B which is able to stimulate the cleavage of membrane bound NCAM isoforms, inhibits NCAM dependent neurite outgrowth. Whether the effect of calmodulin on NCAM dependent neurite outgrowth is mediated via a direct interaction with its binding partner NCAM remains to be clarified. The cell adhesion molecule L1 is proteolytically processed at two distinct sites within the extracellular domain, leading to the generation of different soluble fragments. Evidence was presented that the proprotein convertase PC5A is the protease that cleaves L1 in the third fibronectin type III domain, whereas the proprotein convertases furin, PC1, PC2, PACE4, and PC7 are not effective in cleaving L1. This fragment was present in the hippocampus, which expresses PC5A, but was not detectable in the cerebellum, which does not express PC5A. The 140 kDa L1 fragment was found to be tightly associated with the full-length 200 kDa L1 molecule. The complex dissociated from the membrane upon cleavage by a protease acting at a more membrane-proximal site of full-length L1. This proteolytic cleavage was inhibited by the metalloprotease inhibitor GM 6001 and enhanced by a calmodulin inhibitor. L1-dependent neurite outgrowth of cerebellar neurons was inhibited by GM 6001, suggesting that proteolytic processing of L1 by a metalloprotease is involved in neurite outgrowth. In this study, it was shown that L1 is processed at further proteolytic cleavage sites in its extracellular domain resulting in the release of additional soluble fragments. The metalloprotease MMP9 seems to be one the responsible proteases involved in this process. A lot of transmembrane proteins were identified to be additionally cleaved within their transmembrane domains. The ectodomain shedding of the extracellular domain is the initial step for regulated intramembranous proteolysis. A 15 kDa fragment recognized by an antibody directed against the intracellular domain of L1 is released from the plasma membrane into the cytoplasm via proteolysis and transported to the nucleus. The question whether the intracellular domain of L1 located in the nucleus is involved in regulation of gene transcription is currently under investigation.