Douglas O. Clary

The carbonic anhydrase domain of receptor tyrosine phosphatase β is a functional ligand for the axonal cell recognition molecule contactin

Receptor-type protein tyrosine phosphatase beta (RPTP beta) is expressed in the developing nervous system and contains a carbonic anhydrase (CAH) domain as well as a fibronectin type III repeat in its extracellular domain. Fusion proteins containing these domains were used to search for ligands of RPTP beta. The CAH domain bound specifically to a 140 kDa protein expressed on the surface of neuronal cells. Expression cloning in COS7 cells revealed that this protein is contactin, a GPI membrane-anchored neuronal cell recognition molecule. The CAH domain of RPTP beta induced cell adhesion and neurite growth of primary tectal neurons, and differentiation of neuroblastoma cells. These responses were blocked by antibodies against contactin, demonstrating that contactin is a neuronal receptor for RPTP beta. These experiments show that an individual domain of RPTP beta acts as a functional ligand for the neuronal receptor contactin. The interaction between contactin and RPTP beta may generate unidirectional or bidirectional signals during neural development.

Purification of soluble N-ethylmaleimide-sensitive fusion attachment proteins from bovine brain microsomes.

Publisher Summary Reconstitution of intra-Golgi vesicular transport in vitro has spurred the development of activity assays for required transport components. The first factor to be purified based on the transport assay is N-ethylmaleimide- sensitive fusion protein (NSF), isolated based on its ability to restore activity to an intra-Golgi transport assay inactivated with N-ethylmaleimide. N-Ethylmaleimide-sensitive fusion protein is subsequently found to be a peripheral membrane protein active in the fusion stages of transport. Reconstitution to transport competence of Golgi membrane fractions after extraction with high salt led to the discovery of at least two more peripheral membrane transport activities termed, “fraction 1” and “fraction 2.” Based on the observations addressed in the chapter, the Fr2 proteins are renamed, “SNAPs.” There are at least three SNAP species found in bovine brain, each of which exhibits activity in the in vitro intra-Golgi transport assay and the NSF-membrane binding assay.