Neuronal specific protein NP185 is enriched in nerve endings: Binding characteristics for clathrin light chains, synaptic vesicles, and synaptosomal plasma membrane

Abstract

The neuronal specific protein NP185, found associated with brain clathrin-coated vesicles, formed a complex with unphosphorylated, but not with phosphorylated, clathrin light chains. The NP185-clathrin light chain complex was associated with casein kinase II activity, which, in the presence of polylysine, phosphorylated clathrin light chain b but not the NP185. The dissociation of this complex with 50% ethylene glycol pH 11.5 suggests that NP185 binds to hydrophobic domains of clathrin light chains. When NP185 molecules were retained by monoclonal antibody-linked Sepharose beads, they bound synaptic vesicles, decoated vesicles and synaptosomal plasma membrane. Immunohistochemistry on mouse cerebellar tissue sections using 8G8, a monoclonal antibody raised against NP185, showed neuronal specific labeling closely following synaptic distribution. In immunoblots, NP185 shares similar epitopes to those detected in another assembly polypeptide, AP-180, an indication that both proteins are identical. It appears that NP185 plays a specific role in nerve ending functions through its ability to induce clathrin to polymerize into cages, its interaction with synaptic vesicles, with the plasma membrane and with clathrin coat components.

Neuromuscular junctions contain NP185: The multifunctional protein is located at the presynaptic site

“…In view of NP185's association with CCVs (Puszkin, et al, 1988;Su al., 1991), similarities with other clathrin assembly proteins (Murphy et al, 1991;Su et al, 1991;F'uszkin, et al, 1992), and their binding affinity for synaptic vesicles and the syn-The synaptic boutons are sites where the rapid release and re-uptake of active neurotransmitter molecules occur mediated by complex flow of ions and chemical compounds. Such flow is sustained from a compartment provided by a pool of synaptic terminal vesicles (Betz and Bewick, 1992).…”

“…The protein molecules involved in the reuptake of membrane are similar for many cell types and tissues and are composed of an increasing number of phylogenetically well conserved molecules (Bloom and Puszkin, 1980;Cheng et al, 1980;Puszkin et al, 1983;Kirchhausen and Harrison, 1984;Jackson et al, 1987;Kirchhausen et al, 1987;Puszkin et al, 1989). Nevertheless, there are brain tissue-specific proteins associated with endocytic vesicles, and their abundance in the synaptic terminal would suggest that membrane recycling in synaptosomes differs in mode or regulation from that in other cells or tissues Puszkin, 1988, 1989;Perry and Puszkin, 1991;Su and Puszkin, 1991;Li and Puszkin, 1992).…”

“…Also, this molecule was localized only in mature synaptic terminals . Binding of NP185 to tubulin and to clathrin light chains was found to be tightly regulated by two neuronal kinases, namely, CAM-kinase and Casein kinase I1 Su and Puszkin, 1991). Binding sites for NP185 were detected on synaptic vesicles and synaptic plasma membranes and NP185 Neuromuscular Junctions Contain NP185 409 bryos, NP185-enriched preparations were made immediately after excising the brain tissue and striated muscle fibers.…”

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The NP185 polypeptide (AP,) is a multifunctional component isolated from brain endocytic vesicles, which binds to tubulin and clathrin light chains, decoated vesicles, synaptic vesicles, and the synaptoso-ma1 plasma membrane (Su et al, 1991). The NP185 molecules are expressed during avian cerebellar synaptogenesis and appear to function in CNS regions rich in synaptic terminals .

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Primary Structure of the Neuronal Clathrin-Associated Protein Auxilin and its Expression in Bacteria

“…For nerve-cell-specific functions, such as the retrieval of synaptic vesicle membrane after neurotransmitter release (Heuser, 1989;Maycox et al, 1992), clathrincoated vesicles are equipped with adaptor and clathrin lightchain isoforms (Jackson et al, 1987;Kirchhausen et al, 1987Kirchhausen et al, , 1989Robinson, 1989;Ponnambalam et al, 1990) and two additional proteins. These are AP180 and auxilin (Keen and Black, 1986;Ahle and Ungewickell, 1986;Kohtz and Puszkin, 1988;Ahle and Ungewickell, 1990;Su et al, 1991 ;Perry et al, 1992).…”