Purification and molecular characterization of NP185, a neuronal-specific and synapse-enriched clathrin assembly polypeptide.

NP185, a neuronal-specific protein of 185 kDa, was first discovered when we prepared monoclonal anti-bodies (mAbs) against bovine brain clathrin coated vesicles. Two mAbs, 8G8 and 6G7, permitted us to characterize this protein both biochemically and in development (NP185 is expressed in a NGF-dependent manner in PC12 cells). The expression of NP185 coincides with synaptogenesis.

The synapse-specific phosphoprotein F1-20 is identical to the clathrin assembly protein AP-3.

F1-20 and AP-3 are independently described, synapse-associated, developmentally regulated phosphoproteins with similar apparent molecular masses on SDS-polyacrylamide gel electrophoresis (PAGE). F1-20 was cloned and characterized because of its synapse specificity. AP-3 was purified and studied biochemically because of its function as a clathrin assembly protein.

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

The NP185 polypeptide (AP3) is a multifunctional component isolated from brain endocytic vesicles, which binds to tubulin and clathrin light chains, decoated vesicles, synaptic vesicles, and the synaptosomal 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 (Perry et al.

Systemic lupus erythematosus patients with central nervous system involvement show autoantibodies to a 50-kD neuronal membrane protein.

An antibody was detected in the sera from patients with systemic lupus erythematosus (SLE) and central nervous system (CNS) involvement that reacted with a 50-kD antigen in the plasma membrane of brain synaptic terminals. The 50-kD antigen was solubilized with Triton X-100 from preparations enriched with synaptic plasma membranes, and was partially purified by molecular sieve filtration column chromatography. The sera of 19 of 20 CNS-SLE patients showed strong to moderate immunoreactivity with the 50-kD protein in Western blots.

Neuronal protein NP185 is developmentally regulated, initially expressed during synaptogenesis, and localized in synaptic terminals.

Evidence is presented here that demonstrates the presence of NP185 (AP3) in neuronal cells, specifically within syn-aptic terminals of the central nervous system and in the peripheral nervous system, particularly in the neuro-muscular junction of adult chicken muscle. Biochemical results obtained in our laboratories indicate that NP185 is associated with brain synaptic vesicles, with clathrin-coated vesicles, and with the synaptosomal plasma membrane. Also, NP185 binds to tubulin and clathrin light chains and the binding is regulated by phosphorylation (Su et al.

Neuronal protein NP185 in avian and murine cerebellum: expression during development and evidence for its presence in nerve endings.

The neuronal protein NP185 is a neural tissue-specific protein isolated from clathrin-coated vesicles in brain. Using 8G8, a monoclonal antibody (MAb) characterized in our laboratory, we studied the expression and distribution of neuronal protein NP185 in developing avian cerebellum and in mature murine cerebellum. Furthermore, we compared these parameters to that of synapse-specific neuronal protein, synaptophysin, and an axon-specific (i.

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

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.

Clathrin assembly protein AP-3. The identity of the 155K protein, AP 180, and NP185 and demonstration of a clathrin binding domain.

Three independently isolated clathrin-associated proteins have been reported that have molecular weights of approximately 155,000-185,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis: the 155K protein (Keen, J. H., and Black, M.

Novel NGF-induced proteins in PC12 cells: immunological evidence for their presence in brain nerve endings using a single monoclonal antibody.

A monoclonal antibody, S-11D9, detected a group of novel polypeptides whose expression was induced 6 hr after incubation of PC12 cells with nerve growth factor. The antigens also were visualized by immune precipitation and Western blotting in synaptic vesicles, clathrin-coated vesicles, and synaptic plasma membrane prepared from bovine brain. A large polypeptide was detected on the synaptic plasma membrane; an intermediate size protein was visualized on the plasma membrane and on both synaptic and clathrin-coated vesicles, while a smaller molecule was found only on brain Golgi-enriched membrane preparations.

Novel proteins mediate an interaction between clathrin-coated vesicles and polymerizing actin filaments.

A monoclonal antibody, A-7C11, was generated which reacts with two polypeptides of 40 kDa and 80 kDa associated with the coat proteins of purified brain clathirn-coated vesicles. The 40-kDa antigen was purified and found to display actin-binding properties. Negative-staining electron microscopy showed that one of the antigens reactive with A-7C11 appears to mediate the association of isolated clathrin-coated vesicles with assembling actin filaments in vitro.

Equilibrium kinetics model for the cGMP-stimulated phosphodiesterase of brain coated vesicles.

An equilibrium kinetics model is proposed to described some of the enzymatic properties of the cyclic GMP-stimulated phosphodiesterase activity associated with brain clathrin coated vesicles. The model assumes the presence of pharmacologically distinct regulatory and catalytic domains in the enzyme. The model contemplates that random fashion occupancy of the regulatory site by the substrate, cyclic GMP, induces a conformational change which leads to the generation of an actived catalytic state.

Immunoprecipitation of bovine brain membranes enriched in M1 and M2 muscarinic receptors with monoclonal antibody 10C7.

Monoclonal antibodies prepared against clathrin-coated vesicles cargo molecules were screened for their ability to precipitate the mAChR binding activity present in a light-density smooth membrane fraction from bovine brain called peak I. Only monoclonal antibody 10C7 was able to selectively sediment 38% (+/- 4.2) of the mAChR binding activity of peak I.

Novel regulatory role of phosphorylated clathrin light chain beta in bovine brain coated vesicles.

The 50-kilodalton (kDa) assembly polypeptide of bovine brain clathrin coated vesicles (CCVs) is phosphorylated in a cyclic nucleotide- and Ca2+-independent manner and is dephosphorylated by a Mg2+-ATP-dependent CCV phosphatase. This report provides evidence for modulation of the phosphorylation reaction of the 50-kDa assembly polypeptide by phosphorylated clathrin light chain beta (pLC beta). In vitro, phosphorylated LC beta inhibits phosphorylation of the 50-kDa polypeptide in CCVs.

Clathrin-coated vesicle subtypes in mammalian brain tissue: detection of polypeptide heterogeneity by immunoprecipitation with monoclonal antibodies.

A panel of monoclonal antibodies (mAbs) was developed to identify polypeptides sorted in subtypes of brain coated vesicles (CVs) and to separate these by immunoprecipitation. The corresponding antigen of some of the mAbs elicited by CV components was present also in synaptosomal plasma membrane, synaptic vesicles, or microsomes. On immunoblots the mAbs reacted with constitutive brain CV proteins, with cargo molecules, and with a novel CV component that interacts with the actin cytoskeleton.

Phosphorylation of tubulin by casein kinase II regulates its binding to a neuronal protein (NP 185) associated with brain coated vesicles.

We recently described a new protein associated exclusively with neuronal clathrin-coated vesicles (CCVs), and characterized two monoclonal antibodies that react with it (S-8G8 and S-6G7). In this report, the association of neuronal protein of 185 kilodaltons (NP185) with CCV kinases and its interaction with tubulin are described. The affinity of NP185 for tubulin is significantly enhanced when tubulin is phosphorylated by CCV-associated casein kinase II.

A neuronal protein (NP185) associated with clathrin-coated vesicles. Characterization of NP185 with monoclonal antibodies.

Two monoclonal antibodies (S-8G8 and S-6G7) are characterized that react with an abundant neuronal protein associated with brain clathrin-coated vesicles (CCVs). This 185-kDa polypeptide (NP185) is not a transmembrane cargo molecule and is distinguishable from clathrin by several criteria including neuronal specificity, chymotryptic sensitivity, migration during two-dimensional gel electrophoresis, lack of cross-reactivity of S-8G8 or S-6G7 with purified clathrin, and lack of associated clathrin light chains. When 0.

Immunological and structural homology between human T-cell leukemia virus type I envelope glycoprotein and a region of human interleukin-2 implicated in binding the beta receptor.

The N-terminal segment of human interleukin-2 (hIL-2) appears to mediate binding of the beta hIL-2 receptor (R. Robb, C. Rusk, J.

Brain coated vesicle destabilization and phosphorylation of coat proteins.

Two basic polypeptides, bee venom melittin and poly-L-lysine, induced concentration-dependent destabilization of bovine brain coated vesicles. Ultrastructurally the changes observed were aggregation of clathrin coats and segregation of the vesicle membrane, concomitant with the appearance of elongated cisternae of various sizes. Changes in coated vesicle morphology induced by melittin and poly-L-lysine were concurrent with stimulation of phosphate incorporation in proteins of the coat lattice: Mr 33,000 and 100,000.

Phosphorylation characteristics of brain clathrin-coated vesicle endogenous proteins.

Clathrin-coated vesicles purified from bovine brain express protein kinase activity on two principal endogenous vesicle-associated substrates: a 50,000-Mr polypeptide (pp50) and clathrin-associated protein2 (CAP2; the faster-migrating clathrin light chain). Various exogenous substrates, e.g.

Mapping two functional domains of clathrin light chains with monoclonal antibodies.

The two forms of clathrin light chains (LCA and LCB) or clathrin-associated proteins (CAP1 and CAP2) have presented an immunochemical paradox. Biochemically similar, both possess two known functional parameters: binding the clathrin heavy chain and mediating the action of an uncoating ATPase. All previously reported anti-CAP mAbs, however, react specifically with only CAP1 (Brodsky, F.

Evidence for the presence of muscarinic acetylcholine receptors in bovine brain coated vesicles.

Evidence obtained from quinuclidinylbenzilate binding determinations suggested that muscarinic acetylcholine receptor molecules are present in purified bovine brain coated vesicles. Immunoprecipitates formed from coated vesicles with polyclonal antibodies to clathrin bound on the surface of fixed Staphylococcus aureus cells also showed quinuclidinylbenzilate binding activity. The high purity of coated vesicles was established by assays for biochemical markers and by electron microscopy.

Calmodulin immunoelectron microscopy: redistribution during ram spermatogenesis and epididymal maturation. II.

Affinity-purified monospecific antibodies and indirect immunogold and immunoferritin labeling on ultra-thin sections of low-temperature Lowicryl K4M-embedded samples were used to study the redistribution of calmodulin in ram spermatids and epididymal spermatozoa at the electron microscopic level. Calmodulin appeared as an integral component of well-defined structures or organelles of these cells. In young spermatids, calmodulin was localized in the nucleus, cytoplasm, and developing acrosome.

Immunoelectron microscopic localization of calmodulin and phospholipase A2 in spermatozoa. I.

Using the Lowicryl K4M embedding technique, together with indirect immunoferritin or immunogold labeling on ultra-thin sections, tubulin, calmodulin and phospholipase A2 were distinctly localized in ejaculated bull spermatozoa. Calmodulin was concentrated on the plasma membrane, nucleus, post-acrosomal substance, and, in lesser amounts, between coarse fibers and axonemal microtubules of the flagellum. Phospholipase A2 was distributed evenly along the plasma membrane, nucleus, acrosome, post-acrosomal substance, and in the flagellum, on mitochondria, fibrous sheath, coarse fibers, between coarse fibers and axonemal microtubules.

Brain protein kinase in synaptic vesicles is inhibited by a factor in sera from systemic lupus erythematosus patients with central nervous system manifestations.

Sera from systemic lupus erythematosus patients with clinical central nervous system manifestations had a high mean percent of brain synaptic vesicle protein kinase inhibition (62 +/- 9.3, P less than 0.001).

Cyclic nucleotide phosphodiesterase activity in bovine brain coated vesicles.

Cyclic AMP phosphodiesterase activity in bovine brain coated vesicles displayed a Km of approximately 22 microM for cyclic AMP, a Vmax of 3.2 nmol/min/mg protein, and a Hill coefficient of 1.5, suggesting positive cooperativity.