We have cloned four members of the family of subtilisin-like endoproteases expressed in the bag cell neurons of Aplysia and have demonstrated that two of these enzymes are capable of correctly cleaving the egg-laying hormone precursor. The egg-laying hormone precursor undergoes an ordered series of cleavages, such that different peptides are differentially sorted into distinct secretory vesicles. We have used electron microscopic chemistry to demonstrate that at least one processing enzyme is differentially segregated into a class of secretory vesicles containing the bag cell peptides. The segregation of specific endoproteases, along with specific neuropeptides within a given cell type, may ensure appropriate cleavage and prevent inappropriate cleavage of the polyprotein precursors.

Posttranslational processing of many proteins is essential to the synthesis of fully functional molecules. The ELH (egg-laying hormone) prohormone is cleaved by endoproteases in a specific order at a variety of basic residue processing sites to produce mature peptides. The prohormone is first cleaved at a unique tetrabasic site liberating two intermediates (amino and carboxy) which are sorted to different classes of dense core vesicles in the bag cell neurons of Aplysia. When expressed in AtT-20 cells, the ELH prohormone is also first cleaved at the tetrabasic site. The amino-terminal intermediate is then sorted to the constitutive pathway, and a portion of the carboxy-terminal intermediate is sorted to the regulated pathway. Here, we use mutant constructs of the ELH prohormone expressed in AtT-20 cells to examine the relationship between prohormone processing and consequent sorting. Prohormone which has a dibasic site in place of the tetrabasic site is processed and sorted similarly to wild type. Furthermore, mutant prohormone which lacks the tetrabasic site is processed at an alternative site comprising three basic residues. In these mutant prohormones, mature ELH is still produced and stored in dense core vesicles while amino-terminal products are constitutively secreted. However, deletion of the tetrabasic and tribasic sites results in the rerouting of the amino-terminal intermediate products from the constitutive pathway to the regulated secretory pathway. Thus, in the ELH prohormone, the location of the proteolytic processing events within the secretory pathway and the order of cleavages regulate the sorting of peptide products.

Prohormone structure governs proteolytic processing and sorting in the Golgi complex. In the trans-Golgi network (TGN), neuropeptides are segregated from other secretory proteins and are packaged into dense-core vesicles (DCVs). These vesicles are then transported to the nerve terminal and their contents are released by exocytosis when the appropriate signal is received by the neuron. Processing begins with the removal of the signal sequence as the nascent peptide enters the endoplasmic reticulum . Hence, elucidating the molecular and cellular mechanisms governing these processing events is the key to fully understanding the regulation of neuropeptide biosynthesis. This chapter discusses (1) the structural features of the prohormone important for processing, (2) the processing enzymes, and (3) how the processing sequence of the egg-laying hormone (ELH) prohormone influences the sorting of peptides derived from this molecule. Using the ELH prohormone to study the relationship between processing and sorting has opened up new avenues of thought. In the past, it was learned that cellular mechanisms can control the relative levels and spatial localization of different neuropeptides derived from a common precursor. However, recent results indicate that perturbation of a processing site can cause processing in a different locale in the cell and result in altered pathways of sorting. Defining the hierarchy of processing enzymes and their localization will be crucial to future work in this area and to understand how the design of prohormones complements the repertoire of processing enzymes.

Agrin is a component of the synaptic extracellular matrix and may regulate the organization of acetylcholine receptors and other synaptic molecules in both synapse regeneration and development. Analyses of cDNAs encoding agrin define a number of structural domains, including regions of homology to laminin, Kazal protease inhibitors, and epidermal growth factor repeats.

Potential interactions between membrane components of rat brain synaptic vesicles were analyzed by detergent solubilization followed by size fractionation or immunoprecipitation. The behavior of six synaptic vesicle membrane proteins as well as a plasma membrane protein was monitored by Western blotting. Solubilization of synaptic vesicle membranes in CHAPS resulted in the recovery of a large protein complex that included SV2, p65, p38, vesicle-associated membrane protein, and the vacuolar proton pump. Solubilization in octylglucoside resulted in the preservation of interactions between SV2, p38, and rab3A, while solubilization of synaptic vesicles with Triton X-100 resulted in two predominant interactions, one involving p65 and SV2, and the other involving p38 and vesicle-associated membrane protein. The multicomponent complex preserved with CHAPS solubilization was partially reconstituted following octylglucoside solubilization and subsequent dialysis against CHAPS. Reduction of the CHAPS concentration by gel filtration chromatography resulted in increased recovery of the multicomponent complex. Examination of the large complex isolated from CHAPS-solubilized vesicles by negative stain EM revealed structures with multiple globular domains, some of which were specifically labeled with gold-conjugated antibodies directed against p65 and SV2. The protein interactions defined in this report are likely to underlie aspects of neurotransmitter secretion, membrane traffic, and the spatial organization of vesicles within the nerve terminal.

One of the important events in synapse formation is the accumulation of neurotransmitter receptors beneath the presynaptic nerve terminal. Agrin is a component of the synaptic basal lamina that induces the clustering of acetylcholine receptors when bath-applied to muscle fibers in culture. When a cDNA encoding a putative agrin protein is transfected into cells, the molecule is secreted and concentrated on the extracellular surface. Coculture of transfected cells with muscle fibers induces the formation of receptor patches at contact sites. These results demonstrate that expression of a single gene encoding agrin confers receptor clustering that is restricted to specific sites of cell-muscle contact.

Membrane traffic has been shown to be regulated during cell division. In particular, with the use of viral membrane proteins as markers, endoplasmic reticulum (ER)-to-Golgi transport in mitotic cells has been shown to be essentially blocked. However, the effect of mitosis on other steps in the secretory pathway is less clear, because an early block makes examination of following steps difficult. Here, we report studies on the functional characteristics of secretory pathways in mitotic mammalian tissue culture cells by the use of a variety of markers. Chinese hamster ovary cells were transfected with cDNAs encoding secretory proteins. Consistent with earlier results following viral membrane proteins, we found that the overall secretory pathway is nonfunctional in mitotic cells, and a major block to secretion is at the step between ER and Golgi: the overall rate of secretion of human growth hormone is reduced at least 10-fold in mitotic cells, and export of truncated vesicular stomatitis virus G protein from the ER is inhibited to about the same extent, as judged by acquisition of endoglycosidase H resistance. To ascertain the integrity of transport from the trans-Golgi to plasma membrane, we followed the secretion of sulfated glycosaminoglycan (GAG) chains, which are synthesized in the Golgi and thus are not subject to the earlier ER-to-Golgi block. GAG chains are valid markers for the pathway taken by constitutive secretory proteins; both protein secretion and GAG chain secretion are sensitive to treatment with n-ethyl-maleimide and monensin and are blocked at 19 degrees C. We found that the extent of GAG-chain secretion is not altered during mitosis, although the initial rate of secretion is reduced about twofold in mitotic compared with interphase cells. Thus, during mitosis, transport from the trans-Golgi to plasma membrane is much less hindered than ER-to-Golgi traffic. We conclude that transport steps are not affected to the same extent during mitosis.

The bag cell neurons in the marine snail Aplysia synthesize large amounts of the egg-laying hormone (ELH) prohormone. The ELH precursor is proteolytically processed into 9 peptides making this a useful system for studying prohormone processing and the sorting of proteins destined for the secretory pathway. The peptides derived from the ELH prohormone are differentially packaged into four distinct classes of dense cored vesicles (DVCs). Dense cored vesicles in the large class are greater than 250 nm in diameter, contain the 6 peptides derived from the aminoterminus of the prohormone and are localized to the cell soma and not the neuronal processes. Here we demonstrate that the large DCVs are enriched in prohormone processing intermediates. In addition, many of the large DCVs do not contain acid phosphatase activity suggesting they are an organelle distinct from the lysosomes and that different classes of DCVs may subserve unique functions within the secretory pathway.

We have generated a monoclonal antibody (mAb) 5E10 which recognizes an antigen localized to dense core vesicles (DCVs) in the atrial gland of Aplysia californica. mAb5E10 immunoprecipitates an abundant 57-kDa glycoprotein (atrial gland granule-specific antigen, AGSA) which is a soluble component of atrial gland DCVs. Electron microscopy reveals that AGSA immunoreactivity is restricted to the region between the dense core, which contains neuropeptide immunoreactivity, and the membrane of atrial gland DCVs. AGSA was purified by immunoaffinity chromatography, and the amino acid sequences of both N-terminal and internal cyanogen bromide fragments were determined. This information was used to isolate a 2.8-kilobase cDNA which encodes a 47-kDa protein. The predicted amino acid sequence contains the micro-sequenced peptides, an N-terminal hydrophobic signal sequence, and four N-linked glycosylation sites, but does not contain any significant homologies to database sequences. Northern blots and light level immunocytochemistry demonstrate that the AGSA gene is specifically expressed in the atrial gland. The identification of a protein localized to the cortex of DCVs suggests that this region has a specialized role in the function of these vesicles.

The cellular and synaptic morphology of a component of the feeding motor circuit in Aplysia californica was examined with light and electron microscopic techniques. The circuit consists of a pair of inhibitory premotor interneurons, B4 and B5, as well as two motoneurons, B15 and B16, which innervate the accessory radula closer muscle. The neurons have wide, varicose arborizations in the buccal ganglion neuropil. All four of these neurons are cholinergic, and in addition, B15 contains immunoreactivity to sera raised against small cardioactive peptide B. Varicose processes in the accessory radula closer muscle are immunoreactive with antisera against several neuropeptides. We identified specific neuromuscular junctions by visualizing horseradish peroxidase uptake in recycled synaptic vesicles. Direct innervation of the accessory radula closer muscle by B15 and B16 is demonstrated by experiments in which horseradish peroxidase is transported from motoneuronal soma to the terminals on muscle fibers. In addition, specific synaptic contacts between B4 and B5 and each of the motoneurons are observed in the buccal ganglion neuropil. Finally, multiple contacts consistent with peptidergic, serotoninergic, and cholinergic synapses are made onto the neurons, suggesting that a variety of transmitters modulate motor output at each level of the hierarchical circuit. These results support the physiological evidence suggesting the involvement of neuropeptides as well as "classical" transmitters in the modulation of circuitry governing feeding behavior in Aplysia.

Many neurons in the mollusc Aplysia are identifiable and provide a useful model system for investigating the cellular mechanisms used by the neuroendocrine system to mediate simple behaviors. In this study we determined the subcellular localization of eight Aplysia neuropeptides using immunogold labeling techniques, and analyzed the size distribution of dense core and granular vesicles in peptidergic neurons. Recent observations demonstrate that many neurons use multiple chemical messengers. Thus, an understanding of the functional significance of cotransmitters requires an analysis of their relative subcellular distributions. The peptides are expressed in a subset of neurons, or the exocrine atrial gland, and are primarily localized to dense core vesicles. Multiple regions of precursors which are cleaved into several components are co-localized. Each neuron has a distinct size distribution of peptide-containing dense core vesicles ranging in size from 65 to 600 nm. The atrial gland contains very large (up to 2 micron) peptide-containing granules. Single neurons have multiple populations of granules whose quantal sizes agree with predictions based on physical constraints. Some cells contain very large peptide- containing granules which are found in the cell soma and not in processes. Thus, the genetic determination of neuronal cell type includes not only transmitter choices but also multiple modes of packaging the intercellular messengers.

The neuroactive peptide Phe-Met-Arg-Phe-NH2 (FMRFamide) has a variety of effects on both mammalian and invertebrate tissues; moreover, FMRFamide-like immunoreactivity is found throughout the animal kingdom. Here we describe the isolation and characterization of a cDNA clone from an Aplysia abdominal ganglion cDNA library that encodes a precursor protein that may give rise to as many as 19 individual FMRFamide peptides. Nearly all of the FMRF sequences are flanked on the amino terminus by Lys-Arg residues and on the carboxy terminus by Gly-Lys residues,suggesting that the single lysine residues function to signal cleavage by processing enzymes. The gene is present in a single copy per haploid genome and gives rise to multiple transcripts, at least some of which appear to arise through alternate RNA splicing. Immunohistochemical analysis suggests that the peptide is present in many neurons throughout the Aplysia nervous system and that these neurons send processes to a variety of different tissues.

The Aplysia neuroendocrine system is a particularly advantageous model for cellular and molecular studies because of the relatively small number and large size of its component neurons. Recombinant DNA techniques have been used to isolate the genes that encode the precursors of peptides expressed in identified neurons of known function. The organization and developmental expression of these genes have been examined in detail. Several of the genes encode precursors of multiple biologically active peptides that are expressed in cells which also contain classical transmitters. These studies, as well as immunohistochemical studies and the use of intracellular recording and voltage clamp techniques are the first steps toward revealing the mechanisms by which neuropeptides govern simple behaviors.

We previously isolated and characterized a cDNA clone specifically expressed in neurons R3 to R8 and R14 of the Aplysia abdominal ganglion (Nambu, J.R., R. Taussig, A.C. Mahon, and R.H. Scheller (1983) Cell 35: 47–56). The cDNA nucleotide sequence and the inferred protein amino acid sequence suggest that this gene encodes the precursor for neuroactive peptides used by these cells. Peptides corresponding to three regions of the precursor were synthesized, coupled to a protein carrier, and used to generate antibodies. These antibodies stain a set of cell bodies, R3 to R14, and their processes in the abdominal ganglion; no other cells in the nervous system or the periphery are immunoreactive. R3 to R14 send numerous fine immunoreactive processes into the vascularized sheath that surrounds the ganglion. Each of these cells also has a large axon which exits the ganglion via the branchial nerve and terminates on the heart. In addition, R14 is anatomically distinct from R3 to R13 in that it sends additional immunoreactive processes to the vasculature near the ganglion. Immunoreactive processes and varicosities were observed on the efferent vein of the gill, the abdominal ganglion artery, and the anterior aorta. These data are consistent with previous studies suggesting that one or more neuropeptides released from R3 to R14 function as modulators of cardiovascular physiology.