Gerard J. M. Martens

Secretory granule biogenesis: rafting to the SNARE

Regulated secretion of hormones occurs when a cell receives an external stimulus, triggering the secretory granules to undergo fusion with the plasma membrane and release their content into the extracellular milieu. The formation of a mature secretory granule (MSG) involves a series of discrete and unique events such as protein sorting, formation of immature secretory granules (ISGs), prohormone processing and vesicle fusion. Regulated secretory proteins (RSPs), the proteins stored and secreted from MSGs, contain signals or domains to direct them into the regulated secretory pathway. Recent data on the role of specific domains in RSPs involved in sorting and aggregation suggest that the cell-type-specific composition of RSPs in the trans-Golgi network (TGN) has an important role in determining how the RSPs get into ISGs. The realization that lipid rafts are implicated in sorting RSPs in the TGN and the identification of SNARE molecules represent further major advances in our understanding of how MSGs are formed. At the heart of these findings is the elucidation of molecular mechanisms driving protein--lipid and protein--protein interactions specific for secretory granule biogenesis.

7B2 is a neuroendocrine chaperone that transiently interacts with prohormone convertase PC2 in the secretory pathway

The neuroendocrine polypeptide 7B2 is a highly conserved secretory protein selectively present in prohormone-producing cells equipped with a regulated secretory pathway. We find that the amino-terminal half of 7B2 is distantly related to chaperonins, a subclass of molecular chaperones. When incubated in vitro with newly synthesized pituitary proteins, recombinant 7B2 specifically associates with prohormone convertase PC2. Metabolic cell labeling combined with coimmunoprecipitation studies showed that, in vivo, the precursor form of 7B2 interacts with the proform of PC2. Pulse-chase analysis revealed that this association is transient in that it commences early in the secretory pathway, while dissociation in the later stages appears to coincide with the cleavages of 7B2, proPC2, and prohormone. Our results suggest that 7B2 is a novel type of molecular chaperone preventing premature activation of proPC2 in the regulated secretory pathway.

MicroRNA networks direct neuronal development and plasticity

MicroRNAs (miRNAs) constitute a class of small, non-coding RNAs that act as post-transcriptional regulators of gene expression. In neurons, the functions of individual miRNAs are just beginning to emerge, and recent studies have elucidated roles for neural miRNAs at various stages of neuronal development and maturation, including neurite outgrowth, dendritogenesis, and spine formation. Notably, miRNAs regulate mRNA translation locally in the axosomal and synaptodendritic compartments, and thereby contribute to the dynamic spatial organization of axonal and dendritic structures and their function. Given the critical role for miRNAs in regulating early brain development and in mediating synaptic plasticity later in life, it is tempting to speculate that the pathology of neurological disorders is affected by altered expression or functioning of miRNAs. Here we provide an overview of recently identified mechanisms of neuronal development and plasticity involving miRNAs, and the consequences of miRNA dysregulation.

The p24 family and selective transport processes at the ER-Golgi interface

The secretory pathway is of vital importance for eukaryotic cells and has a pivotal role in the synthesis, sorting, processing and secretion of a large variety of bioactive molecules involved in intercellular communication. One of the key processes in the secretory pathway concerns the transport of cargo proteins from the ER (endoplasmic reticulum) to the Golgi. Type‐I transmembrane proteins of ∼24 kDa are abundantly present in the membranes of the early secretory pathway, and bind the COPI and COPII coat complexes that cover vesicles travelling between the membranes. These p24 proteins are thought to play an important role in the selective transport processes at the ER—Golgi interface, although their exact functioning is still obscure. One model proposes that p24 proteins couple cargo selection in the lumen with vesicle coat recruitment in the cytosol. Alternatively, p24 proteins may furnish subcompartments of the secretory pathway with the correct subsets of machinery proteins. Here we review the current knowledge of the p24 proteins and the various roles proposed for the p24 family members.

Cloning and expression of two mRNAs encoding structurally different crustacean hyperglycemic hormone precursors in the lobster Homarus americanus

The crustacean hyperglycemic hormone (CHH) of the X-organ sinus gland complex is a multifunctional neurohormone primarily involved in the regulation of blood sugar levels. HPLC analysis of lobster sinus glands revealed two CHH-immunoreactive groups, each consisting of two isoforms with identical amino acid sequences and molecular weights. In order to obtain more information concerning the number and sequences of preproCHHs, and to study their expression, we isolated two full-length cDNAs encoding two different CHH preprohormones. Both preprohormone structures consist of a signal peptide, a CHH-precursor-related peptide and a highly-conserved CHH peptide. Expression studies revealed that the X-organ is not the only source of CHH mRNA because the ventral nerve system also expresses this mRNA. Based on these findings and earlier studies on the effect of eyestalk ablation, implantation of thoracic/abdominal ganglia as well as the multifunctionality of CHH, we postulate that CHH, present in the ventral nerve system is a good candidate for a supplementary role in the control of reproduction and molting.

Cloning and expression of two proopiomelanocortin mRNAs in the common carp (Cyprinus carpio L.)

Proopiomelanocortin (POMC) is the precursor for a number of biologically active peptides such as adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH) and beta-endorphin. It is well known that these peptides are involved in the stress response in fish as well as in mammals. We have cloned two different carp POMC cDNAs called, POMC-I and POMC-II. The nucleotide sequences of 955 bp for POMC-I and 959 bp for POMC-II share 93.5% identity in their cDNAs, and the deduced amino acid sequences (both 222 amino acids) are 91.4% identical. In the ACTH and beta-MSH domain, two amino acid substitutions are found, whereas alpha-MSH and beta-endorphin are identical. For beta-MSH, the serine replacement (in POMC-I) by a glycine (in POMC-II) results in a putative amidation site Pro-X-Gly for POMC-II. We used RT-PCR to show that both POMC mRNAs are expressed in the hypophysis, hypothalamus and other parts of the brain of a single fish. Furthermore, in a phylogenetic tree based on POMC sequences the divergence of carp POMC-I and -II from tetraploid animals (salmon, trout and xenopus) is demonstrated.

Cloning and expression of mRNA encoding prepro-gonad-inhibiting hormone (GIH) in the lobster Homarus americanus

The gonad‐inhibiting hormone (GIH) is produced in the eyestalk X‐organ sinus gland complex of male and female lobsters, and plays a prominent role in the regulation of reproduction, e.g. inhibition of vitellogenesis in female animals. To study this neurohormone at the mRNA level, we cloned and sequenced a cDNA which encodes GIH in the lobster Homarus americanus. The structure of preproGIH consists of a signal peptide and the GIH peptide itself. A comparative analysis revealed that lobster GIH, together with crab molt‐inhibiting hormone, belongs to a separate group of the crustacean hyperglycemic hormone (CHH) peptide family which seems to be unique for crustaceans. Expression studies showed that GIH mRNA is expressed in the eyestalk, indicating that the neuroendocrine center in this optic structure is the only source of GIH. As this center modulates the other (neuro)endocrine organs in crustaceans, it is postulated that GIH regulates production and release of hormones involved in reproduction/molting processes.

Cloning and sequence analysis of human pituitary cDNA encoding the novel polypeptide 7B2

Application of a differential hybridization technique led to the isolation of a human pituitary cDNA clone encoding the complete structure of the polypeptide 7B2. This protein of unknown function, which is sorted to secretory granules, appears to be present selectively in neurons and endocrine cells. The polypeptide chain of human 7B2, preceded by a cleaved signal peptide, comprises 185 amino acids (a calculated M r of 20 793). Interesting features of the highly‐conserved 7B2 structure include (i) a serine phosphorylation consensus sequence, (ii) the occurrence of three pairs of dibasic amino acids representing potential proteolytic cleavage sites and, in particular, (iii) the presence of three regions homologous to GTP‐binding domains giving 7B2 structural characteristics of a GTP‐binding protein.

Cell-Autonomous TrkB Signaling in Presynaptic Retinal Ganglion Cells Mediates Axon Arbor Growth and Synapse Maturation during the Establishment of Retinotectal Synaptic Connectivity

BDNF contributes to the activity-dependent establishment and refinement of visual connectivity. In Xenopus, BDNF applications in the optic tectum influence retinal ganglion cell (RGC) axon branching and promote synapse formation and stabilization. The expression patterns of BDNF and TrkB suggest that BDNF specifically regulates the maturation of RGC axons at the target. It is possible, however, that BDNF modulates retinotectal synaptic connectivity by differentially influencing presynaptic RGC axons and postsynaptic tectal cells. Here, we combined single-cell expression of a dominant-negative TrkB–enhanced green fluorescent protein (GFP) fusion protein with confocal microscopy imaging in live Xenopus tadpoles to differentiate between presynaptic and postsynaptic actions of BDNF. Disruption of TrkB signaling in individual RGCs influenced the branching and synaptic maturation of presynaptic axon arbors. Specifically, GFP–TrkB.T1 overexpression increased the proportion of axons with immature, growth cone-like morphology, decreased axon branch stability, and increased axon arbor degeneration. In addition, GFP–TrkB.T1 overexpression reduced the number of red fluorescent protein–synaptobrevin-labeled presynaptic specializations per axon terminal. In contrast, overexpression of GFP–TrkB.T1 in tectal neurons did not alter synaptic number or the morphology or dynamic behavior of their dendritic arbors. Electron microscopy analysis revealed a significant decrease in the number of mature synaptic profiles and in the number of docked synaptic vesicles at retinotectal synapses made by RGC axons expressing GFP–TrkB.T1. Together, our results demonstrate that presynaptic TrkB signaling in RGCs is a key determinant in the establishment of visual connectivity and indicate that changes in tectal neuron synaptic connectivity are secondary to the BDNF-elicited enhanced stability and growth of presynaptic RGCs.

Proton pumping in the secretory pathway.

Protons are pumped into intracellular compartments (e.g., lysosomes, endosomes, coated vesicles, synaptic vesicles and secretory granules) to create a proton motive force across the membrane or to acidify the lumen, which is necessary to perform cellular processes such as hydrolysis of macromolecules, receptor-mediated endocytosis and processing of preproproteins. The proton gradient is established and maintained by the H -pumping vacuolartype ATPase (V-ATPase). Here we discuss this multisubunit enzyme, the importance of proton pumping in the various organelles of the secretory pathway, and the possible types of regulatory mechanisms that may control V-ATPase activity.