A Single Membrane Protein Required for Atrial Secretory Granule Formation

AbstractThe discovery of atrial secretory granules and the natriuretic peptides stored in them identified the atrium as an endocrine organ. Although neither atrial nor brain natriuretic peptide (ANP, BNP) is amidated, the major membrane protein in atrial granules is Peptidylglycine α-Amidating Monooxygenase (PAM), an enzyme essential for amidated peptide biosynthesis. Mice lacking cardiomyocyte PAM (PamMyh6-cKO/cKO) are viable, but a gene dosage-dependent drop in atrial ANP and BNP content occurred. Ultrastructural analysis of adult PamMyh6-cKO/cKO atria revealed a 20-fold drop in the volume fraction of secretory granules and a decrease in peripherally localized Golgi complexes. When primary cultures of Pam0-Cre-cKO/cKO atrial myocytes (PAM floxed, no Cre recombinase) were transduced with Cre-GFP lentivirus, PAM protein levels dropped, followed by a decline in proANP levels. Expression of exogenous PAM in PamMyh6-cKO/cKO atrial myocytes produced a dose-dependent increase in proANP content. Strikingly, rescue of proANP content did not require the monooxygenase activity of PAM. Unlike many prohormones, atrial proANP is stored intact and its basal secretion is stimulated by drugs that inhibit Golgi-localized Arf activators. Increased basal secretion of proANP was a major contributor to its reduced levels in PamMyh6-cKO/cKO myocytes; the inability of these drugs to inhibit basal proANP secretion by PamMyh6-cKO/cKO myocytes revealed a role for COPI-mediated recycling of PAM to the endoplasmic reticulum. Analysis of atrial coated vesicles and the ability PAM to make fluorescently-tagged proANP accumulate in the cis-Golgi region of cells lacking secretory granules revealed a non-catalytic role for PAM in soluble cargo trafficking early in the secretory pathway.SignificanceTransmission electron microscopy of atrial cardiomyocytes revealed dense granules resembling those in endocrine cells and neurons, leading to the discovery of the natriuretic peptides stored in these granules. Subsequent studies revealed features unique to atrial granules, including high level expression of Peptidylglycine α-Amidating Monooxygenase (PAM), an enzyme required for the synthesis of many neuropeptides, but not for the synthesis of natriuretic peptides. The discovery that atrial myocytes lacking PAM are unable to produce granules and that PAM lacking its monooxygenase activity can rescue granule formation provides new information about the proANP secretory pathway. A better understanding of the unique features of atrial cell biology should provide insight into atrial fibrillation, the most common cardiac arrhythmia, atrial amyloidosis and heart failure.

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Peptidylglycine α-amidating monooxygenase is required for atrial secretory granule formation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2004410117 ◽

2020 ◽

Vol 117 (30) ◽

pp. 17820-17831 ◽

Cited By ~ 1

Author(s):

Nils Bäck ◽

Raj Luxmi ◽

Kathryn G. Powers ◽

Richard E. Mains ◽

Betty A. Eipper

Keyword(s):

Gene Dosage ◽

Secretory Granules ◽

Primary Cultures ◽

Atrial Myocytes ◽

Granule Formation ◽

Monooxygenase Activity ◽

Protein Levels ◽

Golgi Region ◽

Copi Vesicle ◽

Amidated Peptide

The discovery of atrial secretory granules and the natriuretic peptides stored in them identified the atrium as an endocrine organ. Although neither atrial nor brain natriuretic peptide (ANP, BNP) is amidated, the major membrane protein in atrial granules is peptidylglycine α-amidating monooxygenase (PAM), an enzyme essential for amidated peptide biosynthesis. Mice lacking cardiomyocyte PAM (PamMyh6-cKO/cKO) are viable, but a gene dosage-dependent drop in atrial ANP and BNP content occurred. Ultrastructural analysis of adultPamMyh6-cKO/cKOatria revealed a 13-fold drop in the number of secretory granules. When primary cultures ofPam0-Cre-cKO/cKOatrial myocytes (no Cre recombinase, PAM floxed) were transduced with Cre-GFP lentivirus, PAM protein levels dropped, followed by a decline in ANP precursor (proANP) levels. Expression of exogenous PAM inPamMyh6-cKO/cKOatrial myocytes produced a dose-dependent rescue of proANP content; strikingly, this response did not require the monooxygenase activity of PAM. Unlike many prohormones, atrial proANP is stored intact. A threefold increase in the basal rate of proANP secretion byPamMyh6-cKO/cKOmyocytes was a major contributor to its reduced levels. While proANP secretion was increased following treatment of control cultures with drugs that block the activation of Golgi-localized Arf proteins and COPI vesicle formation, proANP secretion byPamMyh6-cKO/cKOmyocytes was unaffected. In cells lacking secretory granules, expression of exogenous PAM led to the accumulation of fluorescently tagged proANP in thecis-Golgi region. Our data indicate that COPI vesicle-mediated recycling of PAM from thecis-Golgi to the endoplasmic reticulum plays an essential role in the biogenesis of proANP containing atrial granules.

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Sorting and storage during secretory granule biogenesis: looking backward and looking forward

Biochemical Journal ◽

10.1042/bj3320593 ◽

1998 ◽

Vol 332 (3) ◽

pp. 593-610 ◽

Cited By ~ 363

Author(s):

Peter ARVAN ◽

David CASTLE

Keyword(s):

Secretory Pathway ◽

Molecular Mechanisms ◽

Secretory Granules ◽

Secretory Proteins ◽

Membrane Composition ◽

Granule Formation ◽

Granule Membrane ◽

Regulated Secretory Pathway ◽

Secretory Products

Secretory granules are specialized intracellular organelles that serve as a storage pool for selected secretory products. The exocytosis of secretory granules is markedly amplified under physiologically stimulated conditions. While granules have been recognized as post-Golgi carriers for almost 40 years, the molecular mechanisms involved in their formation from the trans-Golgi network are only beginning to be defined. This review summarizes and evaluates current information about how secretory proteins are thought to be sorted for the regulated secretory pathway and how these activities are positioned with respect to other post-Golgi sorting events that must occur in parallel. In the first half of the review, the emerging role of immature secretory granules in protein sorting is highlighted. The second half of the review summarizes what is known about the composition of granule membranes. The numerous similarities and relatively limited differences identified between granule membranes and other vesicular carriers that convey products to and from the plasmalemma, serve as a basis for examining how granule membrane composition might be established and how its unique functions interface with general post-Golgi membrane traffic. Studies of granule formation in vitro offer additional new insights, but also important challenges for future efforts to understand how regulated secretory pathways are constructed and maintained.

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Neuroendocrine secretory protein 7B2: structure, expression and functions

Biochemical Journal ◽

10.1042/bj3570329 ◽

2001 ◽

Vol 357 (2) ◽

pp. 329-342 ◽

Cited By ~ 78

Author(s):

Majambu MBIKAY ◽

Nabil G. SEIDAH ◽

Michel CHRÉTIEN

Keyword(s):

Secretory Pathway ◽

Secretory Granules ◽

Secretory Protein ◽

Neuroendocrine Cells ◽

High Similarity ◽

Granule Formation ◽

Proteolytic Activation ◽

Polyproline Motif

7B2 is an acidic protein residing in the secretory granules of neuroendocrine cells. Its sequence has been elucidated in many phyla and species. It shows high similarity among mammals. A Pro-Pro-Asn-Pro-Cys-Pro polyproline motif is its most conserved feature, being carried by both vertebrate and invertebrate sequences. It is biosynthesized as a precursor protein that is cleaved into an N-terminal fragment and a C-terminal peptide. In neuroendocrine cells, 7B2 functions as a specific chaperone for the proprotein convertase (PC) 2. Through the sequence around its Pro-Pro-Asn-Pro-Cys-Pro motif, it binds to an inactive proPC2 and facilitates its transport from the endoplasmic reticulum to later compartments of the secretory pathway where the zymogen is proteolytically matured and activated. Its C-terminal peptide can inhibit PC2 in vitro and may contribute to keep the enzyme transiently inactive in vivo. The PC2–7B2 model defines a new neuroendocrine paradigm whereby proteolytic activation of prohormones and proneuropeptides in the secretory pathway is spatially and temporally regulated by the dynamics of interactions between converting enzymes and their binding proteins. Interestingly, unlike PC2-null mice, which are viable, 7B2-null mutants die early in life from Cushing's disease due to corticotropin (‘ACTH’) hypersecretion by the neurointermediate lobe, suggesting a possible involvement of 7B2 in secretory granule formation and in secretion regulation. The mechanism of this regulation is yet to be elucidated. 7B2has been shown to be a good marker of several neuroendocrine cell dysfunctions in humans. The possibility that anomalies in its structure and expression could be aetiological causes of some of these dysfunctions warrants investigation.

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Vesicle-associated Membrane Protein 4 is Implicated inTrans-Golgi Network Vesicle Trafficking

Molecular Biology of the Cell ◽

10.1091/mbc.10.6.1957 ◽

1999 ◽

Vol 10 (6) ◽

pp. 1957-1972 ◽

Cited By ~ 96

Author(s):

Martin Steegmaier ◽

Judith Klumperman ◽

Davide L. Foletti ◽

Jin-San Yoo ◽

Richard H. Scheller

Keyword(s):

Electron Microscopy ◽

Membrane Protein ◽

Rat Brain ◽

Pc12 Cells ◽

Golgi Complex ◽

Subcellular Distribution ◽

Secretory Pathway ◽

Secretory Granules ◽

Golgi Network ◽

Lines Of Evidence

The trans-Golgi network (TGN) plays a pivotal role in directing proteins in the secretory pathway to the appropriate cellular destination. VAMP4, a recently discovered member of the vesicle-associated membrane protein (VAMP) family of trafficking proteins, has been suggested to play a role in mediating TGN trafficking. To better understand the function of VAMP4, we examined its precise subcellular distribution. Indirect immunofluorescence and electron microscopy revealed that the majority of VAMP4 localized to tubular and vesicular membranes of the TGN, which were in part coated with clathrin. In these compartments, VAMP4 was found to colocalize with the putative TGN-trafficking protein syntaxin 6. Additional labeling was also present on clathrin-coated and noncoated vesicles, on endosomes and the medial and trans side of the Golgi complex, as well as on immature secretory granules in PC12 cells. Immunoprecipitation of VAMP4 from rat brain detergent extracts revealed that VAMP4 exists in a complex containing syntaxin 6. Converging lines of evidence implicate a role for VAMP4 in TGN-to-endosome transport.

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Determinants for chromogranin A sorting into the regulated secretory pathway are also sufficient to generate granule-like structures in non-endocrine cells

Biochemical Journal ◽

10.1042/bj20071382 ◽

2009 ◽

Vol 418 (1) ◽

pp. 81-91 ◽

Cited By ~ 26

Author(s):

Hansruedi Stettler ◽

Nicole Beuret ◽

Cristina Prescianotto-Baschong ◽

Bérengère Fayard ◽

Laurent Taupenot ◽

...

Keyword(s):

Chromogranin A ◽

Endocrine Cells ◽

Secretory Pathway ◽

Fluorescent Protein ◽

Secretory Granules ◽

Peptide Hormone ◽

Secretory Proteins ◽

Cell Periphery ◽

Granule Formation ◽

Regulated Secretory Pathway

In endocrine cells, prohormones and granins are segregated in the TGN (trans-Golgi network) from constitutively secreted proteins, stored in concentrated form in dense-core secretory granules, and released in a regulated manner on specific stimulation. The mechanism of granule formation is only partially understood. Expression of regulated secretory proteins, both peptide hormone precursors and granins, had been found to be sufficient to generate structures that resemble secretory granules in the background of constitutively secreting, non-endocrine cells. To identify which segment of CgA (chromogranin A) is important to induce the formation of such granule-like structures, a series of deletion constructs fused to either GFP (green fluorescent protein) or a short epitope tag was expressed in COS-1 fibroblast cells and analysed by fluorescence and electron microscopy and pulse-chase labelling. Full-length CgA as well as deletion constructs containing the N-terminal 77 residues generated granule-like structures in the cell periphery that co-localized with co-expressed SgII (secretogranin II). These are essentially the same segments of the protein that were previously shown to be required for granule sorting in wild-type PC12 (pheochromocytoma cells) cells and for rescuing a regulated secretory pathway in A35C cells, a variant PC12 line deficient in granule formation. The results support the notion that self-aggregation is at the core of granule formation and sorting into the regulated pathway.

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Localization of VIP36 in the Post-Golgi Secretory Pathway Also of Rat Parotid Acinar Cells

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215540305100809 ◽

2003 ◽

Vol 51 (8) ◽

pp. 1057-1063 ◽

Cited By ~ 13

Author(s):

Osamu Shimada ◽

Sayuri Hara-Kuge ◽

Katsuko Yamashita ◽

Hisami Tosaka-Shimada ◽

Li Yanchao ◽

...

Keyword(s):

Morphological Analysis ◽

Secretory Pathway ◽

Secretory Granules ◽

Acinar Cells ◽

Integral Membrane Protein ◽

Madin Darby Canine Kidney ◽

Granule Formation ◽

Rat Parotid ◽

Golgi Network ◽

Darby Canine Kidney

VIP36 (36-kD vesicular integral membrane protein), originally purified from Madin-Darby canine kidney (MDCK) epithelial cells, belongs to a family of animal lectins and may act as a cargo receptor. To understand its role in secretory processes, we performed morphological analysis of the rat parotid gland. Immunoelectron microscopy provided evidence that endogenous VIP36 is localized in the trans-Golgi network, on immature granules, and on mature secretory granules in acinar cells. Double-staining immunofluorescence experiments confirmed that VIP36 and amylase co-localized in the apical regions of the acinar cells. This is the first study to demonstrate that endogenous VIP36 is involved in the post-Golgi secretory pathway, suggesting that VIP36 plays a role in trafficking and sorting of secretory and/or membrane proteins during granule formation.

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Constitutive and basal secretion from the endocrine cell line, AtT-20.

The Journal of Cell Biology ◽

10.1083/jcb.112.5.843 ◽

1991 ◽

Vol 112 (5) ◽

pp. 843-852 ◽

Cited By ~ 63

Author(s):

L Matsuuchi ◽

R B Kelly

Keyword(s):

Cell Line ◽

Secretory Pathway ◽

Secretory Granules ◽

Secretory Protein ◽

Dense Core ◽

Wild Type ◽

Dna Encoding ◽

Basal Secretion ◽

Constitutive Secretion ◽

Regulated Secretory Pathway

A variant of the ACTH-secreting pituitary cell line, AtT-20, has been isolated that does not make ACTH, sulfated proteins characteristic of the regulated secretory pathway, or dense-core secretory granules but retains constitutive secretion. Unlike wild type AtT-20 cells, the variant cannot store or release on stimulation, free glycosaminoglycan (GAG) chains. In addition, the variant cells cannot store trypsinogen or proinsulin, proteins that are targeted to dense core secretory granules in wild type cells. The regulated pathway could not be restored by transfecting with DNA encoding trypsinogen, a soluble regulated secretory protein targeted to secretory granules. A comparison of secretion from variant and wild type cells allows a distinction to be made between constitutive secretion and basal secretion, the spontaneous release of regulated proteins that occurs in the absence of stimulation.

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Synaptotagmin IV is necessary for the maturation of secretory granules in PC12 cells

The Journal of Cell Biology ◽

10.1083/jcb.200506163 ◽

2006 ◽

Vol 173 (2) ◽

pp. 241-251 ◽

Cited By ~ 53

Author(s):

Malika Ahras ◽

Grant P. Otto ◽

Sharon A. Tooze

Keyword(s):

Pc12 Cells ◽

Secretory Granules ◽

Granule Formation ◽

Synaptotagmin Iv ◽

Prohormone Convertase 2 ◽

Granule Maturation ◽

Golgi Network ◽

Interfering Rna

In neuroendocrine PC12 cells, immature secretory granules (ISGs) mature through homotypic fusion and membrane remodeling. We present evidence that the ISG-localized synaptotagmin IV (Syt IV) is involved in ISG maturation. Using an in vitro homotypic fusion assay, we show that the cytoplasmic domain (CD) of Syt IV, but not of Syt I, VII, or IX, inhibits ISG homotypic fusion. Moreover, Syt IV CD binds specifically to ISGs and not to mature secretory granules (MSGs), and Syt IV binds to syntaxin 6, a SNARE protein that is involved in ISG maturation. ISG homotypic fusion was inhibited in vivo by small interfering RNA–mediated depletion of Syt IV. Furthermore, the Syt IV CD, as well as Syt IV depletion, reduces secretogranin II (SgII) processing by prohormone convertase 2 (PC2). PC2 is found mostly in the proform, suggesting that activation of PC2 is also inhibited. Granule formation, and the sorting of SgII and PC2 from the trans-Golgi network into ISGs and MSGs, however, is not affected. We conclude that Syt IV is an essential component for secretory granule maturation.

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Colocalization of chaperone Cpn60, proinsulin and convertase PC1 within immature secretory granules of insulin-secreting cells suggests a role for Cpn60 in insulin processing

Journal of Cell Science ◽

10.1242/jcs.113.11.2075 ◽

2000 ◽

Vol 113 (11) ◽

pp. 2075-2083 ◽

Cited By ~ 1

Author(s):

A.E. Arias ◽

C.S. Velez-Granell ◽

G. Mayer ◽

M. Bendayan

Keyword(s):

Islet Cell ◽

Secretory Pathway ◽

Secretory Granules ◽

Double Labeling ◽

In Vitro Binding ◽

Cell Extracts ◽

Vitro Binding ◽

Preferential Binding ◽

Insulin Secreting Cells

Many of the mechanisms that control insulin processing and packaging by interaction with different elements along the secretory pathway remain poorly understood. We have investigated the possibility that Cpn60, a member of the heat shock protein family, may be present in rat insulin-secreting cells, participating in the proinsulin-insulin maturation process. Immunofluorescence and high resolution immunocytochemical studies revealed the presence of the Cpn60 protein all along the insulin secretory pathway, being particularly abundant over the proinsulin-containing immature secretory granules. Double-labeling experiments showed associations between Cpn60 and proinsulin, as well as between Cpn60 and PC1 convertase, with a preferential binding to proinsulin. These findings paralleled those of coimmunoprecipitation studies showing the Cpn60 chaperone and the mature form of the PC1 convertase in proinsulin immunoprecipitates, as well as the PC1 in Cpn60 immunoprecipitates from total islet cell extracts. In vitro binding of Cpn60 to proinsulin, insulin and glucagon was also documented. Cpn60, significantly abundant in proinsulin-containing secretory granules where conversion of proinsulin to insulin takes place, and the colocalization of the chaperone with proinsulin and PC1 convertase suggest that the Cpn60 protein may play a role directing precise molecular interactions during insulin processing and/or packaging.

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