Granuphilin modulates the exocytosis of secretory granules through interaction with syntaxin 1a

The molecular mechanism for the regulated exocytosis of dense-core granules in endocrine cells remains relatively uncharacterized compared to that of synaptic vesicles in neurons. A novel set of Rab and its effector, Rab27a/granuphilin, which is localized on insulin granules in pancreatic beta cells, was recently identified. Here we demonstrate that granuphilin directly binds to syntaxin 1a on the plasma membrane, and this interaction is regulated by Rab27a. Granuphilin shows affinity to syntaxin 1a with a closed conformation but not to mutant syntaxin 1a, which adopts an open conformation constitutively. Overexpression of granuphilin significantly enhances basal insulin secretion but profoundly inhibits high K(+)-induced insulin secretion. The effect of granuphilin on insulin secretion was impaired by its mutation that disrupts the binding to either Rab27a or syntaxin 1a. Thus, granuphilin is the first regulator in the exocytotic pathway that functions by directly connecting two critical vesicle transport proteins, Rab and SNARE.     

Exophilin4/Slp2-a targets glucagon granules to the plasma membrane through unique Ca2+-inhibitory phospholipid-binding activity of the C2A domain

Rab27a and Rab27b have recently been recognized to play versatile roles in regulating the exocytosis of secretory granules and lysosome-related organelles by using multiple effector proteins. However, the precise roles of these effector proteins in particular cell types largely remain uncharacterized, except for those in pancreatic beta cells and in melanocytes. Here, we showed that one of the Rab27a/b effectors, exophilin4/Slp2-a, is specifically expressed in pancreatic alpha cells, in contrast to another effector, granuphilin, in beta cells. Like granuphilin toward insulin granules, exophilin4 promotes the targeting of glucagon granules to the plasma membrane. Although the interaction of granuphilin with syntaxin-1a is critical for the targeting activity, exophilin4 does this primarily through the affinity of its C2A domain toward the plasma membrane phospholipids phosphatidylserine and phosphatidylinositol-4,5-bisphosphate. Notably, the binding activity to phosphatidylserine is inhibited by a physiological range of the Ca(2+) concentration attained after secretagogue stimulation, which presents a striking contrast to the Ca(2+)-stimulatory activity of the C2A domain of synaptotagmin I. Analyses of the mutant suggested that this novel Ca(2+)-inhibitory phospholipid-binding activity not only mediates docking but also modulates the subsequent fusion of the secretory granules.     

The Rab27a/granuphilin complex regulates the exocytosis of insulin-containing dense-core granules

Recently, we identified and characterized a novel protein, granuphilin, whose domain structure is similar to that of the Rab3 effector protein rabphilin3 (J. Wang, T. Takeuchi, H. Yokota, and T. Izumi, J. Biol. Chem. 274:28542-28548, 1999). Screening its possible Rab partner by a yeast two-hybrid system revealed that an amino-terminal zinc-finger domain of granuphilin interacts with Rab27a. Granuphilin preferentially bound to the GTP form of Rab27a. Formation of the Rab27a/granuphilin complex was readily detected in the pancreatic beta cell line MIN6. Moreover, the tissue distributions of Rab27a and granuphilin are remarkably similar: both had significant and specific expression in pancreatic islets and in pituitary tissue, but no expression was noted in the brain. Analyses by immunofluorescence, immunoelectron microscopy, and sucrose density gradient subcellular fractionation showed that Rab27a and granuphilin are localized on the membrane of insulin granules. These findings suggest that granuphilin functions as a Rab27a effector protein in beta cells. Overexpression of wild-type Rab27a and its GTPase-deficient mutant significantly enhanced high K(+)-induced insulin secretion without affecting basal insulin release. Although Rab3a, another exocytotic Rab protein, has some similarities with Rab27a in primary sequence, intracellular distribution, and affinity toward granuphilin, overexpression of Rab3a caused different effects on insulin secretion. These results indicate that Rab27a is involved in the regulated exocytosis of conventional dense-core granules possibly through the interaction with granuphilin, in addition to its recently identified role in lysosome-related organelles.     

Rab27 effector granuphilin promotes the plasma membrane targeting of insulin granules via interaction with syntaxin 1a

Secretory vesicle exocytosis is a highly regulated process involving vesicle targeting, priming, and membrane fusion. Rabs and SNAREs play a central role in executing these processes. We have shown recently that Rab27a and its effector, granuphilin, are involved in the exocytosis of insulin-containing secretory granules through a direct interaction with the plasma membrane syntaxin 1a in pancreatic beta cells. Here, we demonstrate that fluorescence-labeled insulin granules are peripherally accumulated in cells overexpressing granuphilin. The peripheral location of granules is well overlapped with both localizations of granuphilin and syntaxin 1a. The plasma membrane targeting of secretory granules is promoted by wild-type granuphilin but not by granuphilin mutants that are defective in binding to either Rab27a or syntaxin 1a. Granuphilin directly binds to the H3 domain of syntaxin 1a containing its SNARE motif. Moreover, introduction of the H3 domain into beta cells induces a dissociation of the native granuphilin-syntaxin complex and a marked reduction of newly docked granules. These results indicate that granuphilin plays a role in tethering insulin granules to the plasma membrane by an interaction with both Rab27a and syntaxin 1a. The complex formation of these three proteins may contribute to the specificity of the targeting process during the exocytosis of insulin granules.     

Exocrine granule specific packaging signals are present in the polypeptide moiety of the pancreatic granule membrane protein GP2 and in amylase: implications for protein targeting to secretory granules.

The mechanisms for segregation of secretory and membrane proteins incorporated into storage granules from those transported constitutively have been thought to be conserved in diverse cell types, including exocrine and endocrine cells. However, GP2, the major protein of pancreatic zymogen granule membranes, in its native glycosyl phosphatidylinositol (GPI)-linked form, is incorporated into secretory granules when expressed in exocrine pancreatic AR42J cells, but not in the endocrine cells such as pituitary AtT20. To determine whether the protein moiety of GP2 contains the cell-type specific information for packaging into granules, a secretory form of GP2 (GP2-GPI-), with the GPI attachment site deleted, was generated and introduced into AR42J and AtT20 cells. Like native GP2, GP2-GPI- localized to the zymogen-like granules of AR42J cells and underwent regulated secretion. In AtT20 cells expressing GP2-GPI-, however, the protein was secreted by the constitutive pathway. Thus, a granule packaging signal is present in the luminal portion of GP2 that is functional only in the exocrine cells. However, this cell-type dependent sorting process is not limited to GP2 or membrane proteins. Amylase, a major content protein of pancreatic acinar and serous salivary gland granules, was also secreted exclusively by the constitutive pathway when expressed in AtT20 cells. The cell-type specific targeting of GP2 to granules correlated with its behavior in an in vitro aggregation assay where it co-aggregated more effectively with content proteins from pancreatic zymogen granules than with those from pituitary granules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Urocortin 3 Marks Mature Human Primary and Embryonic Stem Cell-Derived Pancreatic Alpha and Beta Cells

The peptide hormone Urocortin 3 (Ucn 3) is abundantly and exclusively expressed in mouse pancreatic beta cells where it regulates insulin secretion. Here we demonstrate that Ucn 3 first appears at embryonic day (E) 17.5 and, from approximately postnatal day (p) 7 and onwards throughout adult life, becomes a unifying and exclusive feature of mouse beta cells. These observations identify Ucn 3 as a potential beta cell maturation marker. To determine whether Ucn 3 is similarly restricted to beta cells in humans, we conducted comprehensive immunohistochemistry and gene expression experiments on macaque and human pancreas and sorted primary human islet cells. This revealed that Ucn 3 is not restricted to the beta cell lineage in primates, but is also expressed in alpha cells. To substantiate these findings, we analyzed human embryonic stem cell (hESC)-derived pancreatic endoderm that differentiates into mature endocrine cells upon engraftment in mice. Ucn 3 expression in hESC-derived grafts increased robustly upon differentiation into mature endocrine cells and localized to both alpha and beta cells. Collectively, these observations confirm that Ucn 3 is expressed in adult beta cells in both mouse and human and appears late in beta cell differentiation. Expression of Pdx1, Nkx6.1 and PC1/3 in hESC-derived Ucn 3⁺ beta cells supports this. However, the expression of Ucn 3 in primary and hESC-derived alpha cells demonstrates that human Ucn 3 is not exclusive to the beta cell lineage but is a general marker for both the alpha and beta cell lineages. Ucn 3⁺ hESC-derived alpha cells do not express Nkx6.1, Pdx1 or PC1/3 in agreement with the presence of a separate population of Ucn 3⁺ alpha cells. Our study highlights important species differences in Ucn 3 expression, which have implications for its utility as a marker to identify mature beta cells in (re)programming strategies.     

Energy homeostasis and gastrointestinal endocrine differentiation do not require the anorectic hormone peptide YY

The gastrointestinal hormone peptide YY is a potent inhibitor of food intake and is expressed early during differentiation of intestinal and pancreatic endocrine cells. In order to better understand the role of peptide YY in energy homeostasis and development, we created mice with a targeted deletion of the peptide YY gene. All intestinal and pancreatic endocrine cells developed normally in the absence of peptide YY with the exception of pancreatic polypeptide (PP) cells, indicating that peptide YY expression was not required for terminal differentiation. We used recombination-based cell lineage trace to determine if peptide YY cells were progenitors for gastrointestinal endocrine cells. Peptide YY(+) cells gave rise to all L-type enteroendocrine cells and to islet partial differential and PP cells. In the pancreas, approximately 40% of pancreatic alpha and rare beta cells arose from peptide YY(+) cells, suggesting that most beta cells and surprisingly the majority of alpha cells are not descendants of peptide YY(+)/glucagon-positive/insulin-positive cells that appear during early pancreagenesis. Despite the anorectic effects of exogenous peptide YY(3-36) following intraperitoneal administration, mice lacking peptide YY showed normal growth, food intake, energy expenditure, and responsiveness to peptide YY(3-36). These observations suggest that targeted disruption of the peptide YY gene does not perturb terminal endocrine cell differentiation or the control of food intake and energy homeostasis.     

Quinacrine affinity of endocrine cell systems containing dense core vesicles as visualized by fluorescence microscopy

Summary Intravenous injections of low amounts of the fluorescent antimalarial acridine derivative quinacrine into rats and mice lead to selective high affinity binding of the drug, which can be visualized by fluorescence microscopy, to several hormone-producing cell systems. Injection of 1 mg/kg causes strong drug accumulation in the granules of renin-producing juxtaglomerular cells of the kidney and in several types of cells of the pancreatic islets, and a moderate binding to parafollicular cells in the thyroid gland, chromaffin cells of the adrenal medulla, several gastrointestinal cell systems including APUD cells, cells of the anterior pituitary gland, neurosecretory neurons and mast cells. These different cell systems all have large dense-core storage granules.The present results, together with our earlier finding of a population of gastrointestinal nerve fibers which demonstrate a similar selective high affinity binding of quinacrine might all be explained by a binding of quinacrine to large dense-core storage granules, since such granules are known to be present also in certain gastrointestinal nerve fibers. This is further supported by the finding that endocrine cell systems lacking such granules, such as steroid producing cells in the adrenal cortex and testis, do not accumulate quinacrine. Peptide storage, possibly mediated by or accompanied by purines such as ATP within the granules or an acidic intragranular pH constitute possible binding mechanisms.     

Brn-4 transcription factor expression targeted to the early developing mouse pancreas induces ectopic glucagon gene expression in insulin-producing beta cells

The endocrine pancreas is comprised of beta and alpha cells producing the glucostatic hormones insulin and glucagon, respectively, and arises during development by the differentiation of stem/progenitor cells in the foregut programmed by the beta cell lineage-specific homeodomain protein Idx-1. Brain-4 (Brn-4) is expressed in the pancreatic anlaga of the mouse foregut at e10 in the alpha cells and transactivates glucagon gene expression. We expressed Brn-4 in pancreatic precursors or beta cell lineage in transgenic mice by placing it under either Idx-1 or insulin promoter (rat insulin II promoter) control, respectively. Idx-1 expression occurs at developmental day e8.5, and insulin expression occurs at e9.5, respectively. Misexpression of Brn-4 by the Idx-1 promoter results in ectopic expression of the proglucagon gene in insulin-expressing pancreatic beta cells, whereas misexpression by rat insulin II promoter did not. The early developmental expression of Brn-4 appears to be a dominant regulator of the glucagon expressing alpha cell lineage, even in the context of the beta cell lineage.     

Loss of Granuphilin and Loss of Syntaxin-1A Cause Differential Effects on Insulin Granule Docking and Fusion

The Rab27 effector granuphilin/Slp4 is essential for the stable attachment (docking) of secretory granules to the plasma membrane, and it also inhibits subsequent fusion. Granuphilin is thought to mediate these processes through interactions with Rab27 on the granule membrane and with syntaxin-1a on the plasma membrane and its binding partner Munc18-1. Consistent with this hypothesis, both syntaxin-1a- and Munc18-1-deficient secretory cells, as well as granuphilin null cells, have been observed to have a deficit of docked granules. However, to date there has been no direct comparative analysis of the docking defects in those mutant cells. In this study, we morphometrically compared granule-docking states between granuphilin null and syntaxin-1a null pancreatic β cells derived from mice having the same genetic background. We found that loss of syntaxin-1a does not cause a significant granule-docking defect, in contrast to granuphilin deficiency. Furthermore, we newly generated granuphilin/syntaxin-1a double knock-out mice, characterized their phenotypes, and found that the double mutant mice represent a phenocopy of granuphilin null mice and do not represent phenotypes of syntaxin-1a null mice, including their granule-docking behavior. Because granuphilin binds to syntaxin-2 and syntaxin-3 as well as syntaxin-1a, it likely mediates granule docking through interactions with those multiple syntaxins on the plasma membrane.     

Granuphilin molecularly docks insulin granules to the fusion machinery

The Rab27a effector granuphilin is specifically localized on insulin granules and is involved in their exocytosis. Here we show that the number of insulin granules morphologically docked to the plasma membrane is markedly reduced in granuphilin-deficient beta cells. Surprisingly, despite the docking defect, the exocytosis of insulin granules in response to a physiological glucose stimulus is significantly augmented, which results in increased glucose tolerance in granuphilin-null mice. The enhanced secretion in mutant beta cells is correlated with a decrease in the formation of the fusion-incompetent syntaxin-1a-Munc18-1 complex, with which granuphilin normally interacts. Furthermore, in contrast to wild-type granuphilin, its mutant that is defective in binding to syntaxin-1a fails to restore granule docking or the protein level of syntaxin-1a in granuphilin-null beta cells. Thus, granuphilin not only is essential for the docking of insulin granules but simultaneously imposes a fusion constraint on them through an interaction with the syntaxin-1a fusion machinery. These findings provide a novel paradigm for the docking machinery in regulated exocytosis.     

The roles of Rab27 and its effectors in the regulated secretory pathways

Regulated secretory pathways are highly developed in multicellular organisms as a means of intercellular communication. Each of these pathways harbors unique store organelles, such as granules in endocrine and exocrine tissues and melanosomes in melanocytes. It has recently been shown that the monomeric GTPase Rab27 subfamily regulates the exocytosis of these cell-specific store organelles. Furthermore, genetic alterations of Rab27a cause Griscelli syndrome in humans that manifests as pigmentary dilution of the skin and the hair and variable immunodeficiency due to defects in the transport of melanosomes in melanocytes and lytic granules in cytotoxic T-lymphocytes. Rab27 acts through organelle-specific effector proteins, such as granuphilin in pancreatic beta cells and melanophilin in melanocytes. The Rab27 and effector complex then interacts with proteins that are essential for membrane transport and fusion, such as syntaxin 1a and Munc18-1 for granuphilin and myosin Va for melanophilin. Genome information suggests that other putative Rab27 effector proteins, tentatively termed as exophilins or Slp/Slac2, are predicted to exist because these proteins share the conserved N-terminal Rab27-binding domain and show Rab27-binding activity in vitro or when overexpressed in cell lines. These findings suggest that the Rab27 subfamily regulates various exocytotic pathways using multiple organelle-specific effector proteins.     

Ultimobranchial gland of the domestic fowl

Summary The ultimobranchial gland (UBG) of birds is particularly rich in calcitonin, the hypocalcaemic hypophosphataemic hormone, that is secreted by the C-cells of the mammalian thyroid. The principal cells of the UBG have a striking resemblance with the mammalian C-cells, i.e., they possess small intracytoplasmic dense-core secretory granules, 150–300 nm in diameter. The gland also contains a second, morphologically distinct, endocrine cell type with larger granules, 500–800 nm in diameter. A sensitive immunocytochemical reaction was developed with the use of antibodies against salmon calcitonin. By means of this technique the presence of calcitonin-immunoreactive molecules was demonstrated in both secretory cell types of the UB gland of the chicken. This gland can thus be considered as a homogeneous calcitonin-producing tissue. Whether the secretory products are identical is discussed and differences in the secretory pathways are suggested.     

Docking is not a prerequisite but a temporal constraint for fusion of secretory granules

We examined secretory granule dynamics using total internal reflection fluorescence microscopy in normal pancreatic β cells and their mutants devoid of Rab27a and/or its effector, granuphilin, which play critical roles in the docking and recruitment of insulin granules to the plasma membrane. In the early phase of glucose stimulation in wild-type cells, we observed marked fusion of granules recruited from a relatively distant area, in parallel with that from granules located underneath the plasma membrane. Furthermore, despite a lack of granules directly attached to the plasma membrane, both spontaneous and evoked fusion was increased in granuphilin-null cells. In addition to these granuphilin-null phenotypes, Rab27a/granuphilin doubly deficient cells showed the decreases in granules located next to the docked area and in fusion from granules near the plasma membrane in the early phase of glucose-stimulated secretion, similar to Rab27a-mutated cells. Thus, the two proteins play nonoverlapping roles in insulin exocytosis: granuphilin acts on the granules underneath the plasma membrane, whereas Rab27a acts on those in a more distal area. These findings demonstrate that, in contrast to our conventional understanding, stable attachment of secretory granules to the plasma membrane is not prerequisite but temporally inhibitory for both spontaneous and evoked fusion.     

Persistent expression of HNF6 in islet endocrine cells causes disrupted islet architecture and loss of beta cell function

We used transgenesis to explore the requirement for downregulation of hepatocyte nuclear factor 6 (HNF6) expression in the assembly, differentiation, and function of pancreatic islets. In vivo, HNF6 expression becomes downregulated in pancreatic endocrine cells at 18. 5 days post coitum (d.p.c.), when definitive islets first begin to organize. We used an islet-specific regulatory element (pdx1(PB)) from pancreatic/duodenal homeobox (pdx1) gene to maintain HNF6 expression in endocrine cells beyond 18.5 d.p.c. Transgenic animals were diabetic. HNF6-overexpressing islets were hyperplastic and remained very close to the pancreatic ducts. Strikingly, alpha, delta, and PP cells were increased in number and abnormally intermingled with islet beta cells. Although several mature beta cell markers were expressed in beta cells of transgenic islets, the glucose transporter GLUT2 was absent or severely reduced. As glucose uptake/metabolism is essential for insulin secretion, decreased GLUT2 may contribute to the etiology of diabetes in pdx1(PB)-HNF6 transgenics. Concordantly, blood insulin was not raised by glucose challenge, suggesting profound beta cell dysfunction. Thus, we have shown that HNF6 downregulation during islet ontogeny is critical to normal pancreas formation and function: continued expression impairs the clustering of endocrine cells and their separation from the ductal epithelium, disrupts the spatial organization of endocrine cell types within the islet, and severely compromises beta cell physiology, leading to overt diabetes.     

Expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins in the developing pancreas: roles in the adhesion and migration of putative endocrine progenitor cells

Cell-cell and cell-matrix interactions play a critical role in tissue morphogenesis and in homeostasis of adult tissues. The integrin family of adhesion receptors regulates cellular interactions with the extracellular matrix, which provides three-dimensional information for tissue organization. It is currently thought that pancreatic islet cells develop from undifferentiated progenitors residing within the ductal epithelium of the fetal pancreas. This process involves cell budding from the duct, migration into the surrounding mesenchyme, differentiation, and clustering into the highly organized islet of Langerhans. Here we report that alpha(v)beta(3) and alpha(v)beta(5), two integrins known to coordinate epithelial cell adhesion and movement, are expressed in pancreatic ductal cells and clusters of undifferentiated cells emerging from the ductal epithelium. We show that expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins are developmentally regulated during pancreatic islet ontogeny, and mediate adhesion and migration of putative endocrine progenitor cells both in vitro and in vivo in a model of pancreatic islet development. Moreover, we demonstrate the expression of fibronectin and collagen IV in the basal membrane of pancreatic ducts and of cell clusters budding from the ductal epithelium. Conversely, expression of vitronectin marks a population of epithelial cells adjacent to, or emerging from, pancreatic ducts. Thus, these data provide the first evidence for the contribution of integrins alpha(v)beta(3) and alpha(v)beta(5) and their ligands to morphogenetic events in the human endocrine pancreas.     

Identification of glucagon-producing cells (A cells) in dog gastric mucosa

An immunocytochemical technique using specific antiglucagon serum reveals the presence of glucagon-containing cells situated exclusively in the oxyntic glandular mucosa of the dog stomach. Electron microscope examination of the mucosa demonstrated endocrine cells containing secretory granules with a round dense core surrounded by a clear halo, indistinguishable from secretory granules of pancreatic A cells. Like the alpha granules of pancreatic A cells, the granules of these gastric endocrine cells exhibited a peripheral distribution of silver grains after Grimelius silver staining. Moreover, the granules of these cells were found to be specifically labeled with reaction product, using the peroxidase immunocytochemical technique at the ultrastructural level. Accordingly, these cells were named gastric A cells. These data suggest that the gastric oxyntic mucosa contains cells indistinguishable cytologically, cytochemically, and immunocytochemically from pancreatic A cells. It is believed that gastric A cells are responsible for the secretion of the gastric glucagon.