The Granin Family-Its Role in Sorting and Secretory Granule Formation

CELL STRUCTURE AND FUNCTION 20: 415-420 (1995) MINI REVIEW © 1995 by Japan Society for Cell Biology The Granin Family-Its Role in Sorting and Secretory Granule Formation Hitoshi Ozawa*# and Kuniaki Takata Laboratory of Molecular and Cellular MorphologyInstitute for Molecular and Cellular Regulation Gunma University, Showa-machi, Maebashi-shi, Gunma-ken 371, Japan

Key words: chromogranin A (Cg A)/secretogranin I (Sg I, or chromogranin B; Cg B)/secretogranin II (Sg II)/trans-Golgi network (TGN)/secretory granules

ABSTRACT.Twotypes of secretory pathwaysare present in mammaliancells: constitutive secretion and regulated secretion (1). In the constitutive secretory pathway, which is found in all types of cells, secretory products are packed in small vesicles. Mostof the proteoglycans and glycoproteins of the extracellular matrix are secreted in this way. The regulated secretory pathway, found in the more differentiated secretory cells, is mediated by spe- cialized secretory granules. Hormonesand neuropeptides are secreted in this way. Both the constitutive and regulated secretory pathways emanate from the trans-Golgi network (TGN). Recent studies have indicated that the granin family (secretogranins/chromogranins) plays an important role in the sorting and aggregation of secretory products in the trans-Golgi network (TGN), and in the subsequent formation of secretory granules. The granin family is thought to be one of the mediators of the regulated secretory pathway (2-6). This family is widely expressed in endocrine (7-9) and neuronal cells (10-12), where they are stored in secretory granules together with various peptide hormonesand neuropeptides. Three acidic sulfated proteins, chromogranin A (Cg A), secretogranin I (Sg I; also called chromogranin B; Cg B), and secretogranin II (Sg II) are well known as the major proteins in the family. Recently, three acidic secretory proteins, 1B1075 gene product (13), HISL-19 antigen (14) and 7B2 (15), have been thought to be members of the granin family, ac- cordingly termed as Sg III, Sg IV and Sg V, respectively. However, it still remains open to discussion whether 1B1075 gene product, the HISL-19 antigen and 7B2 are true members of the granin family. In this review, the possible roles of granin family, especially Cg A, Sg I (Cg B) and Sg II in the regulated secretory pathway are discussed, focusing on the sorting, aggregation of proteins in the TGN, and subsequent secretory granule formation.

CgA, SgI(CgB) andSgII. The biochemical prop- in some species have been deduced from the correspond- erties of Cg A, Sg I (Cg B) and Sg II are summarized in ing CDNAsequences (20-26). The secretogranins/chro- Table I. The first memberof the granin family identified mogranins (Sgs/Cgs) are highly hydrophilic proteins ex- was Cg A, originally characterized as the major secreto- cept for the cleaved signal peptide region. They have a ry protein of Mr 75,000-85,000 in the bovine adrenal high population of charged, acidic amino acid residues. medulla (16). The second member of the family, Sg II, Sgs/Cgs show very low isoelectric points, heat stability, was identified as Mr 70,000 sulfated secretory protein in the existence of signal peptide and calcium binding ca- the bovine anterior pituitary (17, 18). In the adrenal me- pacity. Some post-translational modifications, such as dulla derived cell line PC12, Mr 86,000-84,000 tyrosine- O-glycosylation, phosphorylation, sulfation and proteo- sulfated secretory protein was also reported (19). Subse- lytic processing, are found in some membersof the quently, it was confirmed that these two proteins were Sgs/Cgs family. the same, and so the protein was namedSg II. The third Granins as markers for neuroendocrine cells. The member of the granin family, Sg I (Cg B) is an Mr Sgs/Cgs family are expressed and present in neuroendo- 113,000-105,000 tyrosine-sulfated secretory protein crine cells in any combination of Cg A, Sg I (Cg B) found in PC12 cells (19). and/or Sg II (3, 4, 7-9). These findings suggest the possi- The primary structures of Cg A, Sg I (Cg B) and Sg II bility of Cgs/Sgs as a marker for endocrine cells and neuronal cells. * To whomcorrespondence and reprints should be addressed. The relative abundance of individual members of the # Present address: Department of Anatomy and Neurobiology, Sgs/Cgs family in a given species differs across species, Kyoto Prefectural University of Medicine, Kawaramachi-Hiroko- and in a given species differs across tissues, suggesting ji, Kamigyo-ku, Kyoto 602, Japan. the differential regulation for the synthesis of the in-

415 H. Ozawa and K. Takata

Table I. Properties of chromogranin A (Cg A), secretogranin I (Sg I, chromogranin B; Cg B) AND SECRETOGRANIN II (Sg II, CHROMOGRANIN Cj Cg C). P r o p e r t i e s C g A S g I (C g B ) S g II ( Cg C)

M r (S D S -P A G E ) 7 5 ,0 0 0 - 8 5 ,0 0 0 10 0, 00 0- 1 20 ,0 00 8 4 ,0 0 0 -8 7 , 00 0 I s o e l e c t r i c p o i n t 5 . 0 - 4 . 8 5.2 - 5. 0 5 .1 -4 .9 m R N A ( k b ) 2 . 1 2 .5 2 .5 D i b a s i c a n d m u l t i b a s i c s i t e s 1 0 1 5 9 S ig n al p ep tid e + + C a ^ - b in d i ng + + H e at-stab ility + + C e l l u la r d i st r i b u ti o n S G * o f n eu r o n an d e n do c r i n e c e l l s SG of ne ur on an d e nd oc rin e cel ls SG of n eu ron a nd en doc ri ne ce ll s N -g ly c o sy la tio n O -g ly c o sy la tio n + + P h o s p h o ry la tio n S er , T h r S e r, T h r S er , T h r S u lfa tio n T y r * * c arb o h y d r ate , T y r, c a rb o h y d ra te , T y r , ca rb o h y d ra te P ro teo ly ti c pr oc es si ng + + S G ; s e cr e to r y g r an u l e * * d et e c te d o n l y i n s o me s pe c ie s dividual Sgs/Cgs. Indeed, Thompson et al. (27) have re- essed peptide from Sg II, secretoneurin, was identified ported that the effect of forskolin (FSK), an activator of (38). The physiological role of secretoneurin, however, adenylate cyclase, on the regulation of Sg I (Cg B) and is still unknown. Recent findings suggest that Sgs/Cgs Sg II mRNAlevels is different in rat PC12 cells. They intracellularly play a role in the packaging and/or sort- demonstrated that Sg I (Cg B) mRNAlevels became ele- ing of peptide hormones and neuropeptides (32-34, 39- vated following treatment with FSK, while Sg II mRNA 41). Immunocytochemical studies have demonstrated levels are rather decreased. Noimmunoreactivity for that Sgs/Cgs are co-localized with certain peptide hor- Sgs/Cgs has been detected in exocrine cells or in cells monesor neuropeptides in the secretory granules of neu- which secrete certain proteins, such as serum albumin, roendocrine cells. These observations suggest the possi- imrnunoglobulinand transferrin, via the constitutive se- bility that Sgs/Cgs and peptide hormones or neuropep- cretory pathway. tide co-aggregate in the condensing and/or packaging Subcellular distribution of Sgs/Cgs has been immuno- process. cytochemically studied by many groups (7-9, 28-34). Co-localization ofgranins with peptides within the se- Sgs/Cgs are present in the matrix of neurosecretory cretory granules. In the cow anterior pituitary, Cg A, granules together with specific peptide hormonesand Cg B (Sg I) and Sg II are sorted and packaged with thy- neuropeptides (3, 4). Double immunoelectron micro- roid stimulating hormone (TSH) and luteinizing hor- scopic studies have revealed the co-localization of mone(LH) into secretory granules of thyrotrophs and Sgs/Cgs with specific hormonesor neuropeptides in neu- gonadotrophs (31). Sgs/Cgs are also co-localized with roendocrine secretory granules (12, 29, 31-34). More- prolactin (PRL) and growth hormone (GH) within the over, immunolabeled Sgs/Cgs have also been detected samesecretory granules of the so-called somatomammo- in the Golgi apparatus together with specific hormones trophs. In the rat anterior pituitary, it has been demon- (29, 33, 34). strated that Sg I (Cg B) and Sg II are co-localized to- Function. The physiological functions of Sgs/Cgs gether with prolactin within the secretory granules of have not been sufficiently elucidated. It has been re- mammotrophs (33, 34). Sg I (Cg B) and Sg II, as well as vealed that Cg A is the precursor of pancreastatin and prolactin, have been also detected in the Golgi appara- chromostatin. Pancreastatin is a peptide which partially tus. The same results have been obtained in GH3B6 inhibits glucose-induced insulin secretion and, in some cells, a subclone of GH3cells (29, 33). In rat gonado- cases, other protein secretion (35). Chromostatin is trophs, on the other hand, Sgs/Cgs were detected in se- known to inhibit exocytosis in isolated chromaffin cells cretory granules together with LHand follicle stimulat- (36). Moreover, it was reported that full length Cg A it- ing hormone (FSH). Ultrastructurally, gonadotrophs self has the capability to inhibit the action of pancre- exhibit two types of secretory granules. Large size secre- astatin (37). Thus, it is suggested that Cg A may act as tory granules (about 500 nmin diameter) show preferen- an autocrine or paracrine regulatory factor in the pan- tial immunoreactivity with LH, FSHand Cg A. Small creatic secretory process. Theextracellular activity of size secretory granules (about 200 nm in diameter) pref- Sg I (Cg B) is Sg II are not well known. Recently, aproc- erentially showed immunoreactivity with LHand Sg II 416 Grain and Secretory Granule Formation (Cg B) (31, 32). These co-localization patterns of gra- duced by millimolar concentration of calcium ion and nins and specific peptide hormones and neuropeptides an acidic pH, which are found in the TGN. Constitutive have also been observed in other cells of neuroendo- secretory proteins, such as immunoglobulin, albumin, crine organs and tissues, such as pancreatic islet cells, transferrin, and a1-acid glycoprotein, are excluded parathyroid, adrenal medulla and hypothalamus (4, 8, from this aggregation process. The next important ac- 9). tion in the sorting and/or packaging of secretory pro- Possible mechanismof sorting and/or packaging teins is the interaction of the TGNmembrane. It has in Golgi apparatus, especially in trans-Golgi network. been revealed that Sg I (Cg B) is exposed to and remains During the course of sorting and/or packaging of pro- associated with the cell surface after the release of secre- teins in TGN, the granins, mainly Cg A, Sg I (Cg B) and tory granules (43, 44). This indicates that a certain Sg II, are thought to selectively form aggregates, which amount of Sg I (Cg B) is insoluble in the matrix of secre- mayfacilitate the condensation of regulated secretory tory granules, and that Sg I may be tightly associated proteins leading to the formation of dense core materi- with the granule membrane. The final step in the forma- als (3, 5, 42) (Fig. 1). The aggregation of granins is in- tion of secretory granules is the envelopmentof the ag-

r\ r^ Regulated secretory protein A/. GraninsConstitutive secretory protein

3 r^

A Constitutive secretory pathway ^ >4 *à" A

Regulated secretory pathway A Ky ^y ^y RER cis mid dle trans Golgi apparatus TGN

Fig. 1. Schematic diagram illustrating the selective aggregation , sorting and packaging process of regulated secretory protein and the granins. The granins lead the regulated secretory protein to the formation of secretory granules, which are secreted by the regulated secretory pathway. RER: rough endoplasmic reticulum. TGN: trans-Golgi network.

417 H. Ozawa and K. Takata

# Regulated secretory protein Conclusion. During the recent few years, it has A Granins been recognized that the granins are co-localized with + Adaptin peptide hormones and neuropeptides within the secreto- -\ Clathrin ry granules of endocrine cells and neurons. Such co-localization strongly suggests that granins play a possible 3 Ligand receptor(?) role in secretory granule formation. Further studies are «- Rab protein (Rab8?) necessary to elucidate the molecular mechanismof secretory granule formation and the role of Sgs/Cgs in the regulated secretory process. Acknowledgment. The authors wish to thank Mrs. Mary Shibuya for her help inmanuscript preparation.

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