The new england journal of medicine review article mechanisms of disease The Chromogranin–Secretogranin Family Laurent Taupenot, Ph.D., Kimberly L. Harper, M.D., and Daniel T. O’Connor, M.D. From the Department of Medicine (L.T., eurons and neuroendocrine cells contain membrane- K.L.H., D.T.O.) and the Center for Molecular delimited pools of peptide hormones, biogenic amines, and neurotransmit- Genetics (D.T.O.), University of California n at San Diego, La Jolla; and the Veterans ters with a characteristic electron-dense appearance on transmission electron Affairs San Diego Healthcare System, San microscopy (Fig. 1). These vesicles, which are present throughout the neuroendocrine Diego, Calif. (L.T., K.L.H., D.T.O.). Address system1,2 and in a variety of neurons, store and release chromogranins and secretogranins reprint requests to Dr. O’Connor at the 3,4 Department of Medicine (9111H), Univer- (also known as “granins”), a unique group of acidic, soluble secretory proteins. The sity of California at San Diego, 3350 La Jolla three “classic” granins are chromogranin A, which was first isolated from chromaffin Village Dr., San Diego, CA 92161, or at cells of the adrenal medulla5,6; chromogranin B, initially characterized in a rat pheochro- [email protected]. mocytoma cell line7; and secretogranin II (sometimes called chromogranin C), which 8,9 N Engl J Med 2003;348:1134-49. was originally described in the anterior pituitary. Four other acidic secretory pro- Copyright © 2003 Massachusetts Medical Society. teins were later proposed for membership in the granin family10: secretogranin III (or 1B1075),11 secretogranin IV (or HISL-19),12 secretogranin V (or 7B2),13 and secretogra- nin VI (or NESP55).14 In this article, we review aspects of the structures, biochemical properties, and clin- ical importance of granins, with particular emphasis on chromogranin A, the granin that was discovered first and that has been studied most extensively. We discuss how granins contribute to the formation of secretory granules and how, as prohormones, they give rise to bioactive peptides through proteolytic processing. Because of their ubiquitous distribution in neuroendocrine and nervous-system tissues and their cose- cretion with resident peptide hormones and biogenic amines, granins are valuable in- dicators of sympathoadrenal activity and clinically useful markers of secretion from normal and neoplastic neuroendocrine cells.1,15-17 Indeed, numerous studies have doc- umented the clinical value of detecting granins in tissues and measuring circulating levels of granins, particularly chromogranin A. In addition to providing information about the neuroendocrine character of various neoplasms, measurement of chromo- granin A has yielded insights into the pathogenesis of essential hypertension. molecular and genetic aspects of granins structural and physicochemical properties Granins consist of single-polypeptide chains of approximately 180 to 700 amino acid residues, bearing an amino-terminal signal peptide that directs the movement of the preproteins from ribosomes to the endoplasmic reticular lumen and, hence, the Golgi complex, where further post-translational modifications occur. Granins tend to bind calcium with low affinity but high capacity and then aggregate in vitro at low pH in the presence of calcium.18-24 These aggregation characteristics suggest that granins have functions within the core of secretory granules. genomic organization and transcriptional regulation The chromosomal positions of all granins except secretogranin IV have been determined in humans, cows, mice, and rats (Table 1); in each case, the loci lie in regions of locally 1134 n engl j med 348;12 www.nejm.org march 20, 2003 Downloaded from www.nejm.org by RODRIGUEZ POVEDA AIXA MILDRED on May 16, 2010 . Copyright © 2003 Massachusetts Medical Society. All rights reserved. mechanisms of disease conserved gene order in homologous chromo- somes (Fig. 2).25 A pH The distribution of granins throughout the neu- 5.8 5.4 5.0 4.6 4.2 roendocrine system has been studied with Northern blot analysis of messenger RNA. Granin messages CgB have been found in cells of various neuroendocrine DBH CgA tissues that have a regulated secretory pathway.9,26,27 97 Moreover, granin synthesis responds differently in different cell types to agents that elevate cyclic 66 AMP (cAMP), steroid hormones, neurotrophins, and phorbol ester. Experiments with transfected granin-gene pro- 45 moters have clarified the mechanism of constitutive and secretagogue-inducible expression of granins in specific cells.28 The proximal promoter regions of chromogranin A, chromogranin B, and secreto- 24 granin II contain a functional cAMP-response ele- Molecular Mass (kD) ment (CRE) upstream of a TATA box (a region of seven nucleotides, mainly thymidine and adenine, located upstream of the starting point of transcrip- 18 tion); otherwise, their promoter sequences dif- fer.29,30 The specificity of expression of chromo- granin A, chromogranin B, and secretogranin III in mouse neuroendocrine cells has been mapped to the CRE site.30-32 Upstream promoter elements may also be important in cell-specific expression of hu- B C man chromogranin A and secretogranin II.33,34 These CRE sites may explain how the expression of chromogranin A, chromogranin B, and secreto- granin II is up-regulated in neuroendocrine cells by nicotinic–cholinergic agonists and the pregangli- onic neuropeptide pituitary adenylyl cyclase–acti- vating polypeptide.31,32,35,36 Moreover, neuronal differentiation of a pheochromocytoma cell line 100 nm 5 mm (PC12) induced by neurotrophin nerve growth fac- tor up-regulates expression of the genes for chro- mogranin A and chromogranin B through an effect Figure 1. Localization of Chromogranins with Dense-Core Secretory Granules on the CRE site.31,37 of Sympathoadrenal Chromaffin Cells. An upstream serum response element also has Panel A shows soluble-core proteins in bovine chromaffin vesicles after two- an important role in the expression of the mouse dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis, fol- 32 lowed by Coomassie blue staining. DBH denotes dopamine b-hydroxylase, CgA secretogranin II gene, and a proximal G- or chromogranin A, and CgB chromogranin B. Panel B shows an electron micro- C-rich region contributes to the expression of the graph of bovine chromaffin cells. The electron-dense spherical and oblong struc- mouse chromogranin B gene.31 Glucocorticoid tures are dense-core secretory chromaffin granules. The black particles, which have a diameter of 8 to 12 nm, represent immunogold labeling of chromogranin sensitivity has been mapped to a novel glucocorti- A with rabbit antibovine chromogranin A antibody. Panel C shows the subcellular coid-response-element variant in the rat chromo- distribution of a human chromogranin A–enhanced green fluorescent protein granin A.38 (EGFP) chimeric photoprotein, in living sympathoadrenal PC12 cells. Chromo- granin A–EGFP expression was examined by three-dimensional deconvolution microscopy. Nuclei were visualized with blue dye (Hoechst 33342). Optical sec- sorting mechanisms tions along the z axis were acquired with increments of 200 nm and the use of Proteins are secreted from cells by exocytosis in ei- a 100¬ oil-immersion objective to generate three-dimensional views of the ther a constitutive or a regulated manner.39,40 The photoprotein distribution. Chromogranin A–EGFP displays a bright, punctate or vesicular fluorescence signal, especially within the subplasmalemmal re- rate of secretion through the constitutive pathway, gion, indicating storage of the chimera in chromaffin secretory granules. which operates in every type of cell, is a simple func- n engl j med 348;12 www.nejm.org march 20, 2003 1135 Downloaded from www.nejm.org by RODRIGUEZ POVEDA AIXA MILDRED on May 16, 2010 . Copyright © 2003 Massachusetts Medical Society. All rights reserved. The new england journal of medicine Table 1. Physicochemical Properties of Granins.* SgIII SgIV SgV SgVI Property CgA CgB SgII (1B1075) (HISL-19) (7B2) (NESP55) Chromosome localization 14 (human), 20 (human), 2 (human), 2 (mouse) ND 15 (human), 20 (human) 21 (bovine), 3 (rat), 9 (rat), 2 (mouse) 6 (rat), 2 (mouse) 1 (mouse) 12 (mouse) Amino acid residues† 431–445 626–657 559–586 449–507 ND 185 241 Molecular mass (kD)‡ Calculated 49–52 48–52 67.5 51–57 ND 21 27.5 Apparent 74–80 100–120 86 57 35 23 55 Acidic residues (%) 25 24 20 19 ND 16 21 Isoelectric point (pHi) 4.5–5.0 5.1–5.2 5.0 5.1 5.6 5.2 4.4–5.2 Multibasic sites§ 8–10 15–18 9 6–10 ND 3 5 Disulfide-bonded loop Yes Yes No No ND No No Calcium binding Yes Yes Yes ND ND Yes ND Thermostability Yes Yes Yes ND ND Yes Yes Phosphorylation Yes Yes Yes ND ND Yes Yes Sulfation Yes Yes Yes Yes ND Yes ND O-glycosylation Yes Yes Yes ND ND ND Yes N-glycosylation No Yes No No No No No Phosphorylation Yes Yes Yes ND ND Yes Yes * CgA denotes chromogranin A, CgB chromogranin B, SgII secretogranin II, SgIII secretogranin III, SgIV secretogranin IV, SgV secretogranin V, SgVI secretogranin VI, and ND not determined. † Amino acid residues are for mature protein without signal peptide. ‡ The molecular mass was calculated from the primary structure. The apparent molecular mass was determined with the use of sodium dodecyl sulfate–polyacrylamide-gel electrophoresis. § Multibasic sites refers to sites with two or more consecutive Arg or Lys residues. tion of the rate of synthesis of the secreted sub- nonretained proteins.41 The mechanism by which stance. In this pathway, newly synthesized proteins granins are sorted at the trans-Golgi network to en- (e.g., albumin and immunoglobulins) continuous- ter the regulated pathway of secretion is unclear.41-48 ly pass through to the trans-Golgi network and are Selective aggregation of regulated secretory then transported in constitutive vesicles to the plas- proteins at the level of the trans-Golgi network ma membrane for immediate release.