Expression and Function of IA-2 Family Proteins, Unique Neuroendocrine-Specific Protein-Tyrosine Phosphatases

Endocr. J./ S. TORII: ROLE OF IA-2 FAMILY PROTEINS doi: 10.1507/endocrj.K09E-157

Advance Publication

ORIGINAL

Expression and Function of IA-2 Family Proteins, Unique Neuroendocrine-specific Protein-tyrosine Phosphatases

Seiji TORII

Secretion Biology Lab, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan

Received June 1, 2009; Accepted June 1, 2009; Released online June 24, 2009

Correspondence to: Seiji Torii, Ph.D., Department of Molecular Medicine, Institute for Molecular and Cellular Regulation, Gunma University, Showa 3-39-15, Maebashi, Gunma 371-8512, Japan

Abstract. IA-2 (also known as islet cell antigen ICA-512) and IA-2β (also known as phogrin, phosphatase homologue in granules of insulinoma) are major autoantigens in insulin-dependent diabetes mellitus (IDDM). Autoantibodies against both proteins are expressed years before clinical onset, and they become predictive markers for high-risk subjects. However, the role of these genes in the IDDM pathogenesis has been reported fairly negative by recent studies. IA-2 and IA-2β are type I transmembrane proteins that possess one inactive protein-tyrosine phosphatase (PTP) domain in the cytoplasmic region, and act as one of the constituents of regulated secretory pathways in various neuroendocrine cell types including pancreatic β-cells. Existence of IA-2 homologues in different species suggests a fundamental role in neuroendocrine function. Studies of knockout animals have shown their involvement in maintaining hormone content, however, their specific steps in the secretory pathway IA-2 functions as well as their molecular mechanisms in the hormone content regulation are still unknown. More recent studies have suggested a novel function showing that they contribute to pancreatic β-cell growth. This review attempts to show the possible biological functions of IA-2 family, focusing on their expression and localization in the neuroendocrine cells.

Key words: Insulin, Secretory granule, Dense-core vesicle, Peptide hormone, Diabetes

THE IA-2 genes constitute an evolutionarily conserved family, and there is one IA-2 gene in Caenorhabditis elegans and in Drosophila, and two in the vertebrates [1-5]. The domain structure is conserved among species as represented in Figure 1. The N-terminal pro domain has been suggested to be implicated in sorting or targeting to hormone-containing secretory granules [6], which is then removed by proteolytic cleavage for the protein maturation [2, 7, 8]. This is followed by an intragranular or extracellular domain called matN domain [6], whose crystallographic structure of human IA-2 resembles that of the SEA domain of mucin [9]. Following the transmembrane region, there is a highly homologous cytoplasmic region that contains one protein tyrosine phosphatase (PTP) domain. IA-2 proteins are synthesized on the rough endoplasmic reticulum as a precursor of 100-120 kDa which is then converted by post-translational modifications such as N-glycosylation into a 110-130 kDa protein, and after exiting from or within the trans-Golgi network (TGN), they are cleaved to generate a mature protein of 60-70 kDa [7, 10]. The dibasic consensus sequence recognized by pro-hormone convertases (PC1/3 or PC2 in mammals, EGL-3 in worm) is commonly present in IA-2 family proteins, however, other mature IA-2β proteins with distinct cleavage sites have recently been detected in mouse brain and pancreatic β-cell [11]. Three spliced transcripts of human IA-2β/phogrin gene have been reported (GenBank database), and

Endocr. J./ S. TORII: ROLE OF IA-2 FAMILY PROTEINS doi: 10.1507/endocrj.K09E-157

Drosophila ia2 has been found to have two isoforms as well [12], although the functional difference of each isoform is unknown. On the other hand, a splice variant of human IA-2/ICA512 lacking exon 13 (encodes the transmembrane region) has been discovered in thymus and spleen as well as in pancreas, and it may play a role in the development of autoimmune response to IA-2 protein [13, 14]. IA-2 and IA-2β genes were designated as protein tyrosine phosphatase receptor type N (PTPRN) and protein tyrosine phosphatase receptor type N2 (PTPRN2), respectively, belonging to the IA-2 family which is a part of the PTP superfamily [15]. These proteins show highest homology in the C-terminal PTP domain, and of note, the PTP active site consensus sequence has several amino acids substitutions at conserved sites critical for enzyme activity. IA-2 and IA-2β fail to show phosphatase activity for general PTP substrates but enzyme activity is restored by changing these amino acids to typical consensus residues [16-19]. The lack of phosphatase activity is elucidated by the crystal structure of the PTP domain of human IA-2, which reveals a canonical PTP domain with the closed WPD loop (WPAE in IA-2) over the active site pocket [20]. Since the amino acid substitutions in the catalytic domain are evolutionarily conserved, inactive PTP structure is likely required for physiological function of IA-2 family proteins. Interestingly, Gross et al. has demonstrated that the PTP portion of IA-2 and IA-2β is able to heterodimerize with other receptor-type PTPs such as RPTPα and RPTPε and prevent their activity in a transient expression system [21], however, this function remains to be established at the physiological level.

Specific localization to dense-core secretory granules The IA-2 family members are predominantly expressed in neuroendocrine cells that possess regulated secretory granules. Transgenic worms have shown an ida-1 (IA-2 in C. elegans) expression in peptidergic neurons [5, 22], whereas in situ hybridization has revealed that ia2 in drosophila is expressed in the central nervous system and in the midgut region [5, 12]. IA-2 protein in mammals has been detected in peptidergic neurons in the central nervous system, endocrine cells in pancreatic islets, adrenal medullary cells, and pituitary cells [2, 22]. Analyses of tissues from IA-2 knockout mice with specific monoclonal antibodies have recently confirmed mature IA-2 expression in the neurites of enteric neuronal cells in the muscular layers of the stomach, small intestine, and colon [23]. IA-2β shows a similar tissue distribution, namely, it is highly expressed in brain and pancreatic islets, and its weak expression in the stomach has been reported by several studies [3, 7, 24, 25]. Consistent with these results, simultaneous expressions of IA-2 and IA-2β have been observed in several neuroendocrine cell lines, such as pancreatic β-cell-derived MIN6 and INS-1, pancreatic α-cell-derived αTC-1, and adrenal medulla-derived PC12 [3, 7, 26, 27]. IA-2 and IA-2β have similar structures and distributions, however their expression is regulated distinctly. IA-2 expression increases in accordance to development in rodent tissues, such as the islet and brain [28-30]. Furthermore, IA-2 expression in pancreatic β-cells is induced by glucose, insulin, and cAMP-generating agents (30, 31), whereas proinflammatory cytokines such as IL-1β, TNF-α, and IFN-γ, cause a down-regulation of the IA-2 level [32]. It is thus suggested that IA-2 expression appears concomitant with the development of cellular secretory responses [33]. In contrast, IA-2β shows a more persistent pattern of expression in the developmental stage of islets than IA-2 [25], and its expression is not significantly affected by glucose levels [30]. Glucose stimulation in β-cells instead causes Ca2+-dependent phosphorylation of IA-2β protein that is mediated by protein kinase A [34]. On the other hand, administration of ghrelin, an appetite-regulating peptide, increases mRNA levels of IA-2β but not those of IA-2 in mouse brain and pancreas [35]. Although the chromosomal localization and structure of several IA-2 genes are found in database, analysis of their transcriptional regulatory region is still unexplored. The IA-2 family proteins are thought to be one of the mediators of the regulated

Endocr. J./ S. TORII: ROLE OF IA-2 FAMILY PROTEINS doi: 10.1507/endocrj.K09E-157 secretory pathway because of their restricted expression. Indeed, IA2 and IA-2β are present on dense-core secretory granules (SGs) together with specific peptide hormones or neuropeptides [2, 6, 23, 27]. Immunoelectron microscopic analyses have revealed the localization of IA-2 on dense-core granules in mouse posterior pituitary [2] and colocalization of IA-2β with insulin in pancreatic β-cells [36] (Figure 2). Although a physiological role is unclear, IA-2 and IA-2β are suggested to form a heterodimer in a cell [21], such as a pancreatic β-cell (Torii, unpublished observation). These localization studies suggest that both proteins appear functionally similar. Segregation of proteins into constitutive secretory vesicles and dense-core granules occurs at the TGN or in immature secretory granule (ISG) (Figure 3). Selective packaging of peptide hormones and their associated proteins into SGs may require molecular mechanisms including several sorting domains for specific protein-protein and protein-lipid interactions and selective aggregation of secretory proteins for retention in the granules [37, 38]. Recent observations on IA-2β suggest that the luminal pro domain contributes to its sorting into SGs [6] and the trafficking signals within the cytoplasmic tail play an essential role in its steady-state localization to SGs [39, 40]. The trafficking signals correspond to a tyrosin-based sorting motif (YQE/DL) and a leucine-based sorting motif (EExxxI/LL), and these sequences are well conserved among the family proteins in the species (Figure 1) [39, 40]. Both cytoplasmic signals are involved in the trafficking of mature IA-2β protein at the plasma membrane (endocytosis), presumably through the interaction with AP-2 clathrin-adaptor complexes [39, 40]. When cultured cells are incubated with the specific antibody directed against luminal domain of IA-2 or IA-2β, it binds to the cell surface protein and then accesses SGs during stimulation of exocytotic events [2, 41]. Therefore, the mature proteins which localize on SGs are distributed to the plasma membrane when hormones are secreted, then enter into endosomes (or related organelles), and recycle back to SGs. Other reports have proposed that upon insulin secretion in β-cells, IA-2 protein is cleaved by calpain at the site specific to IA-2 [42] but absent in IA-2β [40], and the PTP-containing fragment then enters into the nucleus [43, 44]. Although an almost identical fragment (~40 kDa) has been found in the bovine pituitary extracts [10], it remains to be elucidated whether the calpain-mediated cleavage similarly occurs in physiological conditions and whether the cleaved transmembrane protein (~30 kDa) is recycled back to SGs.

Physiological function of IA-2 family proteins To demonstrate a physiological role of the IA-2 family, genetic deletions of the corresponding genes were explored in several animals including nematodes and fruit flies. Gene deletion of ida-1 in C. elegans results in subtle defect in neuroendocrine function, whereas IDA-1 genetically interacts with UNC-31/CAPS [45], a protein suggested to function selectively in exocytosis of dense-core vesicles (DCVs). Direct molecular interaction of IA-2/IA-2β with CAPS is, however, undetected in mouse pancreatic β-cells [27]. The genetic analyses of IDA-1 suggest its close association with the DCV pathway at a step of either hormone processing and maturation processes, hormone sorting and loading to DCVs, or DCV trafficking and exocytosis. Since ida-1 mutants enhance weak alleles in the insulin-like signaling pathway, IDA-1 protein may preferentially function in the insulin-like peptides’ action [45]. The implication of IA-2 in the insulin pathway has also been suggested from the study of ia2 in Drosophila. Disruption of gut-specific ia2 expression reveals a role of IA-2 for modulating both expressions of insulin-like peptide ilp-6 and hexokinase [12]. However, the molecular mechanisms underlying IA-2 regulation of DCV secretion events and/or the insulin pathway have not been demonstrated by these genetic studies. Gene deletion of IA-2 or IA-2β in mice has resulted in mild glucose intolerance and decreased glucose-responsive insulin secretion [46, 47]. These genetic analyses indicated IA-2/IA-2β is an important factor involved in insulin secretion events, however, the

Endocr. J./ S. TORII: ROLE OF IA-2 FAMILY PROTEINS doi: 10.1507/endocrj.K09E-157 resolution to address questions as to where in the secretory pathway IA-2 and IA-2β function is lacking. Although IA-2/IA-2β double KO (DKO) does not cause a much greater inhibition than each single knockout, DKO mice also exhibit an impairment of the rapid increase in plasma insulin after glucose administration [48]. However, an in vitro perfusion experiment has revealed that the secretory response to glucose in DKO islets is essentially normal but is barely decreased only at the sustained phase in insulin secretion when compared with that of control islets [49]. More importantly, the insulin content of DKO islets is 45% less than that of controls [49], which may partly affect the acute secretion defects (by glucose administration) in DKO mice. Thus, IA-2/IA-2β in pancreatic β-cells perhaps contributes to the regulation of insulin content, although the secondary effects and/or extrinsic factors to islets cannot be excluded. On the other hand, enhanced secretory responses induced by arginine were observed in DKO islets as well as in DKO mice [48, 49], even though there was less insulin content. This suggests that neither IA-2 nor IA-2β plays an essential role in DCV trafficking and secretion processes. The phenotype that displays a defect in insulin secretion was not only found in IA-2/IA-2β-deficient mice but also in various gene knockout mice. Other approaches such as biochemical assays and analyses at the cellular level yield more direct and reasonable results. Therefore, overexpression and knockdown experiments with cultured cells lines were applied to show a principal function of IA-2/IA-2β protein. The early studies suggested that IA-2β does not affect hormone secretion by its transduction into AtT-20 mouse anterior pituitary cells and PC12 rat pheochromocytoma cells [34, 50]. Similarly, the authors recently demonstrated the results that basal and glucose-stimulated insulin secretion remained unaffected by adenovirus-mediated IA-2 or IA-2β overexpression in both MIN6 and INS-1E cells [27]. Consistent with overexpression analyses, transient knockdown of IA-2 or IA-2β by shRNA does not cause any change in insulin secretion during 72 h post-infection of adenoviruses [27], suggesting that neither IA-2 nor IA-2β regulates exocytosis of insulin-containing secretory granules in pancreatic β-cells. A previous report by Harashima et al. has shown that the siRNA-directed knockdown of IA-2 caused a marked reduction of insulin content and several SG-related protein (e.g. VAMP2) expressions as well as insulin secretion [51]. However, the possibility cannot be excluded that the siRNA-derived off-target effects influence various gene expressions including insulin and VAMP2, because our shRNAs for IA-2 or IA-2β specifically silenced the endogenous target protein even though shRNAs appeared more effective than their siRNA that were assessed by protein expression levels [27]. They further demonstrated that MIN6 cells stably overexpressing IA-2 show a 3-fold increase in both insulin content and the number of insulin granules [51]. In this case, scrupulous attention should be paid to the possibility that the stable clone obtained such a potency independent of IA-2, because MIN6 cells are heterogenous in hormone contents [52] and the insulin content per cell is changeable during the cloning steps [27, 53, 54]. On the other hand, Mziaut et al. has demonstrated that insulin gene transcription is promoted by prolonging STAT5 activity through the action of calpain-cleaved IA-2 tail [44], although this retrograde pathway does not seem to apply to IA-2β. Taken together with the results by DKO islets [49], these data partly support the idea that IA-2 and IA-2β contribute to the maintenance of insulin content presumably through regulating expression of insulin and/or SG-associated proteins in pancreatic β−cells. It is also plausible that IA-2 family proteins are involved in selective sorting of peptide hormones to SGs. Since IA-2β/phogrin has been shown to interact with AP-1 clathrin adaptor complexes by the dileucine-based trafficking signal [39], IA-2 proteins may act as a sorting receptor for SG-targeting proteins at the TGN or ISG, like a mannose-6-phosphate receptor for lysosomal proteins [55]. However, the authors did not detect early changes in constitutive and regulated insulin secretion by IA-2/IA-2β knockdown, but a slight reduction in insulin content was observed in MIN6 cells that IA-2/IA-2β-silenced for more than 72 h [27], and furthermore, DKO mice exhibited a

Endocr. J./ S. TORII: ROLE OF IA-2 FAMILY PROTEINS doi: 10.1507/endocrj.K09E-157 slight decrease in islet insulin content [49]. Therefore, IA-2 and IA-2β seem to play a supportive role in regulation of insulin granule content rather than function as sole determinants for hormone sorting or SG biogenesis.

Regulation of pancreatic β-cell growth by IA-2 and IA-2β A novel role of IA-2 and IA-2β in the β-cell growth has been recently demonstrated by two independent experiments [27, 56]. Specific knockdown of IA-2 or IA-2β in both MIN6 and INS-1E β-cell lines, and also in isolated mouse islets, caused a marked reduction in the cell proliferation rate, without any changes in insulin secretion [27]. Similarly, siRNA-derived knockdown of IA-2/ICA512 decreases proliferation of INS-1 cells, and furthermore, the regeneration of β-cells at partial pancreatectomy is reduced in IA-2-deficient mice [56]. In contrast with these observations, the morphological and the immunohistological data of islets in IA-2 and/or IA-2β-deficient mice appear to be normal [46-48]. It has been clearly shown that β-cells proliferate by replication [57], and glucose promotes the β-cell mass expansion for compensation of insulin resistance [58]. Thus, IA-2 and IA-2β may positively regulate pancreatic β-cell growth under a specific condition or a kind of pathological state. Regarding the molecular action of IA-2 protein, one group gives the hypothesis that, when insulin exocytosis is promoted, the cytoplasmic tail of IA-2 is cleaved by a Ca2+-dependent calpain and translocates to the nucleus where it promotes transcription of the insulin and other SG genes, and it also promotes up-regulation of cyclin D via STATs activation [43, 44, 56]. However, this series of studies awaits independent examination because most of the experimental condition was demonstrated by overexpression of the cytoplasmic fragment. The authors proposed a different mechanism in which IA-2 and IA-2β in pancreatic β-cells associate with the insulin signaling pathway (Figure 4). When insulin granule exocytosis is induced by glucose, IA-2 and IA-2β translocate to the plasma membrane, where they transiently form a complex with insulin receptor that is activated by secreted insulin in an autocrine/paracrine fashion [27]. The IA-2/IA-2β interaction with insulin receptor in turn leads to stabilization of IRS2 protein, which has been suggested to be a master regulator for β-cell growth [58, 59]. Inspection using the knockout mouse may be required to confirm whether this regulated mechanism works in vivo as well. IA-2 family proteins play an essential role in the regulated secretory pathway in various neuroendocrine cells, presumably through regulating the maintenance of hormone content. This explains the other phenotypes found in the gene-targeting mice, for example, DKO mice have exhibited a female infertility due to defect in the luteinizing hormone surge [60], and they have also exhibited a reduction of plasma rennin concentration likely through the decrease in catecholamine release [61]. For excluding the secondary effect from other IA-2/IA-2β-deficient tissues, conditional knockout animals should be produced in the near future. In pancreatic β-cells, growth-promoting activity of IA-2 and IA-2β appears to be more potent than the general function. In addition to genetic analyses, a number of approaches including biochemical assays will assist with better understanding of the exact function of IA-2 family proteins.

Perspective IA-2 and IA-2β are found to be major autoantigens associated with type-1 diabetes mellitus. At present their physiological functions are incompletely defined, however, extensive studies indicate that they are an excellent marker for cells possessing the reliable neuroendocrine responses, which may be corroborated by SG content. They are also valuable for labeling the DCVs or SGs in intracellular imaging and immunocytochemical studies, and in fact, GFP-tagged IA-2 proteins have been used to visualize SGs for precisely chasing their movement and/or exocytosis processes (8, 62-64). On the other hand, the inactive PTP structure has at least provided some impact on the research of protein phosphatases, since the

Endocr. J./ S. TORII: ROLE OF IA-2 FAMILY PROTEINS doi: 10.1507/endocrj.K09E-157 possibility of an extremely narrow substrate specificity cannot be ruled out [24]. Taken together, the IA-2 family proteins will attract more attention from various research fields continuously in the future.

Acknowledgments I would like to thank Dr. I. Kojima (Gunma Univ.) for giving me an opportunity to write this review article. I also thank to Dr. T. Takeuchi for his generous support and to Hiromi Hashimoto for preparing the EM data. This work is supported by a grant in aid for Young Scientists from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

References 1. Lan MS, Lu J, Goto Y, Notkins AL (1994) Molecular cloning and identification of a receptor-type protein tyrosine phosphatase, IA-2, from human insulinoma. DNA Cell Biol 13: 505-514. 2. Solimena M, Dirkx R Jr., Hermel JM, Pleasic-Williams S, Shapiro JA, Caron L, Rabin DU (1996) ICA 512, an autoantigen of type I diabetes, is an intrinsic membrane protein of neurosecretory granules. Embo J 15: 2102-2114. 3. Lu J, Li Q, Xie H, Chen ZJ, Borovitskaya AE, Maclaren NK, Notkins AL, Lan MS (1996) Identification of a second transmembrane protein tyrosine phosphatase, IA-2beta, as an autoantigen in insulin-dependent diabetes mellitus: precursor of the 37-kDa tryptic fragment. Proc Natl Acad Sci U S A 93: 2307-2311. 4. Kawasaki E, Hutton JC, Eisenbarth GS (1996) Molecular cloning and characterization of the human transmembrane protein tyrosine phosphatase homologue, phogrin, an autoantigen of type 1 diabetes. Biochem Biophys Res Commun 227: 440-447. 5. Cai T, Krause MW, Odenwald WF, Toyama R, Notkins AL (2001) The IA-2 gene family: homologs in Caenorhabditis elegans, Drosophila and zebrafish. Diabetologia 44: 81-88. 6. Wasmeier C, Bright NA, Hutton JC (2002) The lumenal domain of the integral membrane protein phogrin mediates targeting to secretory granules. Traffic 3: 654-665 7. Wasmeier C, Hutton JC (1996) Molecular cloning of phogrin, a protein-tyrosine phosphatase homologue localized to insulin secretory granule membranes. J Biol Chem 271: 18161-18170. 8. Zahn TR Angleson JK, MacMorris MA, Domke E, Hutton JF, Schwartz C, Hutton JC (2004) Dense core vesicle dynamics in Caenorhabditis elegans neurons and the role of kinesin UNC-104. Traffic 5: 544-559. 9. Primo ME, Klinke S, Sica MP, Goldbaum FA, Jakoncic J, Poskus E, Ermacora MR (2008) Structure of the mature ectodomain of the human receptor-type protein-tyrosine phosphatase IA-2. J Biol Chem 283: 4674-4681. 10. Hermel JM, Dirkx R Jr., Solimena M (1999) Post-translational modifications of ICA512, a receptor tyrosine phosphatase-like protein of secretory granules. Eur J Neurosci 11: 2609-2620. 11. Kawakami T, Saeki K, Takeyama N, Wu G, Sakudo A, Matsumoto Y, Hayashi T, Onodera T (2007) Detection of proteolytic cleavages of diabetes-associated protein IA-2 beta in the pancreas and the brain using novel anti-IA-2 beta monoclonal antibodies. Int J Mol Med 20: 177-185. 12. Kim J, Bang H, Ko S, Jung I, Hong H, Kim-Ha J (2008) Drosophila ia2 modulates secretion of insulin-like peptide. Comp Biochem Physiol A Mol Integr Physiol 151: 180-184. 13. Park YS, Kawasaki E, Kelemen K, Yu L, Schiller MR, Rewers M, Mizuta M, Eisenbarth GS, Hutton JC (2000) Humoral autoreactivity to an alternatively spliced variant of ICA512/IA-2 in Type I diabetes. Diabetologia 43: 1293-1301. 14. Diez J, Park Y, Zeller M, Brown D, Garza D, Ricordi C, Hutton J, Eisenbarth GS,

Endocr. J./ S. TORII: ROLE OF IA-2 FAMILY PROTEINS doi: 10.1507/endocrj.K09E-157

Pugliese A (2001) Differential splicing of the IA-2 mRNA in pancreas and lymphoid organs as a permissive genetic mechanism for autoimmunity against the IA-2 type 1 diabetes autoantigen. Diabetes 50: 895-900. 15. Alonso A, Sasin J, Bottini N, Friedberg I, Osterman A, Godzik A, Hunter T, Dixon J, Mustelin T (2004) Protein tyrosine phosphatases in the human genome. Cell 117: 699-711. 16. Lu J, Notkins AL, Lan MS (1994) Isolation, sequence and expression of a novel mouse brain cDNA, mIA-2, and its relatedness to members of the protein tyrosine phosphatase family. Biochem Biophys Res Commun 204: 930-936. 17. Magistrelli G, Toma S, Isacchi A (1996) Substitution of two variant residues in the protein tyrosine phosphatase-like PTP35/IA-2 sequence reconstitutes catalytic activity. Biochem Biophys Res Commun 227: 581-588. 18. Fitzgerald LR, Walton KM, Dixon JE, Largent BL (1997) PTP NE-6: a brain-enriched receptor-type protein tyrosine phosphatase with a divergent catalytic domain. J Neurochem 68: 1820-1829. 19. Drake PG, Peters GH, Andersen HS, Hendriks W, Moller NP (2003) A novel strategy for the development of selective active-site inhibitors of the protein tyrosine phosphatase-like proteins islet-cell antigen 512 (IA-2) and phogrin (IA-2beta). Biochem J 373: 393-401. 20. Kim SJ, Jeong DG, Jeong SK, Yoon TS, Ryu SE (2007) Crystal structure of the major diabetes autoantigen insulinoma-associated protein 2 reveals distinctive immune epitopes. Diabetes 56: 41-48. 21. Gross S, Blanchetot C, Schepens J, Albet S, Lammers R, den Hertog J, Hendriks W (2002) Multimerization of the protein-tyrosine phosphatase (PTP)-like insulin-dependent diabetes mellitus autoantigens IA-2 and IA-2beta with receptor PTPs (RPTPs). Inhibition of RPTPalpha enzymatic activity. J Biol Chem 277: 48139-48145. 22. Zahn TR, Macmorris MA, Dong W, Day R, Hutton JC (2001) IDA-1, a Caenorhabditis elegans homolog of the diabetic autoantigens IA-2 and phogrin, is expressed in peptidergic neurons in the worm. J Comp Neurol 429: 127-143. 23. Takeyama N, Ano Y, Wu G, Kubota N, Saeki K, Sakudo A, Momotani E, Sugiura K, Yukawa M, Onodera T (2009) Localization of insulinoma associated protein 2, IA-2 in mouse neuroendocrine tissues using two novel monoclonal antibodies. Life Sci 84: 678-687. 24. Cui L, Yu WP, DeAizpurua HJ, Schmidli RS, Pallen CJ (1996) Cloning and characterization of islet cell antigen-related protein-tyrosine phosphatase (PTP), a novel receptor-like PTP and autoantigen in insulin-dependent diabetes. J Biol Chem 271: 24817-24823. 25. Chiang MK, Flanagan JG (1996) PTP-NP, a new member of the receptor protein tyrosine phosphatase family, implicated in development of nervous system and pancreatic endocrine cells. Development 122: 2239-2250. 26. Kambayashi Y, Takahashi K, Bardhan S, Inagami T (1995) Cloning and expression of protein tyrosine phosphatase-like protein derived from a rat pheochromocytoma cell line. Biochem J 306 ( Pt 2): 331-335. 27. Torii S, Saito N, Kawano A, Hou N, Ueki K, Kulkarni RN, Takeuchi T (2009) Gene silencing of phogrin unveils its essential role in glucose-responsive pancreatic beta-cell growth. Diabetes 58: 682-692. 28. Roberts C, Roberts GA, Lobner K, Bearzatto M, Clark A, Bonifacio E, Christie MR (2001) Expression of the protein tyrosine phosphatase-like protein IA-2 during pancreatic islet development. J Histochem Cytochem 49: 767-776. 29. Shimizu S, Saito N, Kubosaki A, SungWook S, Takeyama N, Sakamoto T, Matsumoto Y, Saeki K, Matsumoto Y, Onodera T (2001) Developmental expression and localization of

Endocr. J./ S. TORII: ROLE OF IA-2 FAMILY PROTEINS doi: 10.1507/endocrj.K09E-157

IA-2 mRNA in mouse neuroendocrine tissues. Biochem Biophys Res Commun 288: 165-171. 30. Lobner K, Steinbrenner H, Roberts GA, Ling Z, Huang GC, Piquer S, Pipeleers DG, Seissler J, Christie MR (2002) Different regulated expression of the tyrosine phosphatase-like proteins IA-2 and phogrin by glucose and insulin in pancreatic islets: relationship to development of insulin secretory responses in early life. Diabetes 51: 2982-2988. 31. Seissler J, Nguyen TB, Aust G, Steinbrenner H, Scherbaum WA (2000) Regulation of the diabetes-associated autoantigen IA-2 in INS-1 pancreatic beta-cells. Diabetes 49: 1137-1141. 32. Steinbrenner H, Nguyen TB, Wohlrab U, Scherbaum WA, Seissler J (2002) Effect of proinflammatory cytokines on gene expression of the diabetes-associated autoantigen IA-2 in INS-1 cells. Endocrinology 143: 3839-3845 33. Knoch KP, Bergert H, Borgonovo B, Saeger HD, Altkruger A, Verkade P, Solimena M (2004) Polypyrimidine tract-binding protein promotes insulin secretory granule biogenesis. Nat Cell Biol 6: 207-214. 34. Wasmeier C, Hutton JC (2001) Secretagogue-dependent phosphorylation of the insulin granule membrane protein phogrin is mediated by cAMP-dependent protein kinase. J Biol Chem 276: 31919-31928. 35. Doi A, Shono T, Nishi M, Furuta H, Sasaki H, Nanjo K (2006) IA-2beta, but not IA-2, is induced by ghrelin and inhibits glucose-stimulated insulin secretion. Proc Natl Acad Sci U S A 103: 885-890. 36. Klumperman J, Kuliawat R, Griffith JM, Geuze HJ, Arvan P (1998) Mannose 6-phosphate receptors are sorted from immature secretory granules via adaptor protein AP-1, clathrin, and syntaxin 6-positive vesicles. J Cell Biol 141: 359-371. 37. Dikeakos JD, Reudelhuber TL (2007) Sending proteins to dense core secretory granules: still a lot to sort out. J Cell Biol 177: 191-196. 38. Takeuchi T, Hosaka M (2008) Sorting mechanism of peptide hormones and biogenesis mechanism of secretory granules by secretogranin III, a cholesterol-binding protein, in endocrine cells. Curr Diabetes Rev 4: 31-38. 39. Torii S, Saito N, Kawano A, Zhao S, Izumi T, Takeuchi T (2005) Cytoplasmic transport signal is involved in phogrin targeting and localization to secretory granules. Traffic 6: 1213-1224. 40. Wasmeier C, Burgos PV, Trudeau T, Davidson HW, Hutton JC (2005) An extended tyrosine-targeting motif for endocytosis and recycling of the dense-core vesicle membrane protein phogrin. Traffic 6: 474-487. 41. Vo YP, Hutton JC, Angleson JK (2004) Recycling of the dense-core vesicle membrane protein phogrin in Min6 beta-cells. Biochem Biophys Res Commun 324: 1004-1010. 42. Ort T, Voronov S, Guo J, Zawalich K, Froehner SC, Zawalich W, Solimena M (2001) Dephosphorylation of beta2-syntrophin and Ca2+/mu-calpain-mediated cleavage of ICA512 upon stimulation of insulin secretion. Embo J 20: 4013-4023. 43. Trajkovski M, Mziaut H, Altkruger A, Ouwendijk J, Knoch KP, Muller S, Solimena M (2004) Nuclear translocation of an ICA512 cytosolic fragment couples granule exocytosis and insulin expression in {beta}-cells. J Cell Biol 167: 1063-1074. 44. Mziaut H, Trajkovski M, Kersting S, Ehninger A, Altkruger A, Lemaitre RP, Schmidt D, Saeger HD, Lee MS, Drechsel DN, Muller S, Solimena M (2006) Synergy of glucose and growth hormone signalling in islet cells through ICA512 and STAT5. Nat Cell Biol 8: 435-445. 45. Cai T, Fukushige T, Notkins AL, Krause M (2004) Insulinoma-Associated Protein IA-2, a Vesicle Transmembrane Protein, Genetically Interacts with UNC-31/CAPS and Affects Neurosecretion in Caenorhabditis elegans. J Neurosci 24: 3115-3124.

Endocr. J./ S. TORII: ROLE OF IA-2 FAMILY PROTEINS doi: 10.1507/endocrj.K09E-157

46. Saeki K, Zhu M, Kubosaki A, Xie J, Lan MS, Notkins AL (2002) Targeted disruption of the protein tyrosine phosphatase-like molecule IA-2 results in alterations in glucose tolerance tests and insulin secretion. Diabetes 51: 1842-1850. 47. Kubosaki A, Gross S, Miura J, Saeki K, Zhu M, Nakamura S, Hendriks W, Notkins AL (2004) Targeted disruption of the IA-2beta gene causes glucose intolerance and impairs insulin secretion but does not prevent the development of diabetes in NOD mice. Diabetes 53: 1684-1691. 48. Kubosaki A, Nakamura S, Notkins AL (2005) Dense core vesicle proteins IA-2 and IA-2beta: metabolic alterations in double knockout mice. Diabetes 54 Suppl 2: S46-51. 49. Henquin JC, Nenquin M, Szollosi A, Kubosaki A, Notkins AL (2008) Insulin secretion in islets from mice with a double knockout for the dense core vesicle proteins islet antigen-2 (IA-2) and IA-2beta. J Endocrinol 196: 573-581. 50. Tsuboi T, Zhao C, Terakawa S, Rutter GA (2000) Simultaneous evanescent wave imaging of insulin vesicle membrane and cargo during a single exocytotic event. Curr Biol 10: 1307-1310. 51. Harashima S, Clark A, Christie MR, Notkins AL (2005) The dense core transmembrane vesicle protein IA-2 is a regulator of vesicle number and insulin secretion. Proc Natl Acad Sci U S A 102: 8704-8709. 52. Nakashima K, Kanda Y, Hirokawa Y, Kawasaki F, Matsuki M, Kaku K (2009) MIN6 is not a pure beta cell line but a mixed cell line with other pancreatic endocrine hormones. Endocr J 56: 45-53. 53. Sawada Y, Zhang B, Okajima F, Izumi T, Takeuchi T (2001) PTHrP increases pancreatic beta-cell-specific functions in well-differentiated cells. Mol Cell Endocrinol 182: 265-275. 54. Roderigo-Milne H, Hauge-Evans AC, Persaud SJ, Jones PM (2002) Differential expression of insulin genes 1 and 2 in MIN6 cells and pseudoislets. Biochem Biophys Res Commun 296: 589-595. 55. van Meel E, Klumperman J (2008) Imaging and imagination: understanding the endo-lysosomal system. Histochem Cell Biol 129: 253-266. 56. Mziaut H, Kersting S, Knoch KP, Fan WH, Trajkovski M, Erdmann K, Bergert H, Ehehalt F, Saeger HD, Solimena M (2008) ICA512 signaling enhances pancreatic beta-cell proliferation by regulating cyclins D through STATs. Proc Natl Acad Sci U S A 105: 674-679. 57. Dor Y, Brown J, Martinez OI, Melton DA (2004) Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature 429: 41-46. 58. Terauchi Y, Takamoto I, Kubota N, Matsui J, Suzuki R, Komeda K, Hara A, Toyoda Y, Miwa I, Aizawa S, Tsutsumi S, Tsubamoto Y, Hashimoto S, Eto K, Nakamura A, Noda M, Tobe K, Aburatani H, Nagai R, Kadowaki T (2007) Glucokinase and IRS-2 are required for compensatory beta cell hyperplasia in response to high-fat diet-induced insulin resistance. J Clin Invest 117: 246-257. 59. Rhodes CJ (2005) Type 2 diabetes-a matter of beta-cell life and death? Science 307: 380-384. 60. Kubosaki A, Nakamura S, Clark A, Morris JF, Notkins AL (2006) Disruption of the transmembrane dense core vesicle proteins IA-2 and IA-2beta causes female infertility. Endocrinology 147: 811-815. 61. Kim SM, Theilig F, Qin Y, Cai T, Mizel D, Faulhaber-Walter R, Hirai H, Bachmann S, Briggs JP, Notkins AL, Schnermann J (2009) Dense-core vesicle proteins IA-2 and IA-2{beta} affect renin synthesis and secretion through the {beta}-adrenergic pathway. Am J Physiol Renal Physiol 296: F382-389. 62. Pouli AE, Emmanouilidou E, Zhao C, Wasmeier C, Hutton JC, Rutter GA (1998) Secretory-granule dynamics visualized in vivo with a phogrin-green fluorescent protein

Endocr. J./ S. TORII: ROLE OF IA-2 FAMILY PROTEINS doi: 10.1507/endocrj.K09E-157

chimaera. Biochem J 333: 193-199. 63. Taraska JW, Perrais D, Ohara-Imaizumi M, Nagamatsu S, Almers W (2003) Secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells. Proc Natl Acad Sci U S A 100: 2070-2075. 64. Torii S, Takeuchi T, Nagamatsu S, Izumi T (2004) Rab27 effector granuphilin promotes the plasma membrane targeting of insulin granules via interaction with syntaxin 1a. J Biol Chem 279: 22532-22538.

Endocr. J./ S. TORII: ROLE OF IA-2 FAMILY PROTEINS doi: 10.1507/endocrj.K09E-157

Fig.1. Domain structures of IA-2 family proteins. The domain structures of IA-2 family proteins are schematically represented, and the amino acid numbers are shown on the right. SP, signal peptide; TM, transmembrane region; PTP, protein-tyrosine phosphatase homologous region; N, potential N-glycosylation site; Y, tyrosine-based trafficking signal; L, leucine-based trafficking signal; dotted line, putative dibasic cleavage site.

Fig. 2. Immunoelectron micrograph of IA-2β in MIN6 cell. Immunoelectron microscopy of MIN6 β–cell line shows the colocalization of IA-2β/phogrin (represented by 10 nm gold particles) with insulin (5 nm gold particles) on dense-core secretory granules (arrowheads). Bar, 200 nm.

Endocr. J./ S. TORII: ROLE OF IA-2 FAMILY PROTEINS doi: 10.1507/endocrj.K09E-157

Fig. 3. Sorting and transport of secretory granule proteins in neuroendocrine cell. The sorting process of secretory proteins and the trafficking pathway of IA-2/IA-2β are schematically illustrated. The biosynthetic and endocytic pathways are indicated by arrows. A, Constitutive secretion; B, Constitutive-like pathway; C, Regulated secretion; D, Endocytosis; E, Lysosomal pathway.

Endocr. J./ S. TORII: ROLE OF IA-2 FAMILY PROTEINS doi: 10.1507/endocrj.K09E-157

Fig. 4. A model of action of IA-2/IA-2β in autocrine insulin signaling pathway. IA-2/IA-2β colocalizes with insulin on secretory granules in pancreatic β-cells. It translocates to the plasma membrane whenever insulin exocytosis is induced by glucose. IA-2/IA-2β interacts with insulin receptor that is simultaneously activated by secreted insulin, and then promotes the β-cell proliferation through stabilizing IRS2 protein.