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 1 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 2 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].