1OBJECTIVE-Phogrin and IA-2, autoantigens in insulin-dependent diabetes, have been shown to be involved in insulin secretion in pancreatic ␤-cells; however, implications at a molecular level are confusing from experiment to experiment. We analyzed biological functions of phogrin in ␤-cells by an RNA interference technique. RESEARCH DESIGN AND METHODS-Adenovirus-mediated expression of short hairpin RNA specific for phogrin (shPhogrin) was conducted using cultured ␤-cell lines and mouse islets. Both glucose-stimulated insulin secretion and cell proliferation rate were determined in the phogrin-knockdown cells. Furthermore, protein expression was profiled in these cells. To see the binding partner of phogrin in ␤-cells, coimmunoprecipitation analysis was carried out. RESULTS-Adenoviral expression of shPhogrin efficiently decreased its endogenous expression in pancreatic ␤-cells. Silencing of phogrin in ␤-cells abrogated the glucose-mediated mitogenic effect, which was accompanied by a reduction in the level of insulin receptor substrate 2 (IRS2) protein, without any changes in insulin secretion. Phogrin formed a complex with insulin receptor at the plasma membrane, and their interaction was promoted by high-glucose stimulation that in turn led to stabilization of IRS2 protein. Corroboratively, phogrin knockdown had no additional effect on the proliferation of ␤-cell line derived from the insulin receptor-knockout mouse. CONCLUSIONS-Phogrin is involved in ␤-cell growth via regulating stability of IRS2 protein by the molecular interaction with insulin receptor. We propose that phogrin and IA-2 function as an essential regulator of autocrine insulin action in pancreatic ␤-cells. Diabetes 58:682-692, 2009 G lucose is a principle regulator of pancreatic ␤-cell survival and growth as well as insulin secretion (1). It is a potent mitogen on pancreatic ␤-cells and regulates islet ␤-cell mass through their replication (2). Recent studies have suggested that insulin secreted in response to elevated glucose exerts autocrine/paracrine effects, including promotion of insulin biosynthesis and proliferation of ␤-cells (3,4). The importance of insulin signaling in maintaining ␤-cell mass was demonstrated by targeted knockouts of the insulin receptor and insulin receptor substrate 2 (IRS2) (5-8). Although insulin receptor knockout had a restricted effect on ␤-cell mass (7), its mitogenic function on ␤-cells was clearly shown by short interfering RNA (siRNA)-based silencing of insulin receptor in ␤-cell-derived MIN6 cells (9,10). More recently, another pathway was demonstrated showing that glucose metabolism leads to increased ␤-cell mass through the transcriptional activation of IRS2 (11). Calcium/calmodulin-dependent protein kinases and increased cAMP levels were suggested to contribute to IRS2 expression, and this pathway has been shown to be modulated by the incretin hormone glucagonlike peptide 1 (GLP-1) (12,13). In both cases, IRS2 must be a key mediator for glucose-responsive ␤-cell growth (14).Phogrin (IA-2␤) and IA-2 (ICA51...

Phogrin is an integral glycoprotein primarily expressed in neuroendocrine cells. The predominant localization of phogrin is on dense-core secretory granules, and the lumenal domain has been shown to be involved in its efficient sorting to the regulated secretory pathway. Here, we present data showing that a leucine-based sorting signal [EExxxIL] within the cytoplasmic tail contributes its steady-state localization to secretory granules. Deletion mutants in the tail region failed to represent granular distribution in pancreatic b-cell line, MIN6, and anterior pituitary cell line, AtT-20. A sorting signal mutant with two glutamic acids substituted into alanines (EE/AA) is primarily accumulated in the Golgi area instead of secretory granules, and another mutant (IL/AA) is trapped at the plasma membrane due to a defect in endocytosis. We further demonstrate that the leucine-based sorting signal of phogrin specifically interacts with both adaptor protein AP-1 and AP-2 clathrin adaptor complexes in vitro. These observations, along with previous studies, suggest that distinct domains of phogrin mediate proper localization of this transmembrane protein on secretory granules.

Autocrine insulin signaling is critical for pancreatic β-cell growth and activity and is at least partially controlled by protein-tyrosine phosphatases (PTPs) that act on insulin receptors (IRs). The receptor-type PTP phogrin primarily localizes on insulin secretory granules in pancreatic β cells. We recently reported that phogrin knockdown decreases the protein levels of insulin receptor substrate 2 (IRS2), whereas high-glucose stimulation promotes formation of a phogrin-IR complex that stabilizes IRS2. However, the underlying molecular mechanisms by which phogrin affects IRS2 levels are unclear. Here, we found that relative to wildtype mice, IRS2 levels in phogrin-knockout mice islets decreased by 44%. When phogrin was silenced by shRNA in pancreatic β-cell lines, glucose-induced insulin signaling led to proteasomal degradation of IRS2 via a negative feedback mechanism. Phogrin overexpression in a murine hepatocyte cell line consistently prevented chronic insulin treatment-induced IRS2 degradation. , phogrin directly bound the IR without the assistance of other proteins and protected recombinant PTP1B from oxidation to potentiate its activity toward the IR. Furthermore, phogrin expression suppressed insulin-induced local generation of hydrogen peroxide and subsequent PTP1B oxidation, which allowed progression of IR dephosphorylation. Together, these results suggest that a transient interaction of phogrin with the IR enables glucose-stimulated autocrine insulin signaling through the regulation of PTP1B activity, which is essential for suppressing feedback-mediated IRS2 degradation in pancreatic β cells.

Histone variants perform unique functions and are deposited onto DNA by mechanisms distinct from those of canonical histones. The H2A variant, H2A.Z, also known as Htz1 in Saccharomyces cerevisiae, is not uniformly distributed across the genome but facilitates transcriptional activation at target gene promoters and anti-silencing at heterochromatin loci. Htz1 is also involved in DNA replication, DNA repair, chromosome segregation and cell cycle control. Its sequence identity to canonical H2A is only 60%, and it is likely that the nonconserved residues are responsible for Htz1-specific functions. However, precise roles of these variant-specific residues are not well understood. To gain insights into the molecular basis underlying the functional differences between canonical and variant histones, 117 alanine-scanning point mutants of Htz1 were constructed for this study, and chemical genetic screens were carried out. Consequently, seven Htz1 residues that conferred one or more abnormal phenotypes when mutated were identified. Based on primary sequence and functional conservation between H2A and Htz1, two of these residues (F32 and I109) appear to have an Htz1-specific role, whereas the rest seem to have functions shared between H2A and Htz1. This study provides a useful resource for future investigations into functional convergence and divergence between canonical and variant histones.

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