The Diabetes Autoantigen ICA69 and ItsCaenorhabditis elegansHomologue,ric-19, Are Conserved Regulators of Neuroendocrine Secretion

Abstract

ICA69 is a diabetes autoantigen with no homologue of known function. Given that most diabetes autoantigens are associated with neuroendocrine secretory vesicles, we sought to determine if this is also the case for ICA69 and whether this protein participates in the process of neuroendocrine secretion. Western blot analysis of ICA69 tissue distribution in the mouse revealed a correlation between expression levels and secretory activity, with the highest expression levels in brain, pancreas, and stomach mucosa. Subcellular fractionation of mouse brain revealed that although most of the ICA69 pool is cytosolic and soluble, a subpopulation is membrane-bound and coenriched with synaptic vesicles. We used immunostaining in the HIT insulin-secreting ␤-cell line to show that ICA69 localizes in a punctate manner distinct from the insulin granules, suggesting an association with the synaptic-like microvesicles found in these cells. To pursue functional studies on ICA69, we chose to use the model organism Caenorhabditis elegans, for which a homologue of ICA69 exists. We show that the promoter of the C. elegans ICA69 homologue is specifically expressed in all neurons and specialized secretory cells. A deletion mutant was isolated and found to exhibit resistance to the drug aldicarb (an inhibitor of acetylcholinesterase), suggesting defective neurotransmitter secretion in the mutant. On the basis of the aldicarb resistance phenotype, we named the gene ric-19 (resistance to inhibitors of cholinesterase-19). The resistance to aldicarb was rescued by introducing a ric-19 transgene into the ric-19 mutant background. This is the first study aimed at dissecting ICA69 function, and our results are consistent with the interpretation that ICA69/RIC-19 is an evolutionarily conserved cytosolic protein participating in the process of neuroendocrine secretion via association with certain secretory vesicles. INTRODUCTIONTwo main types of specialized secretory vesicles are found in insulin-secreting ␤-cells: dense-core insulin secretory vesicles, which contain a cargo of semicrystalized insulin granules, and lighter synaptic-like microvesicles (SLMVs), which carry a cargo of neurotransmitters such as ␥-aminobutyric acid. Type I diabetes results from the autoimmune destruction of pancreatic ␤-cells, and most of the known protein targets of the diabetogenic autoimmune attack have in common that they are components of the secretory vesicles found in ␤-cells. Autoantigens include proinsulin (Palmer et al., 1983), carboxypeptidase H (Castano et al., 1991, glutamate decarboxylase (GAD) (Baekkeskov et al., 1990), 38-kDa protein (Roep et al., 1990), IA-2/ICA512 (Lan et al., 1994;Rabin et al., 1994;Solimena et al., 1996) and its related protein phogrin (Hawkes et al., 1996), the Glima-38 antigen (Aanstoot et al., 1996), and the GM2-1 ganglioside (Dotta et al., 1998). Although diabetes is primarily a ␤-cell-specific autoimmune disease, expression of the autoantigens is usually not restricted to this cell type but rather exhibits much broader neuroend...

PICK1–ICA69 Heteromeric BAR Domain Complex Regulates Synaptic Targeting and Surface Expression of AMPA Receptors

“…The cellular function of ICA69 is not clear. It has been suggested that ICA69 might be involved in neuron-endocrine secretion, as the mutation of ric-19, the Caenorhabditis elegans homolog of ICA69, leads to resistance to inhibitors of acetylcholinesterase, a phenomenon attributed to defective acetylcholine release (Pilon et al, 2000). ICA69 was found to be located around the Golgi and nearby vesicles (Spitzenberger et al, 2003).…”

“…ICA69 colocalizes with PICK1 except at synapses ICA69 was initially identified from pancreatic ␤ cells and subsequently found to be expressed in many other tissues, including the brain (Pietropaolo et al, 1993;Pilon et al, 2000). PICK1 is known to be highly expressed in the brain, but its expression in the pancreas has not been examined (Xia et al, 1999).…”

Fishing lines, time‐delayed guideposts, and other tricks used by developing pharyngeal neurons in Caenorhabditis elegans

“…They project in opposite directions, differ in their circumferential trajectories (NSM projects in the pharyngeal nerve ring while M2 projects anterior through it, piercing a path through he pm4 cell), and NSM but not M2 is branched. They also offer relatively simple shapes that facilitate scoring of deviant forms, and, importantly, useful green fluorescent protein (GFP) reporters are available for their visualization: pRIC-19::GFP for the M2 neurons (Pilon et al, 2000), and pTPH-1::GFP for the NSM neurons (Sze et al, 2000).…”

Islet Cell Autoantigen of 69 kDa Is an Arfaptin-related Protein Associated with the Golgi Complex of Insulinoma INS-1 Cells

“…Originally identified by immunoscreening of an islet cDNA expression library with prediabetic sera, islet cell autoantigen of 69 kDa (ICA69) 1 is an evolutionary conserved gene with homologues in the nonmammalian model organisms Drosophila melanogaster and Caenorhabditis elegans (1)(2)(3). Like most antigens of type 1 diabetes, ICA69 is enriched in pancreatic ␤-cells and neurons (4).…”

“…Although ICA69 deficiency in mice does not induce any obvious phenotype, the knockout of its C. elegans homologue compromises neurotransmission (3,6). This finding has led to the hypothesis that ICA69, despite lacking membrane anchoring signals, is linked to neuronal synaptic vesicles (SVs) and the related synaptic-like microvesicles (SLMVs) of endocrine cells (3).…”