title: Biochemical, biophysical, and functional properties of ICA512/IA-2 RESP18 homology domain.
abstract: BACKGROUND
  ICA512 (or IA-2/PTPRN) is a transmembrane protein-tyrosine phosphatase located in secretory granules of neuroendocrine cells. Previous studies implied its involvement in generation, cargo storage, traffic, exocytosis and recycling of insulin secretory granules, as well as in β-cell proliferation. While several ICA512 domains have been characterized, the function and structure of a large portion of its N-terminal extracellular (or lumenal) region are unknown. Here, we report a biophysical, biochemical, and functional characterization of ICA512-RESP18HD, a domain comprising residues 35 to 131 and homologous to regulated endocrine-specific protein 18 (RESP18).
  
  
  METHODS
  Pure recombinant ICA512-RESP18HD was characterized by CD and fluorescence. Its binding to insulin and proinsulin was characterized by ELISA, surface plasmon resonance, and fluorescence anisotropy. Thiol reactivity was measured kinetically. Targeting of ΔRESP18HD ICA512-GFP to the membrane of insulinoma cells was monitored by immunofluorescence.
  
  
  RESULTS
  ICA512-RESP18HD possesses a strong tendency to aggregate and polymerize via intermolecular disulfide formation, particularly at pH>4.5. Its cysteine residues are highly susceptible to oxidation forming an intramolecular disulfide between cysteine 53 and 62 and intermolecular disulfides via cysteine 40 and cysteine 47. The regulated sorting of ICA512 to secretory granules in INS-1 cells was impaired by deletion of RESP18HD. ICA512-RESP18HD binds with high-affinity to insulin and proinsulin.
  
  
  CONCLUSIONS
  RESP18HD is required for efficient sorting of ICA512 to secretory granules.
  
  
  CONCLUSIONS
  RESP18HD is a key determinant for ICA512 granule targeting.
authors.affiliation: Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni (IDEHU), Universidad de Buenos Aires-CONICET, Argentina; Cátedra de Inmunología de la Facultad de Farmacia y Bioquímica, IDEHU, Argentina; División Endocrinología del Hospital de Clínicas José de San Martín, Universidad de Buenos Aires-CONICET, Argentina.
authors.forename: Laura
authors.initials: L
authors.lastname: Sosa
authors.affiliation: Paul Langerhans Institute Dresden, Uniklinikum "Carl Gustav Carus", TU Dresden, Germany; German Center for Diabetes Research (DZD e.V.), Neuherberg, Germany; Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
authors.forename: Juha M
authors.initials: JM
authors.lastname: Torkko
authors.affiliation: Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni (IDEHU), Universidad de Buenos Aires-CONICET, Argentina; Cátedra de Inmunología de la Facultad de Farmacia y Bioquímica, IDEHU, Argentina; División Endocrinología del Hospital de Clínicas José de San Martín, Universidad de Buenos Aires-CONICET, Argentina.
authors.forename: María E
authors.initials: ME
authors.lastname: Primo
authors.affiliation: Instituto Multidisciplinario de Biología Celular, CONICET, Argentina.
authors.forename: Ramiro E
authors.initials: RE
authors.lastname: Llovera
authors.affiliation: Instituto Multidisciplinario de Biología Celular, CONICET, Argentina; Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Argentina.
authors.forename: Pamela L
authors.initials: PL
authors.lastname: Toledo
authors.affiliation: Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Argentina.
authors.forename: Antonella S
authors.initials: AS
authors.lastname: Rios
authors.affiliation: Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires-CONICET, Junín 956, 1113, Buenos Aires, Argentina.
authors.forename: F Luis Gonzalez
authors.initials: FL
authors.lastname: Flecha
authors.affiliation: Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni (IDEHU), Universidad de Buenos Aires-CONICET, Argentina; Cátedra de Inmunología de la Facultad de Farmacia y Bioquímica, IDEHU, Argentina; División Endocrinología del Hospital de Clínicas José de San Martín, Universidad de Buenos Aires-CONICET, Argentina.
authors.forename: Aldana
authors.initials: A
authors.lastname: Trabucchi
authors.affiliation: Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni (IDEHU), Universidad de Buenos Aires-CONICET, Argentina; Cátedra de Inmunología de la Facultad de Farmacia y Bioquímica, IDEHU, Argentina; División Endocrinología del Hospital de Clínicas José de San Martín, Universidad de Buenos Aires-CONICET, Argentina.
authors.forename: Silvina N
authors.initials: SN
authors.lastname: Valdez
authors.affiliation: Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni (IDEHU), Universidad de Buenos Aires-CONICET, Argentina; Cátedra de Inmunología de la Facultad de Farmacia y Bioquímica, IDEHU, Argentina; División Endocrinología del Hospital de Clínicas José de San Martín, Universidad de Buenos Aires-CONICET, Argentina.
authors.forename: Edgardo
authors.initials: E
authors.lastname: Poskus
authors.affiliation: Paul Langerhans Institute Dresden, Uniklinikum "Carl Gustav Carus", TU Dresden, Germany; German Center for Diabetes Research (DZD e.V.), Neuherberg, Germany; Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
authors.forename: Michele
authors.initials: M
authors.lastname: Solimena
authors.affiliation: Instituto Multidisciplinario de Biología Celular, CONICET, Argentina; Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Argentina. Electronic address: ermacora@unq.edu.ar.
authors.forename: Mario R
authors.initials: MR
authors.lastname: Ermácora
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D001703:Biophysics; D049109:Cell Proliferation / Q000235:genetics; D006801:Humans; D007328:Insulin / Q000737:chemistry / Q000378:metabolism*; D007515:Islets of Langerhans / Q000737:chemistry / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055099:Neuroendocrine Cells / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics*; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D022142:Secretory Vesicles / Q000737:chemistry / Q000378:metabolism
chemical_list: D007328:Insulin; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C086381:regulated endocrine secretory protein 18; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8
keywords: Diabetes; IA-2; ICA512; Insulin; Protein tyrosine phosphatase; RESP18