Biochemical, biophysical, and functional properties of ICA512/IA-2 RESP18 homology domain

SOSA L, TORKKO JM, PRIMO ME, LLOVERA RE, TOLEDO PL, RIOS AS, GONZALEZ FLECHA FL, TRABUCCHI A, VALDEZ SN, POSKUS E, SOLIMENA M, ERMáCORA MR

BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2015 vol. 1864 p. 511 - 522

1570-9639

Background: ICA512 (or IA-2/PTPRN) is a transmembrane protein-tyrosinephosphatase located in secretory granules of neuroendocrine cells. Previousstudies implied its involvement in generation, cargo storage, traffic, exocytosisand recycling of insulin secretory granules, as well as in â-cell proliferation. While several ICA512 domains have beencharacterized, the function and structure of a large portion of its N-terminalextracellular (or lumenal) region are unknown. Here, we report a biophysical,biochemical, and functional characterization of ICA512?RESP18HD, a domaincomprising residues 35 to 131 and homologous to regulated endocrine-specific protein18 (RESP18).Methods: Pure recombinant ICA512?RESP18HD was characterized by CD andfluorescence. Its binding to insulin and proinsulin was characterized by ELISA,surface plasmon resonance, and fluorescence anisotropy. Thiol reactivity wasmeasured kinetically. Targeting of ÄRESP18HDICA512?GFP to the membrane of insulinoma cells was monitored byimmunofluorescence.Results: ICA512?RESP18HDpossesses a strong tendency to aggregate andpolymerize via intermolecular disulfide formation, particularly at pH N 4.5.Its cysteine residues are highly susceptible to oxidation forming anintramolecular disulfide between cysteine 53 and 62 and intermoleculardisulfides via cysteine 40 and cysteine 47. The regulated sorting of ICA512 tosecretory granules in INS-1 cells was impaired by deletion of RESP18HD. ICA512?RESP18HDbinds with high-affinity to insulin and proinsulin.Conclusions: RESP18HD is required for efficient sorting of ICA512 tosecretory granules.General significance:RESP18HD is a key determinant for ICA512 granule targeting.