Disruption of disulfide bond formation alters the trafficking of prothyrotropin releasing hormone (proTRH)-derived peptides.
Rat prothyrotropin releasing hormone (proTRH) is processed in the regulated secretory pathway (RSP) of neuroendocrine cells yielding five TRH peptides and several non-TRH peptides. It is not understood how these peptides are targeted to the RSP. We show here that a disulfide bond in the carboxy-terminus of proTRH plays an important role in the trafficking of this prohormone. Recombinant proTRH was observed to migrate faster on a native gel when treated with dithiothreitol (DTT) suggesting the presence of a disulfide bond. In vitro disulfide bond formation was prevented either by DTT treatment or by mutating cysteines 213 and 219 to glycines. In both cases the peptides derived from these mutants exhibited increased constitutive release and processing defects when expressed in AtT20 cells, a neuroendocrine cell line used in our prior studies on proTRH processing. Immunocytochemistry revealed that wild-type proTRH and mutant proTRH localized in a punctate pattern typical of proteins sorted to the regulated secretory pathway. These data suggest that the proposed disulfide bond of proTRH is involved in sorting of proTRH-derived peptides and in their retention within maturing secretory granules. This is the first evidence of structural motifs being important for the sorting of proTRH.
Mulcahy
Lawrence R
LR
Department of Molecular and Cellular Biology and Biochemistry, Brown University, Providence, RI 02912, USA.
Barker
Alison J
AJ
Nillni
Eduardo A
EA
D020816:Amino Acid Motifs / Q000235:genetics / Q000502:physiology; D000818:Animals; D002850:Chromatography, Gel; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D007150:Immunohistochemistry; D008954:Models, Biological; D009154:Mutation; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D051381:Rats; D022142:Secretory Vesicles / Q000378:metabolism; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D004220:Disulfides; D010455:Peptides; D011498:Protein Precursors; D013973:Thyrotropin-Releasing Hormone; C046418:pro-thyrotropin releasing hormone; D003545:Cysteine; D011761:Pyrrolidonecarboxylic Acid