An extended tyrosine-targeting motif for endocytosis and recycling of the dense-core vesicle membrane protein phogrin

Traffic. 2005 Jun;6(6):474-87. doi: 10.1111/j.1600-0854.2005.00292.x.

Abstract

Integral membrane proteins of neuroendocine dense-core vesicles (DCV) appear to undergo multiple rounds of exocytosis; however, their trafficking and site of incorporation into nascent DCVs is unclear. Previous studies with phogrin (IA-2beta) identified sorting signals in the luminal domain that is cleaved post-translationally; we now describe an independent DCV targeting motif in the cytosolic domain that may function at the level of endocytosis and recycling. Pulse-chase radiolabeling and cell surface biotinylation experiments in the pituitary corticotroph cell line AtT20 showed that the mature 60/65 kDa form that resides in the DCV is generated by limited proteolysis in a post-trans Golgi network compartment with similar kinetics to the formation of the principal cargo, ACTH. Phogrin is exposed on the cell surface in response to stimuli and progressively internalized to a perinuclear compartment that overlaps with recycling endosomes marked by transferrin. Chimeric molecules of phogrin transmembrane and cytosolic sequences with the interleukin-2 receptor alpha chain (Tac) were sorted to DCVs through the action of an extended tyrosine-based motif Y(654)QELCRQRMA located in a 27aa sequence adjacent to the membrane-spanning domain. A 36aa domain terminating in this sequence conferred DCV localization to Tac in the absence of any other cytosolic or luminal phogrin components. The endocytosis and DCV targeting of phogrin Y(654) > A mutants correlated with the impaired binding of the phogrin cytosolic tail to the micro-subunit of the AP2 adaptor complex in vitro.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Motifs
  • Animals
  • Biotinylation
  • Cell Line
  • Cell Membrane / metabolism
  • Cytoplasm / metabolism
  • Cytosol / metabolism
  • DNA, Complementary / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Endocytosis
  • Endosomes / metabolism
  • Glutathione Transferase / metabolism
  • Golgi Apparatus / metabolism
  • Immunoblotting
  • Interleukin-2 / metabolism
  • Kinetics
  • Membrane Proteins / chemistry*
  • Mice
  • Microscopy, Fluorescence
  • Mutation
  • Protein Binding
  • Protein Processing, Post-Translational
  • Protein Structure, Tertiary
  • Protein Transport
  • Protein Tyrosine Phosphatases / chemistry*
  • Receptor-Like Protein Tyrosine Phosphatases, Class 8
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Proteins / chemistry
  • Secretory Vesicles / metabolism
  • Signal Transduction
  • Time Factors
  • Transfection
  • Tyrosine / chemistry*

Substances

  • DNA, Complementary
  • Interleukin-2
  • Membrane Proteins
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • Tyrosine
  • Glutathione Transferase
  • PTPRN2 protein, human
  • Protein Tyrosine Phosphatases
  • Receptor-Like Protein Tyrosine Phosphatases, Class 8