title: An extended tyrosine-targeting motif for endocytosis and recycling of the dense-core vesicle membrane protein phogrin.
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.
authors.affiliation: Barbara Davis Center for Childhood Diabetes, University of Colorado at Denver and Health Sciences Center, 4200 East 9th Avenue, Box B140, Denver, CO 80262, USA.
authors.forename: Christina
authors.initials: C
authors.lastname: Wasmeier
authors.forename: Patricia V
authors.initials: PV
authors.lastname: Burgos
authors.forename: Tammy
authors.initials: T
authors.lastname: Trudeau
authors.forename: Howard W
authors.initials: HW
authors.lastname: Davidson
authors.forename: John C
authors.initials: JC
authors.lastname: Hutton
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D019921:Biotinylation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D015151:Immunoblotting; D007376:Interleukin-2 / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry*; D051379:Mice; D008856:Microscopy, Fluorescence; D009154:Mutation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry*; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D022142:Secretory Vesicles / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D018076:DNA, Complementary; D007376:Interleukin-2; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D005982:Glutathione Transferase; C102889:PTPRN2 protein, human; D017027:Protein Tyrosine Phosphatases; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8