The neuroendocrine protein VGF is sorted into dense-core granules and is secreted apically by polarized rat thyroid epithelial cells.
We have expressed the neuroendocrine VGF protein in FRT rat thyroid cells to study the molecular mechanisms of its sorting to the regulated and polarized pathways of secretion. By immunoelectron microscopy, we have demonstrated that VGF localizes in dense-core granules. Rapid secretion of VGF is induced by PMA stimulation. Moreover, human chromogranin B, a protein of the regulated pathway, co-localizes in the same granules with VGF. In confluent, FRT monolayers on filters protein secretion occur from the apical cell domain. VGF deletion mutants have been generated. By confocal microscopy, we have found that in transient transfection, all mutant proteins are sorted into granules and co-localize with the full-length VGF. They all retain the apical polarity of secretion. We also found that intracellular VGF and its deletion mutants are largely in an aggregated form. We conclude that FRT thyroid cells correctly decode the sorting information of VGF. The signals present on the protein to enter the granules and to be secreted apically cannot be separated from each other and are not in just one discrete portion of the protein. We propose that selective aggregation might represent the signal for sorting VGF to the regulated, apical route.
Gentile
Flaviana
F
Istituto di Endocrinologia ed Oncologia Sperimentale, CNR, Naples, Italy.
Calì
Gaetano
G
Zurzolo
Chiara
C
Corteggio
Annunziata
A
Rosa
Patrizia
P
Calegari
Federico
F
Levi
Andrea
A
Possenti
Roberta
R
Puri
Claudia
C
Tacchetti
Carlo
C
Nitsch
Lucio
L
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D016764:Cell Polarity / Q000502:physiology*; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000378:metabolism* / Q000648:ultrastructure; D004847:Epithelial Cells / Q000166:cytology / Q000502:physiology*; D017353:Gene Deletion; D016253:Microscopy, Immunoelectron; D016296:Mutagenesis; D009479:Neuropeptides; D021381:Protein Transport; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011994:Recombinant Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology; D013961:Thyroid Gland / Q000166:cytology* / Q000502:physiology*; D014162:Transfection
D009479:Neuropeptides; D011506:Proteins; D011994:Recombinant Proteins; D013873:Thionucleotides; C062040:Vgf protein, rat; D015124:8-Bromo Cyclic Adenosine Monophosphate; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D003994:Bucladesine; D000242:Cyclic AMP; D013755:Tetradecanoylphorbol Acetate