Role of amphiphysin II in somatostatin receptor trafficking in neuroendocrine cells.
Amphiphysins are SH3 domain-containing proteins thought to function in clathrin-mediated endocytosis. To investigate the potential role of amphiphysin II in cellular trafficking of G protein-coupled somatostatin (SRIF) receptors, we generated an AtT-20 cell line stably overexpressing amphiphysin IIb, a splice variant that does not bind clathrin. Endocytosis of (125)I-[d-Trp(8)]SRIF was not affected by amphiphysin IIb overexpression. However, the maximal binding capacity (B(max)) of the ligand on intact cells was significantly lower in amphiphysin IIb overexpressing than in non-transfected cells. This difference was no longer apparent when the experiments were performed on crude cell homogenates, suggesting that amphiphysin IIb overexpression interferes with SRIF receptor targeting to the cell surface and not with receptor synthesis. Accordingly, immunofluorescence experiments demonstrated that, in amphiphysin overexpressing cells, sst(2A) and sst(5) receptors were segregated in a juxtanuclear compartment identified as the trans-Golgi network. Amphiphysin IIb overexpression had no effect on corticotrophin-releasing factor 41-stimulated adrenocorticotropic hormone secretion, suggesting that it is not involved in the regulated secretory pathway. Taken together, these results suggest that amphiphysin II is not necessary for SRIF receptor endocytosis but is critical for its constitutive targeting to the plasma membrane. Therefore, amphiphysin IIb may be an important component of the constitutive secretory pathway.
Sarret
Philippe
P
Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada.
Esdaile
M James
MJ
McPherson
Peter S
PS
Schonbrunn
Agnes
A
Kreienkamp
Hans-Jürgen
HJ
Beaudet
Alain
A
D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D017398:Alternative Splicing; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism; D003346:Corticotropin-Releasing Hormone / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D051379:Mice; D018613:Microscopy, Confocal; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000502:physiology*; D009490:Neurosecretory Systems / Q000378:metabolism*; D017481:Receptors, Somatostatin / Q000378:metabolism*; D011994:Recombinant Proteins; D013004:Somatostatin / Q000378:metabolism / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured
D002966:Clathrin; D009419:Nerve Tissue Proteins; D017481:Receptors, Somatostatin; D011994:Recombinant Proteins; C075637:amphiphysin; D013004:Somatostatin; D000324:Adrenocorticotropic Hormone; D003346:Corticotropin-Releasing Hormone