TY - JOUR
T1 - A single internalization signal from the di-leucine family is critical for constitutive endocytosis of the type II TGF-β receptor
AU - Ehrlich, Marcelo
AU - Shmuely, Avi
AU - Henis, Yoav I.
PY - 2001
Y1 - 2001
N2 - Endocytosis has an important contribution to the regulation of the surface expression levels of many receptors. In spite of the central role of the transforming growth factor β (TGF-β) receptors in numerous cellular and physiological processes, their endocytosis is largely unexplored. Current information on TGF-β receptor endocytosis relies exclusively on studies with chimeric constructs containing the extracellular domain of the GMCSF receptors, following the internalization of the GMCSF ligand; the conformation and interactions of the chimeric receptors (and therefore their endocytosis) may differ considerably from those of the native TGF-β receptors. Furthermore, there are no data on the potential endocytosis motif(s) of the TGF-β receptors or other receptor Ser/Thr kinases. Here, we report the use of type II TGF-β receptors, myc-tagged at their extracellular terminus, to investigate their endocytosis. Employing fluorescent antibody fragments to label exclusively the cell surface myc-tagged receptors exposed to the external milieu, made it possible to follow the internalization of the receptors, without the complications that render labeling with TGF-β (which binds to many cellular proteins) unsuitable for such studies. The results demonstrate that the full-length type II TGF-β receptor undergoes constitutive endocytosis via clathrin-coated pits. Using a series of truncation and deletion mutants of this receptor, we identified a short peptide sequence (I218I219L220), which conforms to the consensus of internalization motifs from the di-leucine family, as the major endocytosis signal of the receptor. The functional importance of this sequence in the full-length receptor was validated by the near complete loss of internalization upon mutation of these three amino acids to alanine.
AB - Endocytosis has an important contribution to the regulation of the surface expression levels of many receptors. In spite of the central role of the transforming growth factor β (TGF-β) receptors in numerous cellular and physiological processes, their endocytosis is largely unexplored. Current information on TGF-β receptor endocytosis relies exclusively on studies with chimeric constructs containing the extracellular domain of the GMCSF receptors, following the internalization of the GMCSF ligand; the conformation and interactions of the chimeric receptors (and therefore their endocytosis) may differ considerably from those of the native TGF-β receptors. Furthermore, there are no data on the potential endocytosis motif(s) of the TGF-β receptors or other receptor Ser/Thr kinases. Here, we report the use of type II TGF-β receptors, myc-tagged at their extracellular terminus, to investigate their endocytosis. Employing fluorescent antibody fragments to label exclusively the cell surface myc-tagged receptors exposed to the external milieu, made it possible to follow the internalization of the receptors, without the complications that render labeling with TGF-β (which binds to many cellular proteins) unsuitable for such studies. The results demonstrate that the full-length type II TGF-β receptor undergoes constitutive endocytosis via clathrin-coated pits. Using a series of truncation and deletion mutants of this receptor, we identified a short peptide sequence (I218I219L220), which conforms to the consensus of internalization motifs from the di-leucine family, as the major endocytosis signal of the receptor. The functional importance of this sequence in the full-length receptor was validated by the near complete loss of internalization upon mutation of these three amino acids to alanine.
KW - Di-leucine
KW - Endocytosis
KW - Immonofluorescence
KW - TGF-β receptors
UR - http://www.scopus.com/inward/record.url?scp=0034996021&partnerID=8YFLogxK
U2 - 10.1242/jcs.114.9.1777
DO - 10.1242/jcs.114.9.1777
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AN - SCOPUS:0034996021
SN - 0021-9533
VL - 114
SP - 1777
EP - 1786
JO - Journal of Cell Science
JF - Journal of Cell Science
IS - 9
ER -