A single internalization signal from the di-leucine family is critical for constitutive endocytosis of the type II TGF-beta receptor.
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 beta (TGF-beta) receptors in numerous cellular and physiological processes, their endocytosis is largely unexplored. Current information on TGF-beta receptor endocytosis relies exclusively on studies with chimeric constructs containing the extracellular domain of the GM-CSF receptors, following the internalization of the GM-CSF ligand; the conformation and interactions of the chimeric receptors (and therefore their endocytosis) may differ considerably from those of the native TGF-beta receptors. Furthermore, there are no data on the potential endocytosis motif(s) of the TGF-beta receptors or other receptor Ser/Thr kinases. Here, we report the use of type II TGF-beta 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-beta (which binds to many cellular proteins) unsuitable for such studies. The results demonstrate that the full-length type II TGF-beta 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 (I(218)I(219)L(220)), 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.
Ehrlich
M
M
Dept of Neurobiochemistry, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
Shmuely
A
A
Henis
Y I
YI
D020816:Amino Acid Motifs; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D017931:DNA Primers; D004705:Endocytosis; D007930:Leucine / Q000737:chemistry / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D016296:Mutagenesis; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
D017931:DNA Primers; D018125:Receptors, Transforming Growth Factor beta; D007930:Leucine