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 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-(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 (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.

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