The ubiquitin-dependent endocytosis motif is required for efficient incorporation of growth hormone receptor in clathrin-coated pits, but not clathrin-coated lattices.

Endocytosis of the growth hormone receptor (GHR) requires an active ubiquitin-conjugation system. In addition, it depends on a 10 amino acid residues motif in the GHR-cytoplasmic tail, the ubiquitin dependent-endocytosis or UbE-motif. To gain insight into the role of ubiquitination in the early steps of endocytosis, we performed an ultrastructural analysis of GH-uptake in Chinese hamster cells expressing wild-type or mutant GHRs. In wild-type GHR cells, GH was found to be exclusively taken up via clathrin-coated pits. In early endosomes it was efficiently sorted from recycling transferrin and targeted to the degradative pathway. Mutation of all lysine residues of a truncated GHR (GHR-399K-) precludes ubiquitination of the receptor, but internalization of GHR-399K- still depends on an active ubiquitin system. We found that GHR-399K- incorporates GH into clathrin-coated vesicles with the same efficiency as wild-type GHR. By contrast, a mutation in the UbE-motif (GHR-F327A) largely abolished incorporation of GH into clathrin-coated vesicles. Notably, access of GH to clathrin-coated lattices was not affected in GHR-F327A cells. These data corroborate and extend previous data that the UbE-motif but not ubiquitination of the receptor itself recruits GHR into clathrin-coated vesicles. Moreover, they suggest that incorporation of GHR into clathrin-coated lattices is differentially regulated from incorporation into clathrin-coated pits.