A beta-turn endocytic code is required for optimal internalization of the growth hormone receptor but not for alpha-adaptin association.
Intracellular trafficking of GH and its receptor, more particularly the chicken GH receptor (cGHR), was examined in COS-7 cells using biochemical and structural studies. Internalization of radioactive GH by the cGHR is reduced as compared with the rat GHR. On the contrary, activation of gene transcription through Janus kinase-2 was similar for both species. Secondary structures of the cytoplasmic domain of chicken and rat GHR were compared, since beta-turns were reported as internalization signals. The substitution of Pro335-Asp335, present in mammalian GH receptors, with Thr307-Gln308 in the cGHR leads to the loss of a beta-turn within a conserved cytoplasmic region. Mutational analysis indicated that the lower rate of internalization of cGHR, as compared with mammalian GHR, was due to this motif. Our data further show that alpha-adaptin, a subunit of adaptor protein AP-2, associates with the GHR upon hormone stimulation. The clathrin-coated pit pathway therefore seems to be involved in the endocytosis of cGHR, as AP-2 is known to intervene in the recruitment of receptors to these pits. Interaction with alpha-adaptin may occur through a common epitope of the chicken and mammalian GHR, since receptors from both species bind similar amounts of alpha-adaptin; alternatively, two different epitopes with similar affinity may be involved. Therefore, not alpha-adaptin but an uncharacterized factor, presumably interacting with the identified beta-turn endocytic code, is responsible for the difference in internalization kinetics. Finally, the present study illustrates that functional amino acid motifs of receptors can be derived from comparative studies.
Vleurick
L
L
INSERM U344, Faculté de Médecine Necker, Paris, France.
Pezet
A
A
Kühn
E R
ER
Decuypere
E
E
Edery
M
M
D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells / Q000378:metabolism; D002645:Chickens; D004268:DNA-Binding Proteins / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D000939:Epitopes; D019382:Human Growth Hormone / Q000378:metabolism; D053614:Janus Kinase 2; D008565:Membrane Proteins / Q000378:metabolism*; D008894:Milk Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011233:Precipitin Tests; D017433:Protein Structure, Secondary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D051381:Rats; D011986:Receptors, Somatotropin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D050799:STAT5 Transcription Factor; D015534:Trans-Activators / Q000378:metabolism
D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D004268:DNA-Binding Proteins; D000939:Epitopes; D008565:Membrane Proteins; D008894:Milk Proteins; D011518:Proto-Oncogene Proteins; D011986:Receptors, Somatotropin; D011994:Recombinant Proteins; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D019382:Human Growth Hormone; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; C507927:Jak2 protein, rat; D053614:Janus Kinase 2