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 chi cken GH receptor (cGHR), was examined in COS-7 cells using biochemical and structural studies. Internalization of radioactive GH by the cGHR is reduce d as compared with the rat GHR. On the contrary, activation of gene transcr iption through Janus kinase-2 was similar for both species. Secondary struc tures of the cytoplasmic domain of chicken and rat GHR were compared, since beta-turns were reported as internalization signals. The substitution of P ro(335)-Asp(336), present in mammalian GH receptors, with Thr(307)-GIn(308) in the cGHR leads to the loss of a beta-turn within a conserved cytoplasmi c region, Mutational analysis indicated that the lower rate of internalizat ion of cGHR, as compared with mammalian GHR, was due to this motif, Our dat a further show that alpha-adaptin, a subunit of adaptor protein AP-2, assoc iates with the GHR upon hormone stimulation. The clathrin-coated pit pathwa y therefore seems to be involved in the endocytosis of cGHR, as AP-2 is kno wn to intervene in the recruitment of receptors to these pits, Interaction with alpha-adaptin may occur through a common epitope of the chicken and ma mmalian GHR, since receptors from both species bind similar amounts of alph a-adaptin; alternatively, two different epitopes with similar affinity may be involved, Therefore, not alpha-adaptin but an uncharacterized factor, pr esumably interacting with the identified beta-turn endocytic code, is respo nsible for the difference in internalization kinetics. Finally, the present study illustrates that functional amino acid motifs of receptors can be de rived from comparative studies.