Endocytosis of the AT1A angiotensin receptor is independent of ubiquitylation of its cytoplasmic serine/threonine-rich region.
Agonist-induced internalisation of the rat type 1A (AT(1A)) angiotensin II receptor is associated with phosphorylation of a serine/threonine-rich region in its cytoplasmic tail. In yeast, hyperphosphorylation of the alpha-factor pheromone receptor regulates endocytosis of the receptor by facilitating the monoubiquitylation of its cytoplasmic tail on lysine residues. The role of receptor ubiquitylation in AT(1A) receptor internalisation was evaluated by deletion or replacement of lysine residues in its agonist-sensitive serine/threonine-rich region. Expression of such receptor mutants in CHO cells showed that these modifications had no detectable effect on the angiotensin II-induced endocytosis of the AT(1A) receptor. Furthermore, fusion of ubiquitin in-frame to an internalisation-deficient AT(1A) receptor mutant with a truncated carboxyl-terminal tail did not restore the endocytosis of the resulting chimeric receptor. No impairment of receptor internalisation was observed after substitution of all lysine residues in the serine/threonine-rich region at saturating angiotensin II concentrations, where endocytosis occurs by a beta-arrestin and dynamin independent mechanism. Taken together, these data demonstrate that ubiquitylation of the cytoplasmic serine/threonine-rich region of the AT(1A) receptor on lysine residues is not required for its agonist-induced internalisation, and suggest that endocytosis of mammalian G protein-coupled receptors (GPCRs) occurs by a different mechanism than that of yeast GPCRs.
Mihalik
Balázs
B
Department of Physiology, Faculty of Medicine, Semmelweis University, P.O. Box 259, H-1444 Budapest, Hungary.
Gáborik
Zsuzsanna
Z
Várnai
Péter
P
Clark
Adrian J L
AJ
Catt
Kevin J
KJ
Hunyady
László
L
D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004705:Endocytosis / Q000502:physiology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D013912:Threonine / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism
D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin; D025801:Ubiquitin; D000804:Angiotensin II; D013912:Threonine; D012694:Serine